CN118019767A

CN118019767A - Optimized MULTABODY constructs, compositions, and methods

Info

Publication number: CN118019767A
Application number: CN202280051709.7A
Authority: CN
Inventors: J·朱利安; E·鲁加斯迭斯; B·特雷纳; T·T·赵
Original assignee: Hospital for Sick Children HSC; University of Toronto
Current assignee: Hospital for Sick Children HSC; University of Toronto
Priority date: 2021-07-12
Filing date: 2022-07-12
Publication date: 2024-05-10

Abstract

In various aspects, the self-assembled polypeptide complex comprises (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, and (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or subunit thereof.

Description

Optimized MULTABODY constructs, compositions, and methods

Cross Reference to Related Applications

The present application claims the benefit and priority of U.S. provisional patent application nos. 63/220,920 (filed on day 12 of 7 in 2021) and 63/289,016 (filed on day 13 of 12 in 2021), each of which is hereby incorporated by reference in its entirety for all purposes.

Sequence listing

The present application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on month 7 and 12 of 2022, named "Sequence Listing Jul-2022 3206-5068.Txt" and was 38,881 bytes in size.

Background

Therapeutic agents based on antibodies or antibody fragments are being developed for a variety of uses, such as for the treatment of various diseases or conditions.

However, antibody-based therapeutics may need to be tailored so that they have desirable properties (e.g., desirable biodistribution, half-life, etc.) after administration to a subject. Some antibody-based therapeutics have a different form than the native immunoglobulin molecule. For example, in some therapeutic agents, an antibody or antibody fragment is fused to another polypeptide; in some therapeutic agents, the antibody or antibody fragment is in a configuration or has a valence not found in nature. These antibody-based therapeutics may also need to be tailored.

Thus, there remains a need for optimized antibody-based therapeutics.

Disclosure of Invention

The present invention meets this need by providing a set of optimized self-assembling polypeptide complexes comprising antibody fragments. Depending on the desired properties (e.g., pharmacokinetic properties), a particular self-assembled polypeptide complex or set of complexes may be selected for use. For example, in certain embodiments, self-assembled polypeptide complexes having half-lives or other pharmacokinetic properties similar to IgG molecules are provided.

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 polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, and

(B) A plurality of second fusion polypeptides, each second fusion polypeptide comprising (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or subunit thereof.

In one aspect, (1) if the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds to SARS-CoV-2; and/or (2) if the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds CD 19.

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

In one aspect, the ferritin 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 ferritin monomer is human ferritin.

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

In one aspect, the Fc polypeptide is an IgG2 Fc polypeptide.

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

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

In one aspect, an antigen-binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

In one aspect, the antigen-binding antibody fragment is a Fab fragment.

In one aspect, each second fusion polypeptide does not comprise any CH2 or CH3 domain.

In one aspect, the one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn.

In one aspect, the mutation or set of mutations comprises a mutation at one or more of the following residues: m252, I253, S254, T256, K288, M428, and N434, or combinations thereof, wherein numbering is according to EU index.

In one aspect, the mutation or set of mutations comprises a mutation at M428 and N434, wherein the numbering is according to the EU index.

In one aspect, the mutation or set of mutations comprises an M428L and an N434S mutation, wherein the numbering is according to the EU index.

In one aspect, the altered binding to FcRn is reduced binding to FcRn.

In one aspect, the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I253V and K288A, and combinations thereof, wherein numbering is according to the EU index.

In one aspect, the one or more mutations comprise a mutation or set of mutations associated with altered effector function.

In one aspect, the Fc polypeptide is an IgG1 Fc polypeptide, and wherein the mutation or set of mutations comprises mutations at one or more of the following residues: l234, L235, G236, G237, P329 and a330 or combinations thereof, wherein numbering is according to EU index.

In one aspect, the altered effector function is a reduced effector function.

In one aspect, the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A/L235A), LALAP (L234A/L235A/P329G), G236R, G237A and a330L, wherein numbering is according to the EU index.

In one aspect, the nanocage monomer or subunit thereof is a ferritin monomer subunit, and

A. Each first fusion polypeptide comprises a ferritin monomeric subunit that is C-hemiferritin, and each second fusion polypeptide comprises a ferritin monomeric subunit that is N-hemiferritin; or alternatively

B. Each first fusion polypeptide comprises a ferritin monomeric subunit that is N-hemiferritin and each second fusion polypeptide comprises a ferritin monomeric subunit that is C-hemiferritin.

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

In one aspect, within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof via an amino acid linker.

In one aspect, within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof.

In one aspect, within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the nanocage monomer or subunit thereof via an amino acid linker.

In one aspect, within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof.

In one aspect, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or subunit thereof, wherein the third fusion polypeptide is different from the second fusion polypeptide.

In one aspect, the antigen-binding antibody fragment within the third fusion polypeptide comprises a light chain variable domain and a heavy chain variable domain.

In one aspect, the antigen-binding antibody fragment within the third fusion polypeptide is a Fab fragment.

In one aspect, each third fusion polypeptide does not comprise any CH2 or CH3 domain.

In one aspect, the antigen-binding antibody fragment of the second fusion polypeptide is capable of binding a first epitope, the antigen-binding fragment of the third fusion polypeptide is capable of binding a second epitope, and the first epitope and the second epitope are different and non-overlapping.

In one aspect, the first epitope and the second epitope are from the same protein.

In one aspect, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides.

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

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

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

In one aspect, the self-assembled polypeptide complex has a half-life of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days when administered to a subject in need thereof.

In one aspect, the self-assembled polypeptide complex is characterized in that, upon administration of a composition comprising the self-assembled polypeptide complex, the self-assembled polypeptide complex has a half-life substantially similar to a reference IgG molecule administered by the same route of administration and in a similar composition.

In one aspect, the reference IgG molecule is an antibody from which an antigen-binding antibody fragment within the second fusion polypeptide is derived, or an antibody from which an antigen-binding antibody fragment within the third fusion polypeptide is derived.

In one aspect, the self-assembled polypeptide complex has a half-life of about 3 days to about 35 days when administered to a subject in need thereof.

In one aspect, the self-assembled polypeptide complex is capable of being detected in serum at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration to a subject in need thereof.

In one aspect, the area under the curve (AUC) of the self-assembled polypeptide complex when administered to a subject in need thereof is at least 10 days, μg/mL, at least 25 days, μg/mL, at least 50 days, μg/mL, at least 100 days, μg/mL, at least 200 days, μg/mL, at least 300 days, μg/mL, at least 400 days, μg/mL, at least 500 days, μg/mL, at least 750 days, at least 1000 days, μg/mL, at least 1500 days, μg/mL, at least 2000 days, μg/mL, at least 2500 days, at least 3000 days, μg/mL, at least 4000 days, μg/mL, at least 5000 days, μg/mL, at least 6000 days, μg/mL, at least 7000, μg/mL, or at least 8000 days, μg/mL.

In one aspect, the area under the curve (AUC) of the self-assembled polypeptide complex is from about 10 days μg/mL to about 8000 days μg/mL when administered to a subject in need thereof.

In one aspect, the maximum concentration of self-assembled polypeptide complex (C _max) when administered to a subject in need thereof is at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 250 μg/mL, at least 500 μg/mL, at least 750 μg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 75mg/mL, at least 100mg/mL, at least 250mg/mL, at least 500mg/mL, or at least 750mg/mL.

In one aspect, the maximum concentration of self-assembled polypeptide complex (C _max) is from about 10 μg/mL to about 750mg/mL when administered to a subject in need thereof.

In one aspect, the subject is a human.

In one aspect, the administration to the subject is by parenteral administration.

In one aspect, administration to a subject is by subcutaneous administration, intravenous administration, intramuscular administration, intranasal administration, or by inhalation.

In one aspect, the self-assembled polypeptide complex is characterized in that the self-assembled polypeptide complex induces Antibody Dependent Cellular Phagocytosis (ADCP) in an in vitro model.

In one aspect, ADCP is induced at a level of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% target internalization.

According to one aspect, there is provided a method comprising administering to a mammalian subject a composition comprising a self-assembled polypeptide complex as described herein.

In one aspect, the subject is a human.

In one aspect, the method comprises administration by a systemic route.

In one aspect, the systemic route includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

In one aspect, the self-assembled polypeptide complex has a half-life in a mammalian subject of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration.

In one aspect, the self-assembled polypeptide complex has a half-life in a mammalian subject of from 3 days to 35 days after administration.

In one aspect, the area under the curve (AUC) of the self-assembled polypeptide complex in a mammalian subject after administration is at least 10 days, μg/mL, at least 25 days, μg/mL, at least 50 days, μg/mL, at least 100 days, μg/mL, at least 200 days, μg/mL, at least 300 days, μg/mL, at least 400 days, μg/mL, at least 500 days, μg/mL, at least 750 days, μg/mL, at least 1000 days, μg/mL, at least 1500 days, μg/mL, at least 2000 days, μg/mL, at least 2500 days, at least 3000 days, μg/mL, at least 4000 days, μg/mL, at least 5000 days, μg/mL, at least 6000 days, μg/mL, at least 7000 days, μg/mL, or at least 8000 days, μg/mL.

In one aspect, the area under the curve (AUC) of the self-assembled polypeptide complex in a mammalian subject after administration is from about 10 days μg/mL to about 8000 days μg/mL.

In one aspect, the maximum concentration (Cmax) of the self-assembled polypeptide complex in the mammalian subject after administration is at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 250 μg/mL, at least 500 μg/mL, at least 750 μg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 75mg/mL, at least 100mg/mL, at least 250mg/mL, at least 500mg/mL, or at least 750mg/mL.

In one aspect, the maximum concentration (Cmax) of the self-assembling polypeptide complex in the mammalian subject after administration is from about 10 μg/mL to about 750mg/mL.

According to one aspect, there is provided a fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, wherein the one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn and/or altered effector function.

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

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

In one aspect, the fusion polypeptide does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

In one aspect, the ferritin monomer is human ferritin.

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

In one aspect, the Fc polypeptide is an IgG2 Fc polypeptide.

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

In one aspect, the altered binding to FcRn is reduced binding to FcRn.

In one aspect, the altered effector function is a reduced effector function.

Drawings

FIG. 1A is a schematic representation of a human ferritin light chain (hFTL) and exemplary N-and C-hemiferritin molecules.

Fig. 1B, 1C, and 1D are illustrations of exemplary Multabody formation of the present disclosure.

FIG. 2A is a negative staining electron micrograph of T-01MB, T-02MB and T-01MB.v2 each containing wild type IgG1 Fc.

FIG. 2B is a negative dye electron micrograph of T-01MB containing various Fc's.

FIGS. 3A and 3B provide Biological Layer Interferometry (BLI) concentration response curves for T-01MB binding to human FcRn at pH 5.6 and pH 7.4, respectively.

FIGS. 3C, 3D and 3E provide BLI concentration response curves for binding of T-01MB containing various Fc to human FcgammaRIIa, human FcgammaRIIb and FcgammaRI, respectively.

FIG. 3F provides a BLI concentration response curve for binding of T-01MB.v2 to human Fc receptor containing wild-type IgG1 Fc or Fc IgG1 having an M428L/N434S (LS) mutation.

FIG. 4A provides BLI concentration response curves for T-01MB, T-02MB and T-01MB.v2 binding to targets/epitopes of PGDM1400, N49P7, 10E8v4 or iMabs (as Fab contained in Multabody).

FIGS. 4B, 4C and 4D provide BLI concentration response curves for T-01MB binding of various Fc's to targets/epitopes of PGDM1400 (BG 5050 SOSIP.664D368R), N49P7 (93 TH 057) and 10E8v4 (gp 41 MPER), respectively.

Figure 4E provides BLI concentration response curves for binding of PGDM1400, N49P7, 10E8v4 or iMab IgG to bg5050sosip.664d368r, 93TH057, gp41 MPER and CD 4.

FIG. 5A illustrates an experiment described in example 4 and performed using CB17/Icr-Prkdc ^scid/IcrIc oCrl immunodeficiency (SCID) mice. FIGS. 5B, 5C, 5D and 5E illustrate serum levels of Multabody or IgG1 controls tested following subcutaneous administration in SCID mice.

FIG. 6A shows an experiment described in example 4 and performed using NOD/Shi-scid/IL-2Rγnull immunodeficiency (NCG) mice. Figure 6B illustrates serum levels of Multabody or IgG1 controls tested following subcutaneous administration in NCG mice. Figure 6C provides the body weight of NCG mice following administration of Multabody or IgG1 controls tested. Mean ± SD of n=3 mice are shown.

Figure 7 illustrates the tested Multabody or control-induced dose-dependent phagocytosis, quantified and expressed as the percentage increase in internalization of 93TH057 coated microspheres compared to uncoated microspheres. * P <0.05, < P <0.001 and P <0.0001. n=four biologically independent samples.

FIGS. 8A, 8B and 8C illustrate widths and median IC ₅₀ values (μg/mL) of Multabody (T-01 MB, T-01MB.v2 and T-02MB, respectively) (diamonds), parent antibodies PGDM1400, N49P7 and 10E8v4 (circles), igG1 control (triangles) and N6/PGDM1400x10E8v4 trispecific antibodies (inverted triangles) tested, as determined using the TZM-bl assay described in example 6.

FIG. 8D illustrates dose-dependent neutralization of HIV-1 by T-01MB containing various Fc, as determined using the TZM-bl assay described in example 6.

FIG. 9A illustrates inhibition of CXCR 4-chemotactic HIV-1 isolate IIIB infection of PBMC by T-01MB, T-01MB.v2 or IgG1 controls. Mean ± SD of three technical replicates is shown. FIG. 9B shows the percentage of viable cells after T-01MB, T-01MB.v2 or IgG1 control relative to untreated control cells.

FIGS. 10A and 10B show the 4-week stability of T-01MB and T-01MB.v2 under temperature stress conditions (40 ℃). See example 8.

FIG. 11 HIV-1bNAb multimerization improves neutralization potency. (A) Schematic representation of self-assembly of apoferritin (24 subunits) and (B) single chain Fab apoferritin fusion. Fab Light (LC) and Heavy (HC) chains are shown in light pink and dark pink, respectively, and are linked to the N-terminus of the human apoferritin light chain (grey) by a GGS-like flexible linker (dark). (C) Schematic representation of the different Fab densities shown on human apoferritin. Co-transfection of scFab-human apoferritin encoding plasmids with unconjugated apoferritin at a ratio of 1:4 (dark yellow), 1:1 (black), 4:1 (blue) and 1:0 (red) resulted in molecules with different scFab valences, as evidenced by less unconjugated apoferritin in size exclusion chromatography, volume of elution, and SDS-PAGE. Negative staining electron micrographs (scale bar 50 nm) of samples with lowest and highest scFab titers are shown. (D) Affinity effects on neutralization of five bNAb against five PsV groups (pvo.04, JRCSF, BG 505T 332N, THRO4156.18, and T278-50). The fold increase in potency was calculated as parent IgG IC ₅₀ (μg/mL) divided by Fab apoferritin fusion IC ₅₀ (μg/mL). Due to neutralization resistance, the fold increase in efficacy analysis was omitted in the following cases: N49P7-T278-50, VRC01-T278-50 and 10-1074-THRO4156.18. Bars (±sd) represent the average of n=3 biologically independent samples.

Characterization of scFab-apoferritin fusion. (A) SDS-PAGE bands corresponding to scFab-apoferritin and unconjugated apoferritin were quantified by densitometry using imageJ software (rsb.info.nih.gov/ij /). Intensity plot (B) of the bands in each lane (yellow box) is shown. The approximate number of scFab shown on the particles of (C) is calculated as follows: intensity/total intensity of scFab bands and compared to the theoretical number deduced from the DNA ratio used for co-transfection.

Fig. 13 design, assembly and neutralization spectra for HIV Multabody of groups 14-PsV. (A) Schematic representation of a human apoferritin cleavage design driving heterodimerization of scFab-human apoferritin subunits. (B) Size exclusion chromatography of 24-mer PGDM1400 scFab-apoferritin particles (black) and T-01MB (dark red) was used in combination with multi-angle light scattering. The molar mass of each elution peak (line under UV absorbance) is expressed as MDa. (C) Negative staining electron micrograph of T-01MB (scale bar 50 nm). (D) concentration response curve of T-01MB binding to multiple epitopes. The PGDM1400, N49P7 and 10E8 binding sites on the surface representation of the HIV-1Env trimer (grey) are colored in red, blue and pink, respectively. Red lines represent raw data; the black line represents the global fit. (E) The width (cut-off IC ₅₀ set to 10 μg/mL) and median IC ₅₀ values (μg/mL) of T-01MB (red diamonds), parent bNAb (white circles), igG combination (grey triangles) and N6/PGDM1400x10E8v4 trispecific antibody (black triangles). Groups 14-PsV were selected based on sensitivity and resistance to the parental IgG. (F) Individual IC ₅₀ values (μg/mL) for each PsV variant. The solid line represents median neutralization IC ₅₀ for all 14 virus strains. Those pseudoviruses that show the highest neutralization resistance are highlighted with red boxes. (D) And (E) IC ₅₀ values were calculated from three biological replicates.

FIG. 14.Multabody affinity purification protocol. Protein a and protein L were successively affinity purified. Binding to protein a was enriched with Multabody having Fc (green), while protein L was enriched with Multabody having kappa chain Fab PGDM1400 (blue). Complementation of the two halves of the apoferritin split design ensures the presence of N49P7/iMab (orange) and 10e8v4 scFab (pink) (fused to C-ferritin) during the protein a purification step. An alanine-to-proline point mutation was introduced at position 12 of the kappa chain of iMab to disrupt binding to protein L. Gel filtration is performed to separate any aggregated material or unassembled components.

FIG. 15. Multabody cross-targeted HIV-1Env and CD4 receptors were generated. Schematic representation of (a) MB component. (B) Size exclusion chromatography of 24-mer PGDM1400 scFab-apoferritin particles (black) and T-02MB (blue) was used in combination with multi-angle light scattering. The molar mass of each sample is shown as MDa. (C) Negative staining electron micrograph (scale bar 50 nm) and (D) binding spectrum of T-02 MB. (E) The width of T-02MB (red diamonds) compared to its parent bNAb (white circles) and IgG combination (grey triangles) (cut-off IC ₅₀ set to 10 μg/mL) and median IC ₅₀ value (μg/mL). The average IC ₅₀ values (μg/mL) of three biological replicates of those pseudoviruses showing the highest neutralization resistance to T-02MB are shown.

FIG. 16 biophysical characterization of HIV-1 Multabody. Comparison of T _m and T _agg temperatures for T-01/T-02MB, 12-mer ferritin fusion, parent IgG and N6/PGDM1400x10E8v4 trispecific antibodies.

Binding properties of igg to four different antigens. BLI response curve of IgG binding to 93TH057 gp120, BG505 sosip.664_d368R, MPER peptide and CD4 immobilized on Ni-NTA biosensor. BG505 sosip.664_d368R trimer and 93TH057 gp120 monomer were selected as epitope specific ligands for PGDM1400 and N49P7, respectively. Red lines represent raw data; the black line represents the global fit.

Engineering and biophysical characterization of multabody v 2. (A) The second generation Multabody design exhibits two different features compared to the original Multabody design: 1) Fc (green) fused to the C-terminus of the other half of apoferritin in the split ferritin design; and 2) the single chain Fc domain fused to the C-terminus of the apoferritin semi-oligomer (green) reverts to the monomeric Fc chain. Dimerization of each Fc in mb.v2 drives the assembly of four fabs (two Fab2 and two Fab 3-bottom rows), whereas each Fc assembles only one Fab into the previous MB version (top row). (B) Negative staining electron micrograph of T-01MB.v2 (scale bar 50 nm). (C) Concentration response curve for T-01mb.v2 binding to multiple epitopes. Red lines represent raw data; the black line represents the global fit. (D) Comparison of T _agg, and (E) long term stability of two different Multabody versions under temperature stress conditions (10 mg/ml;40 ℃). A comparison of PsV neutralization (mean ± SD of two technical replicates) at week 0 and week 4 is shown.

Fig. 19. Multhady v2 feature. (A) The fusion modification of Fc (green) from N-terminal of N-ferritin (top row) to C-terminal of C-ferritin (bottom row) reverses the direction of Fc in Multabody. (B) The Fc dimerization of two Fc chains fused to the C-terminus of two independent ferritin subunits at the 4-fold symmetry axis of the apoferritin nanocage serves as an additional driver for Multabody v2 assembly.

Figure 20 fine tuning of Fc on Multabody to obtain IgG-like properties. (A) Concentration response curves for pH dependent binding of T-01MB and T-01MB.v2 to human FcRn. (B) Comparison of apparent FcRn binding affinity (K _D) of MB and IgG1 at acidic pH. N=3 biologically independent samples are shown. Apparent K _D below 10 ^-12 M (black dashed line) exceeded the instrument detection limit. (C) Concentration response curves for high affinity human fcyri (top) and low affinity human fcyriia (bottom). (D) Dose-dependent phagocytosis, determined as the percentage increase in internalization of 93TH057 coated fluorescent microspheres compared to no antibody control. Anti-human FcR binding inhibitor antibodies were added to block Fc-mediated internalization (dark red). Data were analyzed by two-way ANOVA and Tukey multiple comparison test. Each group was compared to IgG negative control. * P <0.05, < P <0.001 and P <0.0001. IgG and MB samples without affinity for antigen coated beads were added as control samples. n=four biologically independent samples. (E) Serum levels following subcutaneous administration of 5mg/kg Multabody or parental IgG mixture in female NOD/Shi-scid/IL-2Rγnull (NCG) immunodeficient mice. (F) body weight after administration of 5mg/kg of the molecule in NCG mice. The ± average SD of n=3 mice is shown in (E) and (F).

Multhady v2 has broad and potent neutralizing effect on the extended HIV-1PsV group. (A) Version T-01Multabody (red diamonds of different shades), single IgG (black circles) and IgG mixtures (blue triangles) were used for the width and median IC ₅₀ values (μg/mL) of the 25-PsV group (where 56% of the PsV variants were resistant to PGDM1400 neutralization). (B) Individual IC ₅₀ values (μg/mL) for each PsV variant. (A) And (B) IC ₅₀ values were calculated from three biological replicates. (C) The T-01Multabody version and parental IgG and IgG mixtures were compared against efficacy (IC ₅₀) -width (left) and efficacy (IC ₈₀) -width (right) curves of 118 HIV-1PsV variants extension multi-arm groups. (D) Individual IC ₅₀ (left) and IC ₈₀ (right) values for each PsV variant in (C). The yellow dots correspond to IC ₅₀ values for PsV that are highly resistant to neutralization by PGDM 1400. (B) And (D) solid lines represent median IC ₅₀ neutralization titers for all virus strains in each group.

FIG. 22 neutralization of PsV of multhabodies and inhibition of primary PBMC infection. (A) The molar potency (IC ₅₀) -width (left) and molar potency (IC ₈₀) -width (right) images of the T-01Multabody version and parent IgG and IgG mixtures against the 118 HIV-1PsV variant group were compared. (B) HIV-1 replication in PBMC culture supernatants derived from three different blood donors was measured via p24 assay. The data shown represent HIV-1 replication levels 7 days after CXCR 4-chemotactic HIV-1 isolate IIIB infection. Mean ± SD of three technical replicates is shown. (C) The effect of IgG mixtures and Multabody treatment on cell viability was shown as a percentage of surviving cells relative to untreated control cells under the same experimental conditions as (B).

Detailed Description

The inventors have previously described self-assembled polypeptide complexes comprising a fusion polypeptide comprising an antibody fragment. These self-assembling polypeptide complexes can be designed and adapted for a variety of therapeutic purposes. For example, as described herein, self-assembled polypeptide complexes comprising a fusion polypeptide comprising Fab and Fc can be used to target cells expressing an antigen to which Fab can bind, and the Fc portion can mediate interactions with other molecules in the body.

In many cases, it may be desirable to optimize the behavior of these self-assembled polypeptide complexes after administration, for example by modulating properties such as half-life and/or the ability to mediate antibody-mediated effects. Techniques for optimizing IgG molecules are known in the art. For example, modifications of the Fc region of IgG molecules can be used to modulate half-life and certain antibody-mediated effects.

However, the inventors have surprisingly found that in some cases techniques for optimizing IgG molecules may have unexpected effects when applied to the self-assembled polypeptide complexes of the inventors. As one example, the inventors have found that Fc modifications generally associated with reduced FcRn binding (and reduced half-life) in the context of IgG1 molecules actually confer more desirable bioavailability characteristics in the context of the self-assembled polypeptide complexes of the inventors.

With these and other insights, the inventors have developed a set of self-assembling polypeptide complexes, each of which is optimized for a particular desired result, and related methods.

For example, in certain embodiments, the self-assembled polypeptide complexes provided have one or more pharmacokinetic characteristics similar to a reference IgG molecule (e.g., an IgG molecule whose class matches that of an Fc chain within an Fc polypeptide within the self-assembled polypeptide complex) upon administration to a subject. For example, in some embodiments, the self-assembled polypeptide complexes as disclosed herein have similar bioavailability as the reference IgG molecule. In some embodiments, the self-assembled polypeptide complexes as disclosed herein have a half-life similar to a reference IgG molecule.

In certain embodiments, the provided self-assembled polypeptide complexes induce Antibody Dependent Cell Phagocytosis (ADCP) upon administration to a subject.

Definition of the definition

As used herein to refer to values, the terms "about" and "approximately" are used interchangeably and refer to values similar to the reference value. Generally, those skilled in the art who are familiar with the context will understand the relative degree of variation that is covered by the context "about" or "approximately. For example, in some embodiments, the terms "about" and "approximately" may encompass ranges of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the referenced values.

As used herein, the terms "change," "altered," "decrease," "reduced," "increase," "increased," or "decrease," "reduced" (e.g., referring to certain results or effects) have meanings relative to a reference level. In some embodiments, in the context of discussing mutations in an Fc chain or Fc polypeptide, the reference level is a known level or a level determined using IgG that does not contain a reference mutation in the Fc region.

The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) capable of assembling into a ferritin complex, which typically comprises 24 protein subunits. In some embodiments, the ferritin is a human ferritin, e.g., a human ferritin light chain having at least 85% sequence identity to SEQ ID No. 1 or UniProt P02792. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin may be wild-type human ferritin.

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 (e.g., 24 or more ferritin chains).

As used herein, the term "joint" is used to refer to an entity that connects two or more elements to form a multi-element agent. For example, one of ordinary skill in the art will understand that polypeptides whose structure includes two or more functional or tissue domains (e.g., fusion polypeptides) typically include a stretch of amino acids between such domains that connect them to one another. In some embodiments, the polypeptide comprising a linker element has the general structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains that are associated with each other by a linker (L). In some embodiments, the linker is an "amino acid linker," i.e., it comprises amino acid residues, e.g., the amino acid linker may comprise at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100 or more amino acid residues. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide.

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

As used herein, the term "multivalent" refers to a property of having at least two binding sites at which binding partners (e.g., antigens or receptors (e.g., fc receptors)) can bind. The binding partners that can bind to at least two binding sites can be the same or different.

As used herein, the term "nanocage monomer" refers to a single strand of a polypeptide that is capable of self-assembling with other nanocage monomers to form a self-assembled polypeptide complex comprising a plurality of nanocage monomers. In some embodiments, the nanocage monomers are selected from the group consisting of monomers of ferritin, apoferritin, encapsulation protein, thiooxidoreductase (SOR), dioxytetrahydropteridine synthase, pyruvate dehydrogenase, carboxylase, vault protein, groEL, heat shock protein, E2P coat protein, MS2 coat protein, fragments thereof, and variants thereof.

As used herein, the term "polypeptide" generally has the meaning of a polymer of at least three amino acids as recognized in the art, e.g., linked to each other by peptide bonds. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be generic enough to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional fragments (i.e., fragments that retain at least one activity) of such complete polypeptides. Furthermore, one of ordinary skill in the art will appreciate that protein sequences typically tolerate some substitution without disrupting activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity (typically greater than about 50%, 60%, 70% or 80%, and typically also includes at least one region of higher identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99% in one or more highly conserved regions, typically encompassing at least 3-4 and typically as much as 20 or more amino acids) with another polypeptide of the same class is encompassed within the relevant term "polypeptide" as used herein. The polypeptide may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the protein may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

When used in reference to a macromolecular complex (e.g., a polypeptide complex), the term "self-assembled" refers to the spontaneous formation of the complex (e.g., fusion polypeptide) to be formed when there is sufficient component of the complex. In some embodiments, the complex self-assembles under physiological conditions or in a buffer (e.g., solution) corresponding to physiological conditions.

As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is suffering from or susceptible to a related disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a human having one or more characteristics of susceptibility or risk to a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is a subject who is being administered and/or has been administered a diagnosis and/or therapy.

As used herein, the term "treatment" (and "treatment") or "treatment" refers to any administration of a therapy that partially or completely alleviates, ameliorates, alleviates, inhibits, delays onset of, reduces the severity of, and/or reduces the incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be directed to subjects that do not exhibit signs of the relevant disease, disorder, and/or condition and/or subjects that exhibit only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be directed to a subject exhibiting one or more determined signs of the associated disease, disorder, and/or condition. In some embodiments, the treatment may be directed to a subject that has been diagnosed as suffering from a related disease, disorder, and/or condition. In some embodiments, the treatment may be directed to a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and/or condition.

A. fusion polypeptides

In many embodiments, fusion polypeptides compatible with the compositions and methods disclosed herein generally comprise a nanocage monomer or subunit thereof linked to an Fc polypeptide or antigen-binding antibody fragment. Within the fusion polypeptide, the Fc polypeptide or antigen-binding antibody fragment may be linked to the nanocage monomer or subunit thereof at a particular end (e.g., N-terminus or C-terminus) of the nanocage monomer or subunit thereof. In some embodiments, the Fc polypeptide or antigen-binding antibody fragment is linked via an amino acid linker (such as a linker as described herein).

In some embodiments, (1) if the Fc polypeptide is an IgG1 Fc polypeptide, the antigen-binding fragment is not a Fab fragment that binds to SARS-CoV-2; and/or (2) if the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds CD 19.

1. Nanocage monomers and subunits thereof

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

"Subunit" of a ferritin monomer refers to a portion of a ferritin monomer that is capable of spontaneously associating with another, different subunit of a ferritin monomer such that the subunits together form a ferritin monomer that is in turn capable of self-assembling 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-hemiferritin" refers to about half of a ferritin chain, the half comprising the N-terminus of the ferritin chain. As used herein, the term "C-hemiferritin" refers to about half of a ferritin chain, said half comprising the C-terminus of the ferritin chain. The exact point at which the ferritin chains may be separated to form N-and C-hemiferritin may vary depending on the embodiment. In the context of a human ferritin light chain based ferritin monomer subunit, for example, the two halves may be separated at a point corresponding to a position of about position 75 to about position 100 (or a substantial portion thereof) 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 majority 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 majority thereof) of SEQ ID No. 1.

In some embodiments, the two halves are separated at a point corresponding to the position of 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 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 of SEQ ID No. 1.

Fc polypeptide

In certain embodiments, a fragment crystallizable (Fc) polypeptide comprises Fc chains each having one or more mutations relative to a reference Fc chain of the same Ig class. As explained further below, the reference Fc chain may belong to, for example, the IgG1 or IgG2 class.

Unless otherwise indicated, the numbering of mutations within antibody fragments (e.g., fc polypeptides) throughout the disclosure is according to the EU index.

In some embodiments, the Fc polypeptide is a human IgG Fc polypeptide, i.e., the Fc polypeptide comprises an Fc chain substantially similar to an Fc chain within wild-type human IgG, in addition to the mutations noted herein.

In some embodiments, the Fc polypeptide is an IgG1 Fc polypeptide (e.g., a human IgG1 Fc polypeptide), i.e., the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar to a chain within a wild-type IgG1 Fc, in addition to the mutations noted herein. In some embodiments, the wild-type IgG1 Fc is a human IgG1 Fc, wherein each Fc chain has the amino acid sequence of SEQ ID NO. 5.

For example, an IgG1Fc polypeptide 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, an IgG1Fc polypeptide comprises an Fc chain comprising an Fc mutation specifically described for the IgG1Fc polypeptide, but having an amino acid sequence with 100% identity to an Fc chain within a wild-type IgG1 Fc. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence which differs from the sequence of SEQ ID NO. 5 by at least one, at least two, at least three or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO 5 by NO more than ten, NO more than nine, NO more than eight, NO more than seven, NO more than six, NO more than five, or NO more than four amino acid residues.

In some embodiments, the Fc polypeptide is an IgG2 Fc polypeptide (e.g., a human IgG2 Fc polypeptide), i.e., the Fc polypeptide comprises an Fc chain having an amino acid sequence substantially similar to a chain within a wild-type IgG2 Fc, in addition to the mutations noted herein. In some embodiments, the wild-type IgG2 Fc is a human IgG2 Fc, wherein each Fc chain has the amino acid sequence of SEQ ID NO: 46.

For example, an IgG2 Fc polypeptide 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 IgG2a Fc. In some embodiments, an IgG2 Fc polypeptide comprises an Fc chain comprising an Fc mutation specifically described for the IgG2 Fc polypeptide, but having an amino acid sequence with 100% identity to an Fc chain within a wild-type IgG2 Fc. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO. 46 by at least one, at least two, at least three or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO. 46 by NO more than ten, NO more than nine, NO more than eight, NO more than seven, NO more than six, NO more than five, or NO more than four amino acid residues.

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

In some embodiments, the Fc polypeptide is an Fc monomer, e.g., a single Fc chain having only one CH2 domain (the second constant Ig domain of the heavy chain) and only one CH3 domain (the third constant Ig domain of the heavy chain), which is generally capable of dimerizing with another single Fc chain.

In some embodiments, the one or more mutations comprise a mutation or set of mutations associated with altered properties as further described herein. By "related to" is meant that in the context of an antibody (such as an IgG antibody), a mutation or set of mutations has been previously characterized as conferring altered properties (e.g., altered binding to FcRn, altered effector function, etc.). By "altered" is meant that the property (e.g., binding to an Fc receptor (e.g., fcRn)) is different than that observed in the absence of a mutation or group of mutations.

For example, in some embodiments, the altered property comprises altered binding to an Fc receptor.

In some embodiments, the altered property comprises altered binding to FcRn.

For example, the mutation or set of mutations associated with altered FcRn binding may comprise a mutation at one or more residues selected from the group consisting of: m252, I253, S254, T256, K288, M428, N434, or a combination thereof.

In some embodiments, altered binding to an Fc receptor comprises reduced binding to FcRn (e.g., reduced binding relative to a reference level corresponding to that observed in the absence of one or more mutations). For example, in some embodiments, the one or more mutations include a mutation or set of mutations associated with reduced binding to FcRn, e.g., I253A, I253V, K288A or a combination thereof.

In some embodiments, the one or more mutations include a mutation or set of mutations associated with altered effector function, e.g., altered binding to an Fc receptor (e.g., an fcγ receptor, such as fcγri, fcγrii, or fcγriib) associated with effector function.

For example, the one or more mutations may include mutations or groups of mutations at one or more residues selected from the group consisting of: l234, L235, G236, G237, P329, a330, and combinations thereof.

In some embodiments, altered binding to Fc receptors includes reduced effector functions, such as LALA (L234A/L235A), LALAP (L234A/L235A/P329G), G236R, G237A, A L, or a combination thereof.

3. Antigen binding antibody fragments

In certain embodiments, the antigen-binding antibody fragment comprises a heavy chain variable region (e.g., V _H). In certain embodiments, an antigen-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 antigen-binding antibody fragment comprises a Fab comprising 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 antigen-binding antibody fragment does not comprise any domain from the Fc region, e.g., does not comprise any CH2 or CH3 domain.

In some embodiments, the antigen binding fragment binds to an antigen on an infectious agent (e.g., a virus).

In some embodiments, the antigen-binding antibody fragment binds to an antigen on a target cell (e.g., a cancer cell or immune cell).

In embodiments using multiple types of fusion polypeptides with antigen-binding antibody fragments, the antigen-binding antibody fragments in the various types of fusion polypeptides may be capable of binding to the same epitope, or they may be capable of binding to different and non-overlapping epitopes. In some embodiments where the epitopes are different and non-overlapping, the epitopes are from the same protein.

4. Joint

In certain embodiments, the linker is used within a fusion polypeptide and/or within 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, e.g., 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., a (G _nS)_m sequence (e.g., GGS, GGGS (SEQ ID NO: 48) or GGGGS (SEQ ID NO: 49) sequence), that is 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.

B. Self-assembled polypeptide complexes

In one aspect, there is provided a self-assembled polypeptide complex comprising a plurality of fusion polypeptides as disclosed herein. Generally, a self-assembled polypeptide complex is provided comprising (a) a plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, and (b) a plurality of second fusion polypeptides, each second fusion polypeptide comprising (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or subunit thereof.

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

In some embodiments, the nanocage monomers or subunits thereof are ferritin monomer subunits, and (a) each first fusion polypeptide comprises a ferritin monomer subunit that is C-hemiferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is N-hemiferritin; or (b) each first fusion polypeptide comprises a ferritin monomeric subunit that is N-hemiferritin, and each second fusion polypeptide comprises a ferritin monomeric subunit that is C-hemiferritin.

In some embodiments, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides. In some embodiments, the self-assembled polypeptide complex comprises a total of 24 fusion polypeptides. In some embodiments, the self-assembled polypeptide complex comprises a total of greater 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 about 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 a ratio of the first fusion polypeptide to all other fusion polypeptides of about 1:1, 1:2, 1:3, or 1:4.

Pharmacokinetic properties

In certain embodiments, the provided self-assembled polypeptide complexes have one or more pharmacokinetic characteristics similar to a reference IgG molecule (e.g., an IgG molecule whose class matches the class of Fc chains within the Fc polypeptide of the first fusion polypeptide within the self-assembled polypeptide complex) when administered to a subject in need thereof. In some embodiments, upon administration of the self-assembled polypeptide complex to a human subject, a range of pharmacokinetic properties discussed herein (e.g., half-life, AUC, and/or C _max) is obtained. In some embodiments, the range of pharmacokinetic properties discussed herein is obtained when the self-assembled polypeptide complex is administered via a systemic route (e.g., via intravenous or subcutaneous administration).

In some embodiments, the self-assembled polypeptide complexes as disclosed herein have a half-life similar to a reference IgG molecule. The reference IgG molecule can be, for example, an antibody from which the antigen-binding antibody fragment within the second fusion polypeptide and/or the third fusion polypeptide within the self-assembled polypeptide complex is derived. For example, if the antigen binding fragment within the second fusion polypeptide and/or the third fusion polypeptide comprises a variable region from "antibody a", then in some embodiments the reference IgG molecule may be "antibody a".

In some embodiments, the self-assembled polypeptide complex has a half-life of about 3 to 35 days, about 3 to about 28 days, about 3 to about 21 days, about 3 to about 14 days, about 3 to about 10 days, about 3 to about 7 days, about 3 to about 5 days, about 5 to about 35 days, about 5 to about 28 days, about 5 to about 21 days, about 5 to about 14 days, about 5 to about 10 days, about 5 to about 7 days, about 7 to about 35 days, about 7 to about 28 days, about 7 to about 21 days, about 7 to about 14 days, about 7 to about 10 days, about 10 to about 35 days, about 10 to about 28 days, about 10 to about 21 days, about 10 to about 14 days, about 14 to about 35 days, about 14 to about 28 days, about 14 to about 21 days, about 21 to about 35 days, or about 21 to about 28 days after administration to a subject in need thereof. In some embodiments, the self-assembled polypeptide complex has a half-life of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration to a subject in need thereof. In some embodiments, the self-assembled polypeptide complex is capable of being detected in serum after at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration to a subject in need thereof.

In some embodiments, the self-assembled polypeptide complexes as disclosed herein have similar bioavailability to a reference IgG molecule (e.g., an antibody from which Fab fragments contained in the self-assembled polypeptide complexes are derived). For example, in some embodiments, after administration to a subject in need thereof, the self-assembled polypeptide complex has a concentration of about 10 to about 8000 day/μg/mL, about 10 to about 7000 day/μg/mL, about 10 to about 6000 day/μg/mL, about 10 to about 5000 day/μg/mL, about 10 to about 4000 day/μg/mL, about 10 to about 3000 day/μg/mL, about 10 to about 2500 day/μg/mL, about 10 to about 1000 day/μg/mL, about 10 to about 1500 day/μg/mL, about 10 to about 1000 day/μg/mL, about 10 to about 750 day/μg/mL, about 10 to about 500 day/μg/mL, about 10 to about 400 day/μg/mL, about 10 to about 300 day/μg/mL, about 10 to about 200 day/μg/mL, about 10 to about 100 day/μg/mL, about 10 to about 50 day/μg/mL, about 10 to about 1000 day/μg/mL about 10 to about 25 days · μg/mL, about 25 to about 8000 days · μg/mL, about 25 to about 7000 days · μg/mL, about 25 to about 6000 days · μg/mL, about 25 to about 5000 days · μg/mL, about 25 to about 4000 days · μg/mL, about 25 to about 3000 days · μg/mL, about 25 to about 2500 days · μg/mL, about 25 to about 1000 days · μg/mL, about 25 to about 1500 days · μg/mL, about 25 to about 1000 days · μg/mL, about 25 to about 750 days · μg/mL, about 25 to about 500 days · μg/mL, about 25 to about 400 days · μg/mL, about 25 to about 300 days · μg/mL, about 25 to about 200 days · μg/mL, about 25 to about 100 days · μg/mL, about 25 to about 50 days · μg/mL, about 50 to about 8000 day. Mu.g/mL, about 50 to about 7000 day. Mu.g/mL, about 50 to about 6000 day. Mu.g/mL, about 50 to about 5000 day. Mu.g/mL, about 50 to about 4000 day. Mu.g/mL, about 50 to about 3000 day. Mu.g/mL, about 50 to about 2500 day. Mu.g/mL, about 50 to about 2000 day. Mu.g/mL, about 50 to about 1500 day. Mu.g/mL, about 50 to about 1000 day. Mu.g/mL, about 50 to about 750 day. Mu.g/mL, about 50 to about 500 day. Mu.g/mL, about 50 to about 400 day. Mu.g/mL, about 50 to about 300 day. Mu.g/mL, about 50 to about 200 day. Mu.g/mL, about 50 to about 100 day. Mu.g/mL, about 100 to about 8000 day. Mu.g/mL, about 100 to about 7000 day. Mu.g/mL, about 100 to about 6000 day. Mu.g/mL about 100 to about 5000 day. Mu.g/mL, about 100 to about 4000 day. Mu.g/mL, about 100 to about 3000 day. Mu.g/mL, about 100 to about 2500 day. Mu.g/mL, about 100 to about 1000 day. Mu.g/mL, about 100 to about 1500 day. Mu.g/mL, about 100 to about 1000 day. Mu.g/mL, about 100 to about 750 day. Mu.g/mL, about 100 to about 500 day. Mu.g/mL, about 100 to about 400 day. Mu.g/mL, about 100 to about 300 day. Mu.g/mL, about 100 to about 200 day. Mu.g/mL, about 200 to about 8000 day. Mu.g/mL, about 200 to about 7000 day. Mu.g/mL, about 200 to about 6000 day. Mu.g/mL, about 200 to about 5000 day. Mu.g/mL, about 200 to about 4000 day. Mu.g/mL, about 200 to about 3000 day. Mu.g/mL, about 100 to about 300 day. Mu.g/mL, about 200 to about 300 day. Mu.g/mL, about 200 to about 2000 day. Mu.g/mL, about 200 to about 1000 day. Mu.g/mL, about 200 to about 1500 day. Mu.g/mL, about 200 to about 1000 day. Mu.g/mL, about 200 to about 750 day. Mu.g/mL, about 200 to about 500 day. Mu.g/mL, about 200 to about 400 day. Mu.g/mL, about 200 to about 300 day. Mu.g/mL, about 300 to about 8000 day. Mu.g/mL, about 300 to about 7000 day. Mu.g/mL, about 300 to about 6000 day. Mu.g/mL, about 300 to about 5000 day. Mu.g/mL, about 300 to about 4000 day. Mu.g/mL, about 300 to about 3000 day. Mu.g/mL, about 300 to about 2500 day. Mu.g/mL, about 300 to about 2000 day. Mu.g/mL, about 300 to about 1500 day. Mu.g/mL, about 300 to about 1000 day. Mu.g/mL, about 300 to about 750 day. Mu.g/mL, about 300 to about day. Mu.g/mL about 300 to about 500 days. Mu.g/mL, about 300 to about 400 days. Mu.g/mL, about 400 to about 8000 days. Mu.g/mL, about 400 to about 7000 days. Mu.g/mL, about 400 to about 6000 days. Mu.g/mL, about 400 to about 5000 days. Mu.g/mL, about 400 to about 4000 days. Mu.g/mL, about 400 to about 3000 days. Mu.g/mL, about 400 to about 2500 days. Mu.g/mL about 400 to about 2000 days. Mu.g/mL, about 400 to about 1500 days. Mu.g/mL, about 400 to about 1000 days. Mu.g/mL, about 400 to about 750 days. Mu.g/mL, about 400 to about 500 days. Mu.g/mL, about 500 to about 8000 days. Mu.g/mL, about 500 to about 7000 days. Mu.g/mL, about 500 to about 6000 days. Mu.g/mL, about 500 to about 5000 days. Mu.g/mL, about 500 to about 4000 days/g/mL, about 500 to about 3000 days/g/mL, about 500 to about 2500 days/g/mL, about 500 to about 2000 days/g/mL, about 500 to about 1500 days/g/mL, about 500 to about 1000 days/g/mL, about 500 to about 750 days/g/mL, about 750 to about 8000 days/g/mL, about 750 to about 7000 days/g/mL, about 750 to about 6000 days/g/mL, about 750 to about 5000 days/g/mL, about 750 to about 4000 days/g/mL, about 750 to about 3000 days/g/mL, about 750 to about 2500 days/g/mL, about 750 to about 2000 days/g/mL, about 750 to about 1500 days/g/mL, about 750 to about 1000 days/g/mL, about 1000 to about 8000 days/g/mL, about 1000 to about 7000/g/mL about 1000 to about 6000 day. Mu.g/mL, about 1000 to about 5000 day. Mu.g/mL, about 1000 to about 4000 day. Mu.g/mL, about 1000 to about 3000 day. Mu.g/mL, about 1000 to about 2500 day. Mu.g/mL, about 1000 to about 2000 day. Mu.g/mL, about 1000 to about 1500 day. Mu.g/mL, about 1500 to about 8000 day. Mu.g/mL, about 1500 to about 7000 day. Mu.g/mL, about 1500 to about 6000 day. Mu.g/mL, about 1500 to about 5000 day. Mu.g/mL, about 1500 to about 4000 day. Mu.g/mL, about 1500 to about 3000 day. Mu.g/mL, about 1500 to about 2500 day. Mu.g/mL, about 1500 to about 2000 day. Mu.g/mL, about 2000 to about 8000 day. Mu.g/mL, about 2000 to about 7000 day. Mu.g/mL, about 2000 to about 6000 day. Mu.g/mL, about 1500 to about 3000 day. Mu.g/mL, about 1500 to about 5000 day. Mu.g/mL, about 2000 to about 5000 days. Mu.g/mL, about 2000 to about 4000 days. Mu.g/mL, about 2000 to about 3000 days. Mu.g/mL, about 2000 to about 2500 days. Mu.g/mL, about 2500 to about 8000 days. Mu.g/mL, about 2500 to about 7000 days. Mu.g/mL, about 2500 to about 6000 days. Mu.g/mL, about 2500 to about 5000 days. Mu.g/mL, about 2500 to about 4000 days. Mu.g/mL, about 2500 to about 3000 days. Mu.g/mL, about 3000 to about 8000 days. Mu.g/mL, about 3000 to about 7000 days. Mu.g/mL, about 3000 to about 3000 days. Mu.g/mL, about 4000 to about 4000 days. Mu.g/mL, about 4000 to about 6000 days. Mu.g/mL, about 4000 to about 5000 days. Mu.g/mL, about 8000 to about 8000 days. Mu.g/mL, about 7000 to about 6000 days. Mu.g/mL. In some embodiments, the self-assembled polypeptide complex has an AUC of at least 10, 25, 50, 6000, 7000, or 8000 days μg/mL after administration to a subject in need thereof.

In some embodiments, the self-assembled polypeptide complexes as disclosed herein have similar bioavailability as the reference IgG molecule. For example, in some embodiments, after administration to a subject in need thereof, the self-assembled polypeptide complex has a concentration of about 10 μg/mL to about 750mg/mL, about 25 μg/mL to about 750mg/mL, about 50 μg/mL to about 750mg/mL, about 75 μg/mL to about 750mg/mL, about 100 μg/mL to about 750mg/mL, about 250 μg/mL to about 750mg/mL, about 500 μg/mL to about 750mg/mL, about 750 μg/mL to about 750mg/mL, about 1mg/mL to about 750mg/mL, about 10mg/mL to about 750mg/mL, about 25mg/mL to about 750mg/mL, about 50mg/mL to about 750mg/mL, about 75mg/mL to about 750mg/mL, about 100mg/mL to about 750mg/mL, about 250mg/mL to about 750mg/mL, about 500mg/mL to about 750mg/mL, about about 10 μg/mL to about 500mg/mL, about 25 μg/mL to about 500mg/mL, about 50 μg/mL to about 500mg/mL, about 75 μg/mL to about 500mg/mL, about 100 μg/mL to about 500mg/mL, about 250 μg/mL to about 500mg/mL, about 500 μg/mL to about 500mg/mL, about 750 μg/mL to about 500mg/mL, about 1mg/mL to about 500mg/mL, about 10mg/mL to about 500mg/mL, about 25mg/mL to about 500mg/mL, about 50mg/mL to about 500mg/mL, about 75mg/mL to about 500mg/mL, about 100mg/mL to about 500mg/mL, about 250mg/mL to about 500mg/mL, about 10 μg/mL to about 250mg/mL, about 25 μg/mL to about 250mg/mL, about 50 μg/mL to about 250mg/mL, about 75 μg/mL to about 250mg/mL, about 100 μg/mL to about 250mg/mL, about 250 μg/mL to about 250mg/mL, about 500 μg/mL to about 250mg/mL, about 750 μg/mL to about 250mg/mL, about 1mg/mL to about 250mg/mL, about 10mg/mL to about 250mg/mL, about 25mg/mL to about 250mg/mL, about 50mg/mL to about 250mg/mL, about 75mg/mL to about 250mg/mL, about 100mg/mL to about 250mg/mL, about 10 μg/mL to about 100mg/mL, about 25 μg/mL to about 100mg/mL, about 50 μg/mL to about 100mg/mL, about 75 μg/mL to about 100mg/mL, about 25mg/mL to about 100mg/mL about 100 to about 100mg/mL, about 250 to about 100mg/mL, about 500 to about 100mg/mL, about 750 to about 75mg/mL, about 100 to about 75mg/mL, about 1 to about 100mg/mL, about 10 to about 100mg/mL, about 25 to about 100mg/mL, about 50 to about 100mg/mL, about 75 to about 100mg/mL, about 10 to about 75mg/mL, about 25 to about 75mg/mL, about 50 to about 75mg/mL, about 75 to about 75mg/mL, about 100 to about 75mg/mL, about 250 to about 75mg/mL, about 500 to about 75mg/mL, about 750 to about 75mg/mL, about 1mg/mL to about 75mg/mL, about 10mg/mL to about 75mg/mL, about 25mg/mL to about 75mg/mL, about 50mg/mL to about 75mg/mL, about 10 μg/mL to about 50mg/mL, about 25 μg/mL to about 50mg/mL, about 50 μg/mL to about 50mg/mL, about 75 μg/mL to about 50mg/mL, about 100 μg/mL to about 50mg/mL, about 250 μg/mL to about 50mg/mL, about 500 μg/mL to about 50mg/mL, about 750 μg/mL to about 50mg/mL, about 1mg/mL to about 50mg/mL, about 10mg/mL to about 50mg/mL, about 25mg/mL to about 50mg/mL, about 10 μg/mL to about 25mg/mL, about 25 μg/mL about 50 μg/mL to about 25mg/mL, about 75 μg/mL to about 25mg/mL, about 100 μg/mL to about 25mg/mL, about 250 μg/mL to about 25mg/mL, about 500 μg/mL to about 25mg/mL, about 750 μg/mL to about 25mg/mL, about 1mg/mL to about 25mg/mL, about 10 μg/mL to about 10mg/mL, about 25 μg/mL to about 10mg/mL, about 50 μg/mL to about 10mg/mL, about 75 μg/mL to about 10mg/mL, about 100 μg/mL to about 10mg/mL, about 250 μg/mL to about 10mg/mL, about 500 μg/mL to about 10mg/mL, about 750 μg/mL to about 10mg/mL, about 1mg/mL, about 10mg/mL, about 10 μg/mL to about 1mg/mL, about 25 μg/mL to about 1mg/mL, about 50 μg/mL to about 1mg/mL, about 75 μg/mL to about 1mg/mL, about 100 μg/mL to about 1mg/mL, about 250 μg/mL to about 1mg/mL, about 500 μg/mL to about 1mg/mL, about 750 μg/mL to about 1mg/mL, about 10 μg/mL to about 750 μg/mL, about 25 μg/mL to about 750 μg/mL, about 50 μg/mL to about 750 μg/mL, about 75 μg/mL to about 750 μg/mL, about 100 μg/mL to about 750 μg/mL, about 250 μg/mL to about 750 μg/mL, about 500 μg/mL to about 750 μg/mL, about about 10 to about 500. Mu.g/mL, about 25 to about 500. Mu.g/mL, about 50 to about 500. Mu.g/mL, about 75 to about 100. Mu.g/mL, about 100 to about 500. Mu.g/mL, about 250 to about 500. Mu.g/mL, about 10 to about 250. Mu.g/mL, about 25 to about 250. Mu.g/mL, about 50 to about 250. Mu.g/mL, about 75 to about 250. Mu.g/mL, about 100 to about 250. Mu.g/mL, about 10 to about 100. Mu.g/mL, about 25 to about 100. Mu.g/mL, about 50 to about 100. Mu.g/mL, about 75 to about 100. Mu.g/mL, A maximum concentration (C _max) of about 10 μg/mL to about 75 μg/mL, about 25 μg/mL to about 75 μg/mL, about 50 μg/mL to about 75 μg/mL, about 10 μg/mL to about 50 μg/mL, about 25 μg/mL to about 50 μg/mL, or about 10 μg/mL to about 25 μg/mL. In some embodiments, the self-assembled polypeptide complex has a maximum concentration (C _max) of at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 250 μg/mL, at least 500 μg/mL, at least 750 μg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 75mg/mL, at least 100mg/mL, at least 250mg/mL, at least 500mg/mL, or at least 750mg/mL after administration to a subject in need thereof.

Functional effects

In certain embodiments, the provided self-assembled polypeptide complexes are capable of antibody-dependent cell phagocytosis (ADCP). In some embodiments, ADCP is induced at a level of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% target internalization. Methods for measuring ADCP are known in the art and include, for example, in vitro assays using macrophage cell lines and targets.

C. Therapeutic method

In one aspect, methods are provided that can be used to treat, ameliorate, or prevent a disease or condition (e.g., an infectious disease, cancer, or autoimmune disease), which generally include the step of administering to a subject a composition comprising a self-assembled polypeptide complex of the disclosure.

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

Compositions for administration to a subject generally comprise 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 administration by any of a variety of routes of administration, including systemic routes (e.g., oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

Examples

Example 1 expression and analysis of representative Multabody

In this example, multabody (MB) is formed from a fusion protein generated by fusing together the N-terminal fragment (residues 1-90) (N_hFTL, SEQ ID NO: 2) of human ferritin light chain (hFTL, SEQ ID NO: 1), hFTL, or the C-terminal fragment (residues 91-175) (C_hFTL, SEQ ID NO: 3) of hFTL with a single chain Fab (scFab) and/or fragment crystallizable region (Fc) or single chain Fc dimer (scFc) via a linker, such as the (Gly _n-Ser)_m peptide linker) as described herein.

T-01MB: preparing a gene encoding a fusion protein (1) scFab of HIV neutralizing antibody PGDM1400 fused to the N-terminus of hFTL (PGDM 1400-hFTL, SEQ ID NO: 27), (2) scFc fused to the N-terminus of n_ hFTL (scFc-n_hftl, SEQ ID NO: 34), (3) scFab of HIV neutralizing antibody N49P7 fused to the N-terminus of C hFTL (N49P 7-c_hftl, SEQ ID NO: 28), and (4) scFab of HIV neutralizing antibody 10E8v4 fused to the N-terminus of C hFTL (10E 8v4-c_hftl, SEQ ID NO: 30), mixed in a molar ratio of 4:2:1:1, and transiently transfected into HEK 293F cells to generate and form T-01MB. See, fig. 1B.

T-02 MB: the genes encoding the fusion proteins were prepared (1) PGDM1400-hFTL (SEQ ID NO: 27), (2) scFc-N_ hFTL (SEQ ID NO: 34), (3) scFab of the anti-CD 4 antibody ibalizumab (iMab), fused to the N-terminus of C_ hFTL (iMab-C_hFTL, SEQ ID NO: 31), and (4) 10E8v4-C_ hFTL (SEQ ID NO: 30), mixed in a molar ratio of 4:2:1:1, and transiently transfected into HEK 293F cells to produce and form T-02MB. See, fig. 1B.

T-01 MB.v2: preparation of the Gene encoding the fusion protein (1) PGDM1400-hFTL (SEQ ID NO: 27), (2) scFab of N49P7 fused to N-terminus of N_ hFTL (N49P 7-N_ hFTL (SEQ ID NO: 29), and (3) Fc monomer fused to C-terminus of 10E8-C_ hFTL (10E 8v4-C_ hFTL-Fc, SEQ ID NO: 32) mixed in a molar ratio of 3:1:1 and transiently transfected into HEK 293F cells to produce and form T-01MB.v2 see FIG. 1D (as described above for "T-01MB", 10E8-C_ hFTL contains scFab. Of HIV neutralizing antibody 10E8v4 fused to N-terminus of C_ hFTL, thus construct 10E8v4-C_ hFTL-Fc (SEQ ID NO: 32) contains C-half ferritin with C-half at the N-terminus of C-half ferritin and C-half at the C-terminus of 3-half ferritin Fc.)

Multabody described in this example has a wild-type (WT) Fc or an engineered IgG1 Fc. Such engineered IgG1 Fc contains any one or more mutations in L234A, L235A, K A, I253V, I253A, P329G, M428L, N434S or any combination thereof (according to EU numbering scheme). For example, T-01MB IgG1K 288A has an engineered IgG1 Fc with the K288A mutation; t-01MB IgG1 LALAP has an engineered IgG1 Fc with the L234A, L A and P329G mutations; t-01 MB.v2IgG1LS has an engineered IgG1 Fc with mutations M428L and N434S.

Multabody was purified using protein a affinity chromatography, optionally followed by protein L affinity chromatography. The Multabody containing fractions were concentrated and further purified by Size Exclusion Chromatography (SEC) in sodium phosphate buffer. After SEC purification, the size of Multabody formed was assessed using negative dye Electron Microscopy (EM) and/or SEC with inline multi-angle light scattering (SEC-MALS).

EXAMPLE 2 determination of Multabody binding to Fc receptor by biological layer interferometry

The binding kinetics and affinities of Multabody containing different Fc mutations to Fc receptors (human fcγ receptor I (hfcyri), hfcyriia, and hfcyriib) were determined by Biological Layer Interferometry (BLI) using the Octet RED96 BLI system (Pall ForteBio).

Briefly, his-tagged Fc receptors were loaded onto Ni-NTA biosensors to achieve a signal response of 0.8 nm. The association rate was measured by transferring the loaded biosensor into serially diluted wells containing test Multabody (20-10-5-2.5-1.25-0.65 nM) or IgG1 control (250-125-62.5-31.2-15.6-7.8 nM) for a contact time of 180 seconds. The IgG1 control was a mixture of PGDM1400, N49P7 and 10E8v4 antibodies, all with wild type IgG1 backbone. To assess the potential of Multabody to undergo endosomal recycling, their binding to the hFcRn/β2-microglobulin complex was measured at physiological pH (7.4) and acidic pH (5.6).

The Fc mutations of the IgG1 backbone evaluated in Multabody included: K288A, I253V and I253A, which reduce binding of antibodies to FcRn; P329G, LALA (L234A, L235A) and LALAP (L234A, L235A and P329G), which reduce binding of antibodies to fcγr; and combinations thereof. (numbering is according to EU numbering scheme.)

Representative examples of relevant fragments of the resulting sensorgrams are provided in fig. 3A, 3B, 3C, 3D, 3E, and 3F. The determined K _on、k_off values and the resulting Multabody equilibrium dissociation constants (K _D) are summarized in tables 1 and 2.

At acidic pH (5.6), T-01MB with wild-type IgG1 Fc bound FcRn with 1000-fold higher binding affinity than the IgG1 control; the same is observed for T-01MB having the K288A, I253V, P329G, LALA, LALAP, K288A+P329G or K288A+ LALAP IgG1 Fc mutation. The binding affinity of T-01MB with either the I253A or the I253a+ LALAP IgG1 Fc mutation to FcRn was similar to IgG1 control at pH 5.6. T-01MB with wild IgG1 Fc, P329G IgG1 Fc, LALA IgG1 Fc, or LALAP IgG Fc mutations showed measurable binding to FcRn at physiological pH (7.4).

T-01MB with wild-type IgG1 Fc bound to hFcγRI, hFcγRIIa and hFcγRIIb with 1000-fold higher affinity than the IgG1 control. Multabody with the P329G, LALA or LALAP IgG1 Fc mutation showed reduced or no binding to the tested fcγ receptor.

T-01MB.v2 with wild-type IgG1 Fc or with LS Fc mutations has a more similar FcRn binding profile to IgG1, has comparable binding capacity to human FcRn at acidic pH, but does not bind at physiological pH. In addition, T-01MB.v2 with mutations of the IgG1 Fc or LS type showed reduced binding to the high affinity Fc gamma RI and did not bind to the low affinity Fc gamma receptor tested, similar to T-01MB containing the LALAP mutation.

Among the Fc mutations and combinations of mutations tested, the i257a+ LALAP IgG Fc mutation combination adjusted the Fc receptor binding profile of Multabody (T-01 MB format) to IgG 1-like.

TABLE 1 kinetic constants and affinities for FcRn of Multabody determined by BLI

* Multabody showed residual binding to FcRn at pH 7.4

TABLE 2 kinetic constants and affinities for the Fcγ receptor determined by BLI Multabody

/>

Example 3 target binding of Multabody as determined by biological layer interferometry

The binding kinetics and affinity of PGDM 1400, N49P7, 10E8v4 or iMab (as Fab included in Multabody) to the respective targets were determined by BLI using the Octet RED96 BLI system (Pall ForteBio).

The experiment was performed similarly as described in example 2, except that the Hi-labeled targets PGDM1400, N49P7, 10E8v4 and BG5050 sosip.664d368r of iMab, gp120 subunit 93TH057, gp41 membrane proximal outer region (MPER) and soluble CD4 were loaded onto the Ni-NTA biosensor, respectively, to achieve a signal response of 0.8 nm. The loaded biosensors were then titrated with different concentrations of test Multabody, PGDM, N49P7, 10E8v4, or iMab antibodies (with wild-type IgG1 scaffold) or a mixture of PGDM1400, N49P7, and 10E8v4 antibodies (all with wild-type IgG1 scaffold) (IgG 1 control).

Representative examples of relevant fragments of the resulting sensorgrams are provided in fig. 4A, 4B, 4C, 4D, and 4E. The determined K _on、k_off values and the resulting Multabody equilibrium dissociation constants (K _D) are summarized in tables 3and 4.

TABLE 3 kinetic constants and affinities for target binding by Multabody determined by BLI

TABLE 4 kinetic constants and affinities for target binding by Multabody determined by BLI

EXAMPLE 4 pharmacokinetics of Multabody in mice

Analysis was performed in mice on the pharmacokinetics of Multabody with wild-type Fc or engineered IgG1 Fc.

On day 0, test Multabody or IgG1 control was injected into five CB17/Icr-Prkdc ^scid/IcrIcoCrl immunodeficient (SCID) mice/group at a dose of 5 mg/kg. The IgG1 control was a mixture of PGDM1400, N49P7 and 10E8v4 antibodies, all with wild type IgG1 backbone. Serum samples were collected starting on day 1 and were collected every two days for 9 days. An additional 5mg/kg dose was administered on day 10, and serum samples were collected on days 11 and 15.

Furthermore, multabody containing wild-type Fc, LALALAP+I253A IgG1 Fc mutant combination or M428L+N434S (LS) Fc mutant combination was subcutaneously injected into NOD/Shi-scid/IL-2Rγnull immunodeficient (NCG) mice (3 mice/group) at a single dose of 5 mg/kg. Blood samples were collected at various time points after injection. IgG1 controls were tested in parallel.

The level of circulation Multabody was assessed by ELISA. Briefly, 96-well plates were coated with 50. Mu.L of His-tagged antigen, which was recognized by Fab in Multabody. Mu.g/mL. Serum/blood samples were diluted and added to wells. Binding agents were detected using HRP-protein a as a secondary molecule. Chemiluminescent signals were quantified using a microplate reader. A calibration curve of a standard protein dilution was prepared.

Figures 5B-5E and 6B show graphs of plasma concentration of Multabody tested over time. The LALAP +i235A, LALAP +k288A and k288a+p329G mutant combination was able to restore serum levels of T-01MB to similar levels to IgG1 controls; LS mutant combinations were able to restore serum levels of T-01MB.v2 to levels similar to the IgG1 control. Thus, T-01MB IgG1LALAP I235A, T-01MB IgG1LALAP K288A, T-01MB IgG1 K288A P329G and T-01MB.v2 IgG1 LS exhibit antibody-like pharmacokinetics or favorable pharmacokinetic profiles.

EXAMPLE 5 evaluation of Multabody-induced antibody-dependent cellular phagocytosis

The potential of the selected Multabody to induce antibody-dependent cell phagocytosis (ADCP) was assessed using the THP-1 cell line.

Red fluorescent FluoSpheres NeutrAvidin microspheres were coated with biotinylated 93TH057 gp120 (antigen of N49P 7) and incubated with various concentrations of T-01MB IgG1 LALAP I253A or T-01mb.v2 igg1ls, or IgG1 mixtures of PGDM1400, N49P7 and 10e8v4 IgG1 antibodies at 37 ℃ for 2 hours before 200 μl THP-1 cells were added at 5x 10 ⁴ cells/well. After 16 hours, cells were pelleted and washed with PBS. Viability of cells was determined using a viable/dead fixable purple stain. Cells washed with PBS were fixed with 2% paraformaldehyde for 20min at room temperature, precipitated, and washed once with FACS buffer (pbs+10% FBS,0.5mM EDTA). The cells were then analyzed using a BD LSR II flow cytometer and the data was analyzed using FlowJo. IgG1 and Multabody controls without affinity for 93TH057 gp120 were tested in parallel; fcR binding inhibitor antibodies (Invitrogen, 14-9161-73) were used to block Fc-mediated internalization.

The results are depicted in fig. 7. Phagocytosis was quantified and expressed as the percentage of increased internalization of 93TH057 coated microspheres compared to uncoated microspheres. T-01MB.v2 IgG1LALAP I253A induced dose-dependent ADCP comparable to the IgG1 control, although binding to FcgammaRI was lower (see example 2).

EXAMPLE 6 evaluation of Multabody-mediated neutralization of HIV-1

The ability of selected Multabody to neutralize HIV-1 was assessed using a TZM-bl assay that measures HIV-1 neutralization as a function of reduced expression of the firefly luciferase (Luc) reporter gene modulated by HIV-1Tat after one round of infection with Env pseudotype virus.

Briefly, HIV-1 pseudotyped virus was generated by co-transfecting 293T cells with an HIV-1 subtype B backbone NL4-3.Luc.R ^- E plasmid (plasmid encoding full-length Env clone). Antibodies to Fab included in test Multabody, multabody, igG1 control-1 (mixture of PG DM1400, N49P7 and 10E8V4 IgG1 antibodies), igG1 control-2 (mixture of PG DM1400, iMab and 10E8V4 IgG1 antibodies) or N6/PGDM1400x10E8V4 trispecific antibodies (against CD4bs, V1V2 apex and MPER binding sites) were incubated with 10-15% tissue culture infectious doses of pseudovirus for 1 hour at 37 ℃ and then 44-72 hours with cells transfected with pseudotype virus. Virus neutralization was monitored by adding Brit elite plus reagent (PerkinElmer) to the cells and measuring luminescence relative to light units (RLU) using a Synergy Neo2 multi-function assay microplate reader. Test preparations were assayed against a single pseudovirus or against a group of 14 or 25 pseudoviruses (14-or 25-PsV groups). The 25-PsV group included strains in the 14-PsV group, and 11 HIV-1 strains with high resistance to PDGM1400 were added in the 14-PsV group. Thus, groups 25-PsV contained 56% of PsV variants resistant to PDGM1400 IgG neutralization (cut-off IC ₅₀ set to 10 μg/mL).

Exemplary results are shown in fig. 8A, 8B, 8C, and 8D, and the determined IC ₅₀ values and neutralization widths are summarized in tables 5 and 6. The median IC ₅₀ values of Multabody showed about one and two orders of magnitude reduction, respectively, compared to the IC ₅₀ values of IgG mixtures and trispecific antibodies. T-01MB.v2 (where the neutralization profile of antibodies N49P7 and 10E8v4 is more advantageous than other Multabody) achieved 100% neutralization in groups 25-PsV.

When tested against the extended multi-branched group of 118PsV, T-01mb.v2 matched the flood width of the corresponding IgG mixture (100% viral coverage, cut-off IC ₅₀ set to 10 μg/mL), but exhibited significant neutralization efficacy (fig. 21C-21D, fig. 22A and table 9). Specifically, igG mixture and T-01MB only neutralized 9% and 8% of PsV, respectively, with IC ₅₀ value of 0.001 μg/mL, while in the case of T-01MB.v2, 50% of PsV was still neutralized and IC ₅₀ value was only 0.001 μg/mL (FIG. 21C). Notably Multabody achieved median IC ₅₀ values of only 0.0009 μg/mL (0.4 pM), thus achieving 32-fold and 490-fold more efficient pan-neutralization in mass and molar concentration, respectively, compared to IgG mixtures (fig. 21D). Furthermore, IC ₈₀ of T-01mb.v2 demonstrated its propensity to neutralize better than that of individual IgG and IgG mixtures, neutralizing 96% of all strains tested, with a median IC ₈₀ value of 0.005 μg/mL (2.2 pM) (fig. 21C-21D, fig. 22A and table 9). Importantly, multabody also blocked infection of primary Peripheral Blood Mononuclear Cells (PBMC) by replication competent CXCR 4-chemotactic HIV-1IIIB strains (fig. 22B), showing enhanced efficacy over matched IgG mixtures, and no effect on cell viability (fig. 22C).

Table 5 HIV neutralization of multhabodies

TABLE 6 HIV neutralization by Multabody

Multabody	IC₅₀
		T-01MB IgG1	0.014nM
T-01MB IgG2	0.018nM
		T-01MB IgG1 P329G	0.0094nM
T-01MB IgG1 LALA	0.0041nM
		T-01MB IgG1 LALAP	0.012nM
T-01MB IgG1 K288A	0.014nM
		T-01MB IgG1 I253A	0.015nM
T-01MB IgG1 K288A P329G	0.0042nM
		T-01MB IgG1 LALAP K288A	0.0060nM
T-01MB IgG1 LALAP I253A	0.0074nM

EXAMPLE 7 evaluation of inhibition of HIV-1 infection by Multabody

Selected Multabody was evaluated for its ability to inhibit HIV-1 infection using human Peripheral Blood Mononuclear Cells (PBMCs).

Briefly, PBMC were obtained from three healthy blood donors and activated with Phytohemagglutinin (PHA) in the presence of recombinant human IL-2 in complete RPMI medium supplemented with 10% Fetal Bovine Serum (FBS) for 72 hours, followed by HIV-1 infection. Laboratory CXCR 4-chemotactic HIV-1 isolate IIIB was incubated with the Multabody or IgG1 control tested for 1 hour at room temperature and then added to activated PBMC in triplicate. The IgG1 control was a mixture of PGDM1400, N49P7 and 10E8v4 antibodies, all with wild type IgG1 backbone. The infected cells were cultured at a dose of 0.01-10ug/mL with or without the test Multabody or antibody control. HIV-1 replication levels were assessed by measuring extracellular release of p24 Gag protein in cell free culture supernatants using a high sensitivity ALPHALISA P detection kit on a BioTEK Synergy read plate apparatus at day 7 post infection according to the manufacturer's protocol. Cell viability was also assessed on day 7 of infection by fixing cells in 2% PFA and the absolute number of cells was counted by flow cytometry using BD LSRFortessa (Becton Dickinson).

Exemplary results are shown in fig. 9A and 9B. Multabody (T-01 MB and T-01 MB.v2) are capable of inhibiting infection of primary PBMC by CXCR 4-chemotactic HIV-1 isolate IIIB with replication capacity, with enhanced efficacy compared to IgG1 controls and without any effect on cell viability.

Example 8 characterization of the thermal stability of multabodies

The UNit system was used to determine the melting temperature (T _m) and aggregation temperature (T _agg) of Multabody and reference molecules (parent antibodies PGDM1400, N49P7, 10E8v4 and iMab; PGDM1400x10E8v4 trispecific antibodies). The sample was concentrated to 1.0mg/mL and warmed from 25 ℃ to 95 ℃ in 1 ℃ increments. The temperature at which T _m;T_agg was determined to be 50% increase in static light scattering relative to baseline was observed at 266nm wavelength was obtained by measuring center of gravity average fluorescence. The mean and standard error of 3 independent measurements were calculated using UNit analysis software. Table 7 summarizes T _m and T _agg.

The stability of Multabody was further analyzed under accelerated conditions. The samples were concentrated to 10mg/mL and incubated at 40 ℃ for four weeks. The percentage of correctly folded protein was calculated weekly based on the soluble content of SEC. Multabody are highly stable under these conditions, with over 70% of the sample remaining soluble for 30 days. (see, FIG. 10A).

In addition, samples before (week 0) and after (week 4) the incubation period were evaluated in PsV and in the assay to compare the biological functions of the molecules. The neutralization potency was only slightly lost at week 4 compared to the potency at week 0, further confirming stability. (see, FIG. 10B).

The Multabody tested had similar thermal stability compared to the reference molecule and was stable for at least four weeks with minimal loss of neutralizing efficacy when stored at 40 ℃.

TABLE 7 melting temperature (T _m) and aggregation temperature (T _agg) of Multabody

Example 9 engineering of Undersystem HIV-1 neutralizing efficacy by multispecific antibody affinity engineering

Abstract

Deep mining of the B cell pool of HIV-1 infected individuals has led to the isolation of tens of broadly neutralizing antibodies to HIV-1 (bNAb). However, it is still uncertain whether any such bNAb is broad and effective enough for treatment. Here we engineered HIV-1bNAb, combined on a single multispecific and affinity molecule via direct gene fusion of its Fab fragment to the human apoferritin light chain. The resulting molecules exhibited a significant median IC ₅₀ value of 0.0009 μg/mL and had 100% neutralization coverage-virus neutralization potency at a cut-off concentration of 4 μg/mL for a broad range of HIV-1 pseudovirions (118 isolates) 32-fold enhancement compared to the corresponding HIV-1bNAb mixture. Importantly, fc is incorporated into molecules and engineered to modulate Fc receptor binding, resulting in IgG-like bioavailability in vivo. This robust plug-and-play antibody design is suitable for indications that use both multi-specificity and avidity to mediate optimal biological activity.

The high genetic diversity of HIV-1 remains a major obstacle to the development of therapeutic agents for prophylaxis and treatment. Here we describe the design of an antibody platform that allows the assembly of high affinity multi-specific molecules that simultaneously target the most conserved epitopes on HIV-1 envelope glycoproteins. The combined multivalent and multispecific conversion into extraordinary neutralization potency and ubiquity neutralization of HIV-1 strains exceeds the most effective anti-HIV broadly neutralizing antibody mixtures.

Introduction to the invention

Despite decades of research, there is still no effective vaccine or therapeutic approach against the human immunodeficiency virus type I (HIV-1). However, the fact that a small fraction of HIV-1 infected individuals produce antibodies with superior neutralizing potency in circulating HIV-1 isolates highlights the potential for antibody-mediated HIV-1 control. Since the isolation of the first generation of broadly neutralizing antibodies (bNAbs) 2F5 (1), 4E10 (2, 3), 2G12 (4) and B12 (5, 6), the number of bnabs has increased dramatically due to the implementation of new technologies such as Env-specific single B cell sorting (7-9), antibody cloning and high throughput neutralization assays (10-13) and more recently proteomic deconvolution (14). Several HIV-1bNAb have now been described that target mainly six conserved sites on trimeric HIV envelope glycoproteins (Env), including the V1/V2 loop at the trimeric vertex, V3 cycloglycans, CD4 binding sites (CD 4 bs), gp120-g41 interface, env silencing surface and perimembranous outer region (MPER) (7, 9, 11-20).

The interest in bNAb as a therapeutic against HIV-1 stems from the potent antiviral activity observed in challenge studies in macaques (21-25) and humanized mice (26-29), as well as the reduction of viremia in infected humans after infusion of bNAb (30-34). Furthermore, antibodies have key advantages over oral antiretroviral therapy (ART): they have a longer circulatory half-life and can form immune complexes that enhance the host's immunity to the virus. These observations led to clinical evaluation of antibody-based therapies, conferring protection against HIV-1 infection by passive administration of bNAb (35), and striving to control and/or clear HIV-1 in infected individuals (31-33).

Recent antibody-mediated prevention (AMP) assays explore the ability of bNAb VRC01 to confer passive immunity against HIV-1. In these studies, the antibody width and potency deduced from TZM-bl neutralization assays were proposed as effective predictors of human antibody efficacy. In particular, IC ₈₀ values below 1 μg/ml are determined to be the efficacy threshold that biological therapeutics need to achieve to confer protection against a particular HIV-1 strain (35). VRC01 only reached a threshold for 30% of HIV-1 strains in the assay and therefore failed to confer broad protection, highlighting the urgent need for more potent, broadly acting molecules. While such coverage widths can be achieved by administering multiple bNAb, efficacy may still limit the therapeutic efficacy of the antibody cocktail despite recent efforts in IgG engineering (36-40).

Here, we overcome the huge sequence diversity of HIV-1 with remarkable neutralizing efficacy by engineering human apoferritin subunits to drive multimerization of three different HIV-1bNAb on a single molecule. The resulting multispecific, multi-affinity antibody (Multabody) was able to achieve ubiquitination (100% viral coverage), with a median IC ₅₀ value of 0.0009 μg/mL (0.4 pM). The Multabody design described herein represents a robust and powerful plug-and-play platform that multimerizes antibodies to enhance their neutralization by HIV-1 in the broadest range of isolates.

Materials and methods

Expression and purification of only Fab apoferritin multimers. The genes encoding the human apoferritin light chain and scFab-human apoferritin fusion were synthesized and cloned into the pHLsec expression vector by GeneArt (Life Technologies). 200mL of HEK 293F cells (Thermo FISHER SCIENTIFIC) were inoculated at a density of 0.8X10 ⁶ cells/mL in Freshtyle expression medium and incubated in a Multitron Pro shaker (Infos HT) at 37℃with 8% CO ₂ and 70% humidity at 125rpm shaking. Cells were transiently transfected within 24 hours after inoculation using 50 μg of filtered DNA pre-incubated with transfection reagent FectoPRO (Polyplus Tr ansfections) at a ratio of 1:1 for 10 minutes at Room Temperature (RT). The plasmid encoding scFab-human apoferritin and human apoferritin were mixed in a ratio of 1:4, 1:1, 4:1 and 1:0. After 6-7 days, the cell suspension was harvested by centrifugation at 5000 Xg for 15 minutes and the supernatant was filtered through a 0.22. Mu. m Steritop filter (EMD Millipore). The particles were purified by Fab affinity chromatography and eluted after washing. The protein-containing fractions were pooled, concentrated and loaded onto a Superose 6 10/300GL size exclusion column (GE HEATHCARE) in 20mM sodium phosphate, pH 8.0, 150 mM NaCl.

Multabody design, expression and purification. Genes encoding scFab and scFc fragments linked to hemiferritin were generated by deleting residues 1 to 90 (C-ferritin) and 91 to 175 (N-ferritin) of the human apoferritin light chain. Furthermore, the binding specificity of protein L for iMab-C-ferritin was destroyed by site-directed mutagenesis of alanine 12 of the antibody light chain to a proline residue (69). Transient transfection of T-01MB in HEK 293F cells was obtained by mixing 66. Mu.g of plasmid PGDM1400 scFab-human apoferritin, scFc-N-ferritin, N49P7 scFab-C-ferritin, 10E8v4 scFab-C-ferritin in a ratio of 4:2:1:1. In the case of T-02MB, plasmid N49P7 scFab-C-ferritin was replaced by iMab scFab-C-ferritin. In the case of T-01MB.v2, 63. Mu.g of plasmid PGDM1400 scFab-human apoferritin N49P7 scFab-N-ferritin 10E8v4 scFab-C-ferritin-Fc was used in a ratio of 3:1:1. The DNA mixture was filtered and incubated with 60. Mu.l FectoPRO at room temperature and then added to the cell culture. Purification of Multabody with four components was achieved by two-step affinity purification based on the hetero-oligomerization required to drive self-assembly: protein a HP column (GE HEALTHCARE) with 20mm Tris pH 8.0, 3M MgCl ₂ and 10% glycerol elution buffer (Fc binding), and protein L (GE Healthcare) (PGDM 1400 binding because 10E8 and N49P7 do not bind to protein L, and iMab-protein L binding is disrupted by a12P mutation). A buffer exchange step was performed between the two affinity chromatography steps using a PD-10 desalting column (GE HEALTHCARE). The protein containing fractions were concentrated and further purified by gel filtration on a Superose 6 10/300GL column (GE HEALTHCARE) in 20mM sodium phosphate, pH 8.0, 150mM NaCl.

Negative staining electron microscope. Mu L Multab ody, at a concentration of about 0.02mg/mL, was added to the carbon coated copper mesh for 30 seconds and stained with 3. Mu.l of 2% uranyl formate. Immediately the excess stain was 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 field emission FE I TECNAI F electron microscope operating at 200kV and equipped with a Orius charge-coupled device (CCD) camera (Gatan Inc).

Biological layer interferometry. Binding kinetics measurements were performed in PBS pH 7.4,0.01% BSA and 0.002% Twee n using the Octet RED96 BLI system (Pall ForteBio) system. A unique His-tagged ligand was selected for each Multabody component and Fc receptor and loaded onto the Ni-NTA biosensor to achieve a signal response of 0.8 nm. The association rate was measured by transferring the loaded biosensor into serial diluted wells containing Multabody (10-5-2.5-1.25-0.65-0.32 nM) or IgG (500-250-125-62.5-31.2-15.6 nM). The rate of dissociation is measured by immersing the biosensor in a well containing buffer. The duration of both steps was 180 seconds. To achieve selective binding to PGDM1400, the D368R mutation was introduced in CD4b of BG5050 sosip.664 trimer, thus disrupting the binding of N49P7 to this antigen. Similarly, gp120 subunit 93TH057, soluble CD4 and hFcRn, which are complexed with β2-microglobulin, were produced as ligands for N49P7, iMab and Fc, respectively. Binding to 10E8 was tested using a His-tagged MPER peptide (HHHHH HNEQELLELDKWASLWNWFNITNWLWYIKKKK (SEQ ID NO: 47), purchased from GenScript). The binding affinities of IgG and Multabody with silent mutations of effector function were measured using recombinantly expressed hfcyri and hfcyriia. Purification using Ni-NTA followed by size exclusion chromatography in 20mM phosphate, pH 8.0, 150mM NaCl buffer to purify BG5050 sosip.664d368r, CD4, 93TH057, hFcRn, hfcyri and hfcyriia.

Size exclusion chromatography is combined with multi-angle light scattering (SEC-MALS). MiniDA WN TREOS and Optilab T-rEX refractor (Wyatt) were used in combination with Agilent Technologies1260INFINITY II HPLC. 50 μg of 24-mer PGDM1400 scFab-ferritin fusion, T-01MB and T-02MB were loaded onto a Superose 6 10/300 (GE HEALTHCARE) column in 20mM sodium phosphate, pH 8.0, 150mM NaCl. Data collection and analysis was performed using astm a software (Wyatt).

Stability measurement. The melting temperature (T _m) and aggregation temperature (T _agg) of Multabody, parent IgG, 12-mer homo-oligomeric Fab and Fc, and N6/PGDM1400x10E8v4 trispecific antibodies were determined using UNit system (Unchained Labs). T _m was obtained by measuring the center of gravity average (BCM) fluorescence, while T _agg was determined as the temperature at which a 50% increase in static light scattering relative to baseline was observed at 266nm wavelength. The sample was concentrated to 1.0mg/mL and warmed from 25 ℃ to 95 ℃ in1 ℃ increments. The mean and standard error of three independent measurements were calculated using UNit analysis software.

Stability was further analyzed under accelerated stress conditions. Multabody was diluted in 20mM sodium phosphate, pH 8.0, 150mM NaCl, concentrated to 10mg/mL, and incubated at 40℃for four weeks. The percentage of correctly folded protein was calculated weekly based on the soluble content of SEC. Samples before (week 0) and after (week 4) incubation periods were evaluated in PsV neutralization assays to compare the functional activity of the molecules.

Virus production and TZM-bl neutralization assay. A set of 14 HIV-1 pseudotyped viruses was generated by co-transfecting 293T cells with HIV-1 subtype B backbone NL4-3.Luc.R ^- E plasmid (AIDS research and reference reagent program (ARRRP)) and the plasmid encoding the full-length Env clone, as described above (45). HIV isolates x2088.c09, ZM106.9 and 3817.v2.c59 were provided by aids vaccine discovery partnership organization (CAVD) friends, and pCNE8, 1632_s2_b10, THRO4156.18, 278-50, ZM197m.pb7, SF162, t257-31, du422.1 and BG505 were from NIH ARRRP. Mutant T332N was introduced into the BG505 Env expression vector by site-directed mutagenesis using the KOD-Plus mutagenesis kit (Toyobo, osaka, japan). An expanded set of 25 pseudotyped HIV-1 was generated by adding HIV isolates p1054.tc4.1499, 6535, zm214m.pl15, AC10.29, P16845, p6244_13.b5.4576, pM246f_c1G, TRJO4551, QH0692 and pCAAN5342 obtained from NIH ARRRP. Neutralization was determined in a single cycle neutralization assay using a standard TZM-bl neutralization assay (45). Briefly, igG and Multabody were incubated with 10-15% tissue culture infectious dose of pseudovirus for 1 hour at 37℃and then 44-72 hours with TZM-bl cells. Virus neutralization was monitored by adding Britelite plus reagents (PerkinElmer) to the cells and measuring luminescence in Relative Light Units (RLU) using a Synergy Neo2 multi-function assay microplate reader (Biotek Instruments). HIV-1Env pseudoviruses in the expanded multi-branched group of 118 PsV were generated by transfection of an Env expression plasmid with the full-length, env-defective HIV genome SG3dEnv into 293T cells. HIV-1 pseudovirus was incubated with Multabody (initial concentration 10. Mu.g/ml and titrated 6 times seven) for 1 hour at 37℃before TZM-bl cells were added. At 48 hours post infection, cells were lysed and luciferase expression was quantified by addition of luciferin substrate (Promega). For neutralization assays with parental IgG, historical data from the harvard medical institute virology and vaccine research center (initial concentration 50 μg/ml, titration 5-fold seven times) was used. A cutoff limit of 10 μg/mL was used to determine antibody width.

Antibody dependent phagocytosis. mu.L of red fluorescent Neutravidin microspheres (Invitrogen, F8775) were washed twice with PBS+0.1% BSA and incubated with 10. Mu.g of biotinylated 93TH057 antigen. Biotinylation was performed using the EZ-link Sulfo-NHS biotinylation kit (Thermo Scientific, 2143) according to the manufacturer's instructions. The final volume was adjusted to 200 μl with PBS/0.1% bsa and incubated overnight with rotation at 4 ℃. The beads were washed twice to remove unbound protein prior to use and resuspended in 200 μl per 5 μl unlabeled bead volume.

Immune complexes were formed by incubating 93TH057 coated fluorescent beads (10 μl of each sample) with 10 μl of 1,5 and 10 μg Multabody or antibody preparation at 37 ℃ for 2 hours. THP-1 cells (ATCC TIB-202) were maintained in RMPI+10% FBS (Wisent) at less than 5x 10 ⁵ cells/mL; and added to the immunocomplexes at a concentration of 5x 10 ⁴ cells/well (200 μl) and then incubated at 37 ℃ for 16 hours at 5% CO ₂. After incubation, cells were pelleted and washed with PBS, then stained with Live read fixable purple stain (Invitrogen, L34995) according to the manufacturer's protocol. Cells were washed with PBS and fixed with 2% paraformaldehyde for 20 min at room temperature. The fixed cells were pelleted and washed once with FACS buffer (pbs+10% FBS,0.5mM EDTA) and analyzed on LSRII flow cytometry (BD Biosciences). Data were analyzed in FlowJo (BD Biosciences, ashland, OR) and phagocytosis was quantified as the percentage of increase compared to 93TH057 coated beads in the absence of antibody. Anti-human FcR binding inhibitor antibodies (Invitrogen, 14-9161-73) were added to the indicated samples at the recommended concentrations as additional controls.

PBMC infection. Peripheral Blood Mononuclear Cells (PBMCs) were obtained from three healthy blood donors, all of whom provided written informed consent. The study was approved by the university of Toronto research ethics Committee (protocol # 00037384). Blood was collected in heparinized vacuum blood collection tubes (BD Biosciences) followed by density centrifugation using Lymphoprep (StemCell Technologies, cat. No. 07861) to isolate PBMCs. PBMC were activated with phytohemagglutinin (PHA; gibco) in the presence of recombinant human IL-2 (50U/mL) in complete RPMI medium (Wisent) containing 10% fetal bovine serum (FBS, wisent), 100. Mu.g/mL streptomycin and 100U/mL penicillin for 72 hours before HIV-1 infection. Three days after activation, HIV-1 cell infection was performed by adding CXCR4 trending laboratory isolate IIIB (150 pg per well of p24 Gag antigen) to triplicate cultures of activated PBMCs in round bottom 96 well plates seeded with 2 x 10 ⁵ cells/well in rpmi+10% fbs+25U/mL IL-2. Multabody (T-01 MB and T-01 MB.v2) or IgG mixtures were pre-incubated with virus for 1 hour at room temperature before covering the cells with virus. As shown, the infected cells were cultured at a dose of 0.01-10ug/mL in the presence/absence Multabody or antibody control. HIV-1 replication levels were assessed by measuring extracellular release of p24 Gag protein in cell free culture supernatants using a high sensitivity ALPHALISA P detection kit (PerkinElmer, waltham, MA) on BioTEK Synergy read plate apparatus at day 7 post infection according to manufacturer's protocol.

Cell viability and flow cytometry. On day 7 of infection, cells were fixed in 2% PFA and harvested for viability testing via absolute counting by flow cytometry performed using BD LSRFortessa (Becton Dickinson). Cell viability was determined by comparing the total number of viable gated cells in Multabody or antibody-treated wells to the number of cells recovered from untreated control wells. Cell viability data were analyzed using FACSDiva.

Pharmacokinetic studies. In vivo studies were performed using three 6 week old female NOD/Shi-scid/IL-2Rγnull (NCG strain code 572,Charles River Laboratories) immunodeficient mice/group. Mice were housed in groups of 4/6 individuals. Each mouse has a unique identifier. Animals were housed in ventilated cages (type II (16x19 x35 cm, floor area=500 cm ²)) under the following controlled conditions: 22 ℃, 55% humidity and 12:12 hours light-dark period, 7 am, 7 pm. The study was reviewed and approved by the local ethics Committee (CELEAG). T-01MB, consisting of scFab fragments of antibodies PGDM1400, N49P7 and 10E8v4, and scFc fragment containing the following IgG1 Fc were used in this study: i) No mutation, and ii) effector function silencing mutations L234A, L a and P329G (LALAP) and I253A mutation. In addition, T-01MB.v2 consisted of identical antibody specificities, with i) no Fc mutation, and ii) a half-life extending mutation in IgG1 Fc (M428L/N434S). Mice received a single subcutaneous injection of 5mg/kg Multabody in 200 μl PBS (pH 7.5) or control sample (IgG mixture matched to Fab specificity of Multabody). Blood samples were collected at various time points and serum samples were assessed for circulating antibody levels by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were coated with 50. Mu.L of each of His _6x -labeled antigen, gp120 subunit 93TH057, and MPER peptide recognized by MB:BG 5050D 368R SOSIP.664 trimer at a concentration of 0.5. Mu.g/ml, to determine circulating sample concentrations using reagent-specific standard curves for IgG and Multabody. HRP-protein A (Invitrogen) was used as the secondary molecule and chemiluminescent signal was quantified using an Epoch 2 microplate spectrophotometer and software Biotek Gen 5.3.03.

Results

The efficacy of HIV-1bNAb can be enhanced by avidity

Apoferritin is a spherical nanocage with hydrodynamic radius of about 6nm formed by self-oligomerization of 24 identical subunits (fig. 11A). To investigate the effect of multivalent on neutralization potency, we used the self-assembly properties of human apoferritin light chains to multimerize antigen binding (Fab) fragments derived from the most potent and broad HIV-1bNAb, which target different HIV-1Env epitopes. The apoferritin subunit is genetically fused to a single chain Fab (scFab). scFab is generated using a flexible linker between the light and heavy chains to ensure proper Fab heterodimerization. Apoferritin self-assembly driven multimerization of scFab and displayed antibody fragments at the nanocage periphery (fig. 11B). Different densities of multimerized Fab were achieved by co-transfection of scFab-human apoferritin encoding plasmids with different ratios of non-genetically modified human apoferritin (fig. 11C, fig. 12). The ability of scFab-apoferritin fusion to block HIV-1 infection was compared to the corresponding IgG using a small HIV-1 pseudovirus (PsV) group (FIG. 11D). Remarkably, PGDM1400 is one of the most potent anti-HIV bNAb described so far, showing 10 to 40 fold higher neutralization potency when multimerized via the apoferritin light chain compared to the traditional IgG format. bNAb 10-1074 also showed a considerable increase in neutralization potency (4 to 40 fold), while bNAb 10E8, N49P7 and VRC01 showed no effect or a milder increase.

Multabody effectively and extensively neutralize HIV-1

In view of these results, we attempted to increase the coverage of PGDM1400 using the Multabody platform based on the apoferritin split design we previously described (41). The strategy involved separating the four helix apoferritin subunits in half (N-ferritin and C-ferritin) and fusing their N-termini to scFab of different specificities (fig. 13A). This approach allows for the inclusion of a greater number of Fab's on the nanocage surface, resulting in a final molecule with higher affinity. Furthermore, the design allows for an efficient combination of three different antibody specificities and crystallizable fragments (fcs) to confer IgG-like properties to the molecule, such as ease of purification with protein a affinity (fig. 14). In particular, we combined scFab PGDM1400 with a single chain construct of Fc of the near-ubiquitously neutralizing antibody 10E8v4 (modified 10E8 (42) with improved solubility) and scFab of N49P7 and human IgG1 isotype (scFc) (fig. 13A). To explore whether it was also possible to design Multabod y that cross-target HIV-1Env and its primary receptor CD4, we replaced N49P7 with ibalizumab (iMab), a CD4 directed post-attachment inhibitor, which has been shown to be effective in inhibiting HIV-1 entry (43, 44) (fig. 15A). The resulting trispecifics Multabody, designated T-01MB and T-02MB, respectively, formed highly decorative and homogeneous particles of about 2.4MDa (fig. 13B-13C, 15B-15C) with similar thermal stability as the corresponding IgG (fig. 16). Use of epitope-specific molecules: BG505 SOSIP D368R (PGDM 1400), 93TH057 gp120/C D4 (N49P 7/iMab) and MPER peptide (10E 8v 4), epitope binding of trispecific Multabody was assessed in a binding kinetics experiment (figure 17). Binding to the three epitope-specific antigens with high apparent binding affinity and no detectable dissociation confirm the presence of the three antibody specificities in Multabody (fig. 13d, fig. 15 d).

The neutralization potency and width of Multabody were first evaluated in a standardized in vitro TZM-bl neutralization assay for a set of 14 PsV (45). Groups 14-PsV were designed to include low sensitivity PsV, at least one of which PsV was resistant to each bNAb evaluated (cut-off IC ₅₀ set to 10 μg/mL). The IC ₅₀ value and width of Multabody were compared to the following: (i) each individual IgG, (ii) an IgG mixture containing the same relative amounts of each IgG present in Multabody, (iii) N6/PGDM1400x10E8v4 trispecific antibody (46). T-01MB and T-02MB exhibited 93% and 100% width (cutoff IC ₅₀ set to 10 μg/mL), respectively, for this group, with median IC ₅₀ values of 0.009 μg/mL (3.9 pM) and 0.008 μg/mL (3.5 pM), respectively (FIG. 13E, FIG. 15E, and Table 8). Thus, there was approximately one and two orders of magnitude decrease in median IC ₅₀ values compared to IC ₅₀ values for IgG mixtures and trispecific antibodies, respectively, when Multabody were calculated in μg/mL and nM. Examination of the individual IC ₅₀ values revealed that PsV, which was resistant to PGDM1400 IgG neutralization, was also less sensitive to Multabody (fig. 13F, 15E and table 8). These data indicate that the neutralizing properties of Multabody are largely dependent on one of the three antibody specificities within the particle, PGDM1400 in this example.

/>

Engineering apoferritin scaffolds

To further improve the neutralization properties of Multabody, we introduced some modifications to its design and made the second generation version (mb.v2). In the original MB, scFc was located at the N-terminus of the N-ferritin half and only one Fab (Fab 2 or Fab 3) was incorporated into Multabody of each functional Fc homodimer (fig. 18A, top). In contrast, optimized mb.v2 contained a greater number of Fab per Fc homodimer. To achieve this, the monomeric Fc fragment (i.e., one Fc chain) and scFab are located at the C-terminal and N-terminal ends of the C-ferritin half, respectively (bottom of fig. 18A, fig. 19A). As a result, dimerization of functional Fc homodimers drives assembly of mb.v2 particles and split ferritin complementation and ferritin subunit oligomerization (fig. 18A, fig. 19B). Importantly, homodimerization to form one functional Fc ensures assembly of four fabs (i.e., two fabs 2 and two fabs 3) other than PGDM1400, thereby facilitating a more balanced avidity of each of the three fabs in a fully assembled mb.v2.

The optimized Multabody design was tested in the T-01 background (PGDM 1400, N49P7, 10E8v 4) which targets three epitopes on HIV-1 Env. The resulting Multabody (T-01 mb.v2) assembled into well-formed spherical particles, with no significant morphological differences compared to the previously characterized T-01MB (fig. 18B). Binding of antigen to BG505 SOSIP D368R, 93TH057 gp120 and MPER peptide confirmed the correct folding of three Fab specificities in T-01mb.v2 (figure 18C). In addition, the new Multabody version retained the same high thermal stability reported for T-01MB, with a T _agg value of 67 ℃ (fig. 18D). Multabody was concentrated to 10mg/mL and accelerated stability testing was performed by incubation at 40 ℃ for four weeks. Evaluation of the amount of soluble protein over time revealed that Multabody was highly stable under these conditions, with more than 70% of the samples remaining soluble for 30 days. Only a slight loss in neutralization potency of Multabody was observed at week 4 compared to the potency at week 0, further confirming stability (fig. 18E).

Multabody has pharmacokinetics similar to that of corresponding IgG

Antibody Fc domains are capable of interacting with a variety of receptors, including fcγ receptor (fcγr) and neonatal Fc receptor (FcRn), which confer effector function and in vivo half-life, respectively. However, fc avidity can negatively impact the cycle time of molecules with multiple Fc fragments (41, 47). Indeed, T-01MB shows strong binding to Fc receptors, including binding to human FcRn at physiological pH (fig. 20A-20B), as well as binding to high and low affinity fcγr (fig. 20C). Thus, we introduced a unique combination of LALAP (L234A, L235A and P329G) and I253A mutations in the Fc of T-01MB to reduce binding to fcγr and FcRn, respectively, and achieved comparable binding observed for IgG1 molecules (fig. 20A-20C). T-01MB.v2 showed a more similar binding profile to IgG1, with comparable binding to human FcRn at acidic pH and no binding at physiological pH, even in the case of half-life extension mutation LS (M428L/N434S) (FIGS. 20A-20B). Binding of T-01mb.v2 to fcγr resulted in a low binding profile similar to that obtained using LALAP FcR silent mutations in T-01MB (figure 20C). The different binding patterns observed for the two MB versions may be due to the different arrangement of Fc fragments within the molecule (fig. 18A, 19A). Although fcyri binding was low, phagocytosis experiments using antigen coated beads showed that both Multabody forms induced Fc-dependent internalization in THP-1 cells at levels similar to those achieved with the corresponding IgG mixtures (fig. 20D).

Next, we examined the in vivo bioavailability of both Multabody forms with and without engineered Fc. The NOD/Shi-scid/IL-2Rγnull (NCG) immunodeficient mice were subcutaneously administered a single dose of 5mg/kg and the amount of each molecule in serum was measured every two days for 15 consecutive days. As expected from the in vitro characterization, only Fc engineered Multabody with IgG-like binding profile showed several days of in vivo exposure with a similar decay rate as the parental IgG mixture (fig. 20E). Multabody administration was well tolerated with no weight loss (figure 20F) or visible side effects.

Extraordinary potency and ubiquity breadth achieved by MB.v2

We assessed the neutralization profile of T-01mb.v2 against group PsV generated by adding 11 HIV-1 strains with high resistance to PGDM1400 to our previous group. The resulting 25-PsV panel contained 56% of PsV variants resistant to PGDM1400 IgG neutralization (cut-off IC ₅₀ set to 10 μg/mL) (fig. 21A-21B). As expected, the width and potency of T-01MB is greatly affected in the presence of PGDM1400 resistance PsV (FIGS. 21A-21B, table 8). However, according to engineering, the neutralization profile of antibodies N49P7 and 10E8v4 is more dominant in T-01mb.v2, allowing this optimized Multabody to achieve ubiquity while retaining the enhanced neutralization potency of this type of molecule observed previously (fig. 21A-21B, table 8). When tested against the extended multi-branched group of 118PsV, T-01mb.v2 matched the flood width of the corresponding IgG mixture (100% viral coverage, cut-off IC ₅₀ set to 10 μg/mL), but exhibited significant neutralization efficacy (fig. 21C-21D, fig. 22A and table 9). Specifically, igG mixture and T-01MB only neutralized 9% and 8% of PsV, respectively, with IC ₅₀ value of 0.001 μg/mL, while in the case of T-01MB.v2, 50% of PsV was still neutralized and IC ₅₀ value was only 0.001 μg/mL (FIG. 21C). Notably Multabody achieved median IC ₅₀ values of only 0.0009 μg/mL (0.4 pM), thus achieving 32-fold and 490-fold more efficient pan-neutralization in mass and molar concentration, respectively, compared to IgG mixtures (fig. 21D). Furthermore, IC ₈₀ of T-01mb.v2 demonstrated its propensity to neutralize better than that of individual IgG and IgG mixtures, neutralizing 96% of all strains tested, with a median IC ₈₀ value of 0.005 μg/mL (2.2 pM) (fig. 21C-21D, fig. 22A and table 9). Importantly, multabody also blocked infection of primary Peripheral Blood Mononuclear Cells (PBMC) by replication competent CXCR 4-chemotactic HIV-1IIIB strains (fig. 22B), showing enhanced efficacy over matched IgG mixtures, and no effect on cell viability (fig. 22C).

/>

Discussion of the invention

Recent AMP clinical trials have highlighted the expected importance of the efficacy and breadth of bNAb, making it a therapeutic agent capable of protecting against HIV-1 infection. Using the principles of antibody affinity used in the Multabody technology we previously described to increase the potency of antibodies against SARS-CoV-2 (41), we designed the second generation Multabody platform here to provide superior neutralization breadth and potency against the vast sequence diversity of HIV-1.

The most notable feature of the optimized Multabody design compared to the first generation Multabody format (41) is the relative number of Fab per functional Fc domain self-association. In the design of T-01mb.v2, two N49P7 fabs and two 10e8v4 fabs were incorporated into the MB of each dimer Fc, as compared to the 1:1fc:10e8v4/N49P7 ratio imposed by the design of the Multabody platform described previously. The higher number of these two Fab's in the optimized Multabody favors their affinity and therefore their contribution to the neutralising characteristics of the particles is greater. This is in contrast to T-01MB, which is primarily dependent on the neutralizing properties of PGDM 1400. The more balanced contribution of each antibody was reflected in the better functional properties of T-01mb.v2, which showed 100% cross-over branch neutralization coverage with a median IC ₅₀ value of 0.0009 μg/mL. In addition, 83% of the tested 118 pseudoviruses had their viral infection blocked by T-01mb.v2, with IC ₈₀ values below 1 μg/ml, which has recently been proposed as the efficacy threshold required to confer in vivo protection in humans (35). However, it is still unclear whether the predictors of protection should be considered in mass or molar concentration. Indeed, although Multabody has similar hydrodynamic radii and geometry (41) compared to IgM, the molar mass of Multabody is about 10 times greater than IgG. Thus, if the molar concentration is an in vivo measurement associated with protection, T-01mb.v2 exhibits an extremely low median IC ₅₀ of 0.4 pM. Correspondingly, IC ₈₀ efficacy of less than 6.7nM (molar equivalent of IgG 1. Mu.g/ml) was achieved in 96% of the 118-HIV-1PsV strain. These remarkable neutralizing properties exceeded those obtained using the bispecific and trispecific antibodies described previously (46, 48-50). In these antibody formats, limited avidity prevents a combination of high avidity and multi-specificity, and therefore potency and breadth are limited to the parent mAb.

The field of biological therapy is increasingly prone to the development of high-valent molecules. The strategy ranges from the generation of a decabivalent IgM-like molecule (51, 52) after addition of the mu-tail end of IgM to the constant region of IgG, to the design of alternative antibody formats. Including fusions of Fab in a linear head-to-tail fashion (53), additional IgG (54-56) or tandem (Tamdab) (57) or diabody combinations fused to CH3 of IgG (diabody) (58). In addition, multimerization scaffolds such as p53 (59), leucine zipper helix (60), streptavidin (61), barnase-barstar module (62), virus-like nanoparticles (63), and more recently de novo antibody cage forming proteins (de novo antibody cage-forming proteins) (64) were used to overcome the bivalent limitations of IgG and to increase the biological activity of antibodies. Although these approaches are attractive, their successful development as therapeutic agents faces different challenges. Multimeric antibody forms that rely on antibody variable fragments (Fv) are generally associated with low stability and therefore have a higher propensity to aggregate (65). Furthermore, dissociation of non-covalent fusions, which are determined by the affinity constant of the complex, can limit the long-term stability of the molecule in vivo. In sharp contrast, multabody builds on the intact IgG components (Fab and Fc) which fuse with the thermostable, functionally silent human apoferritin light chain scaffold and are therefore highly stable IgG-like molecules even under thermal stress. The mouse replacement Multabody, previously administered subcutaneously in immunocompetent C57BL/6 mice, showed undetectable levels of anti-drug antibodies similar to their parent IgG, which provides proof of principle for the potential low intrinsic immunogenicity of the Multabody platform (41). Future studies on higher organisms will help determine the immunogenicity of Multabody encoded by human sequences, which we believe may be determined primarily by the nature of the underlying antibody sequence.

Bioavailability of large biological agents is another challenge associated with engineering approaches to improve avidity (63). Multabody have been engineered to contain an Fc domain to effect FcRn-mediated recycling of molecules. Because of Fc affinity, T-01MB binding to FcRn is improved by several orders of magnitude at pH 6.0, however, the simultaneous improvement in affinity at pH 7.4 limits the application of this strategy in extending half-life, and several mutations are required to reduce Fc binding to FcRn and fcγr so as not to exceed the observed binding affinity of IgG. This enhanced Fc receptor affinity was not observed in the case of T-01mb.v2, where the Fc chain fusion at the C-terminus of apoferritin resulted in the formation of particles with inverted and more located Fc domains, and therefore, decreased Fc affinity. Similar to previous studies using mouse substitution Multabody (41), the Fc affinity modulation strategy successfully resulted in a very similar decay rate of Multabody molecule over time to the parental IgG mixture. In addition to the favorable pharmacokinetic profile, multabody with residual binding to fcγr induced Fc-mediated phagocytosis in vitro to similar levels as the parent IgG mixture, at least in THP-1 systems co-expressing both fcγri and fcγriia (66). Future experiments are required to fully characterize Multabody the ability to trigger immune effector functions and their in vivo effects.

From the limited number of antibody specificities we characterized in this study we observed that antibodies targeting epitopes located at the apex of the HIV-1 Env trimer (such as PGDM 1400) appear to have the greatest benefit in neutralizing efficacy when formulated as Multabody. This increase in potency is less pronounced when the epitope is closer to the viral membrane, as is the case with 10E 8. The dependence of the enhanced potency on epitope location may be further affected by low surface spike density (67), arrangement of those sparse Env trimers on the HIV-1 surface (68) or accessibility of certain epitopes, which may be more or less spatially blocked. In view of this, it would be interesting to explore how antibody efficacy against viruses with higher surface densities and closely spaced spikes could be enhanced by the Multabody platform, and to further determine the impact of epitope location on affinity-mediated efficacy enhancement.

Replacement of N49P7 with iMab, a CD4 directed post-attachment inhibitor, resulted in a functional Multabody with potent neutralizing activity, demonstrating that this type of particle can achieve cross-targeting of viral epitopes and cellular receptors. These data present an interesting possibility that Multabody technology can also be implemented in other fields that promote binding of receptors between different entities, such as intercellular interactions in immunotherapy. In general, our protein engineering studies demonstrated the versatility of human apoferritin as a modular nanocage to co-construct antibodies avidity and multi-specificity to enhance function.

Reference to the literature

1.A.J.Conley,et al.,Neutralization of divergent human immunodeficiency virus type 1variants and primary isolates by IAM-41-2F5,an anti-gp41 human monoclonal antibody.Proc.Natl.Acad.Sci.U.S.A.91,3348-3352(1994).

2.G.Stiegler,et al.,A Potent Cross-Clade Neutralizing Human Monoclonal Antibody against a Novel Epitope on gp41 of Human Immunodeficiency Virus Type 1.AIDS Res.Hum.Retroviruses 17,1757-65(2001).

3.M.B.Zwick,et al.,Broadly Neutralizing Antibodies Targeted to the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1Glycoprotein gp41.J.Virol.75,10892-905(2001).

4.A.Buchacher,et al.,Generation of Human Monoclonal Antibodies against HIV-1 Proteins;Electrofusion and Epstein-Barr Virus Transformation for Peripheral Blood Lymphocyte Immortalization.AIDS Res.Hum.Retroviruses 10,359-69(1994).

5.C.F.Barbas,et al.,Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.Proc.Natl.Acad.Sci.U.S.A.89,9339-9343(1992).

6.D.R.Burton,et al.,Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody.Science 266,1024-1027(1994).

7.X.Wu,et al.,Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1.Science 329,856-861(2010).

8.J.F.Scheid,et al.,Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals.Nature 458,636-640(2009).

9.D.Sok,et al.,Recombinant HIV envelope trimer selects for quatemary-dependent antibodies targeting the trimer apex.Proc.Natl.Acad.Sci.U.S.A.111,17624-17629(2014).

10.L.M.Walker,et al.,Broad neutralization coverage of HIV by multiple highly potent antibodies.Nature 477,466470(2011).

11.N.A.Doria-Rose,et al.,Developmental pathway for potent V 1V2-directed HIV-neutralizing antibodies.Nature 509,55-62(2014).

12.J.Huang,et al.,Broad and potent neutralization of HIV-1 by a gp41-specific human antibody.Nature 491,406-412(2012).

13.L.M.Walker,et al.,Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target.Science 326,285-289(2009).

14.M.M.Sajadi,et al.,Identification of Near-Pan-neutralizing Antibodies against HIV-1 by Deconvolution of Plasma Humoral Responses.Cell 173,1783-1795.e14(2018).

15.J.F.Scheid,et al.,Sequence and Structural Convergence of Broad and Potent HIV Antibodies That Mimic CD4 Binding.Science 333,1633-1637(2011).

16.T.Schoofs,et al.,Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope.Immunity 50,1513-1529.e9(2019).

17.J.Huang,et al.,Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth.Immunity 45,1108-1121(2016).

18.R.Pejchal,et al.,A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.Science 334,10971103(2011).

19.C.Blattner,et al.,Structural delineation of a quaternary,cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 env trimers.Immunity 40,669-680(2014).

20.H.Mouquet,et al.,Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies.Proc.Natl.Acad.Sci.U.S.A.109,E3268-77(2012).

21.T.W.Baba,et al.,Human neutralizing monoclonal antibodies of the IgG1subtype protect against mucosal simian-human immunodeficiency virus infection.Nat.Med.6,200-206(2000).

22.A.J.Hessell,et al.,Effective,low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques.Nat.Med.15,951-954(2009).

23.A.J.Hessell,et al.,Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers.PLoS Pathog.5,e1000433(2009).

24.R.Hofmann-Lehmann,et al.,Postnatal pre-and postexposure passive immunization strategies：Protection of neonatal macaques against oral simian-human immunodeficiency virus challenge.J.Med.Primatol.31,109-119(2002).

25.R.Hofmann-Lehmann,et al.,Postnatal passive immunization of neonatal macaques with a triple combination of hunan monoclonal antibodies against oral simian-human immunodeficiency virus challenge.J.Virol.75,7470-7480(2001).

26.Y.U.Van Der Velden,et al.,Short Communication：Protective Efficacy of Broadly Neutralizing Antibody PGDM 1400 Against HIV-1 Challenge in Humanized Mice.AIDS Res.Hum.Retroviruses 34,790-793(2018).

27.F.Klein,et al.,HIV therapy by a combination of broadly neutralizing antibodies in humanized mice.Nature 492,118-122(2012).

28.M.Deruaz,et al.,Protection of humanized mice from repeated intravaginal HIV challenge by passive immunization：A model for studying the efficacy of neutralizing antibodies in vivo.J.Infect.Dis.214,612616(2016).

29.J.A.Horwitz,et al.,HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice.Proc.Natl.Acad.Sci.U.S.A.110,16538-16543(2013).

30.S.Mehandru,et al.,Adjunctive Passive Immunotherapy in Human Immunodeficiency Virus Type 1-Infected Individuals Treated with Antiviral Therapy during Acute and Early Infection.J.Virol.81,11016-11031(2007).

31.M.Caskey,et al.,Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.Nature 522,487-491(2015).

32.M.Caskey,et al.,Antibody 10-1074 suppresses viremia in HIV-1-infected individuals.Nat.Med.23,185-191(2017).

33.R.M.Lynch,et al.,Virologic effects of broadly neutralizing antibody VRC01administration during chronic HIV-1 infection.Sci.Transl.Med.7,319ra206(2015).

34.J.E.Ledgerwood,et al.,Safety,pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.Clin.Exp.Immunol.182,289-301(2015).

35.L.Corey,et al.,Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition.N.Engl.J.Med.384,10031014(2021).

36.R.S.Rudicell,et al.,Enhanced Potency of a Broadly Neutralizing HIV-1Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo.J.Virol.88,12669-12682(2014).

37.Y.D.Kwon,et al.,Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody.Cell Rep.22,1798-1809(2018).

38.E.Rujas,et al.,Functional Optimization of Broadly Neutralizing HIV-1Antibody 10E8 by Promotion of Membrane Interactions.J.Virol.92,e02249-17(2018).

39.E.Rujas,et al.,Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments.Cell Rep.32,108037(2020).

40.R.Diskin,et al.,Increasing the potency and breadth of an HIV antibody by using structure-based rational design.Science 334,12891293(2011).

41.E.Rujas,et al.,Multivalency transforms SARS-CoV-2 antibodies into broad and ultrapotent neutralizers.Nat Commun12,3661(2021).

42.Y.D.Kwon,et al.,Optimization of the Solubility of HIV-1-Neutralizing Antibody 10E8 through Somatic Variation and Structure-Based Design.J.Virol.90,5899-5914(2016).

43.J.M.Jacobson,et al.,Safety,pharmacokinetics,and antiretroviral activity of multiple doses of ibalizumab(formerly TNX-355),an anti-CD4 monoclonal antibody,in human immunodeficiency virus type 1-infected adults.Antimicrob.Agents Chemother.53,450-457(2009).

44.D.R.Kuritzkes,et al.,Antiretroviral Activity of the Anti-CD4 Monoclonal Antibody TNX-355 in Patients Infected with HIV Type 1.J.Infect.Dis.189,286-291(2004).

45.D.C.Montefiori,Measuring HIV neutralization in a luciferase reporter gene assay.Methods Mol.Biol.485,395-405(2009).

46.L.Xu,et al.,Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques.Science 358,85-90(2017).

47.O.S.Qureshi,et al.,Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.Sci.Rep.7(2017).

48.M.Asokan,et al.,Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization.J.Virol.89,12501-12512(2015).

49.S.N.Khan,et al.,Targeting the HIV-1 Spike and Coreceptor with Bi-and Trispecific Antibodies for Single-Component Broad Inhibition of Entry.J.Virol.92,e00384-18(2018).

50.J.J.Steinhardt,et al.,Rational design of a tfispecific antibody targeting the HIV-1 Env with elevated anti-viral activity.Nat.Commun.9(2018).

51.R.I.Smith,M.J.Coloma,S.L.Morfison,Addition of a mu-tailpiece to IgG results in polymeric antibodies with enhanced effector functions including complement-mediated cytolysis by IgG4.J.Immunol.154,2226-36(1995).

52.T.Olafsen，B.I.Rasmussen，L.Norderhaug，S.Bruland,I.Sandlie,IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells.Immunotechnology 4,141-153(1998).

53.K.Miller,et al.,Design,Construction,and In Vitro Analyses of Multivalent Antibodies.J.Immunol.170,48544861(2003).

54.C.Wu,et al.,Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin.Nat.Biotechnol.25,1290-1297(2007).

55.C.Klein,W.Schaefer,J.T.Regula,The use of CrossMAb technology for the generation of bi-and multispecific antibodies.MAbs 8,1010-1020(2016).

56.A.Steinmetz,et al.,CODV-Ig,a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications.MAbs 8,867-878(2016).

57.S.M.Kipriyanov,et al.,Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinefics.J.Mol.Biol.293,41-56(1999).

58.D.Lu,et al.,Di-diabody：A novel tetravalent bispecific antibody molecule by design.J.Immunol.Methods 279,219-232(2003).

59.S.Kubetzko,E.Balic,R.Waibel,U.Zangemeister-Wittke,A.Plückthun,PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5：Effects on tumor targeting.J.Biol.Chem.281,35186-35201(2006).

60.P.Pack,K.Müller,R.Zahn,A.Plückthun,Tetravalent miniantibodies with high avidity assembling in Escherichia coli.J.Mol.Biol.246,28-34(1995).

61.S.M.Kipriyanov,et al.,Affinity enhancement of a recombinant antibody：Formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.Protein Eng.9,203-211(1996).

62.S.M.Deyev,R.Waibel,E.N.Lebedenko,A.P.Schubiger,A.Plückthun,Design of multivalent complexes using the barnase·barstar module.Nat.Biotechnol.21,1486-1492(2003).

63.M.A.G.Hoffmann,et al.,Nanoparticles presenting clusters of CD4 expose a universal vulnerability of HIV-1 by mimicking target cells.Proc.Natl.Acad.Sci.U.S.A.117,18719-18728(2020).

64.R.Divine,et al.,Designed proteins assemble antibodies into modular nanocages.Science 372,cabd99947(2021).

65.A.A.Plückthun,Different equilibrium stability behavior of scFv fragments：Identification,classification,and improvement by protein engineering.Biochemistry 38,8739-8750(1999).

66.H.B.Fleit,C.D.Kobasiuk,The Human Cell Line THP-1 Expresses Fc-ganmma-RI and Fc-gamma-RII.J Leukoc Biol 49,556-565(1991).

67.P.Zhu,et al.,Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions.Proc.Natl.Acad.Sci.U.S.A.100,15812-15817(2003).

68.J.Chojnacki,et al.,Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy.Science 338,524-528(2012).

69.M.Graille,et al.,Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.Structure 9,679-687(2001).

70.T.Zhou,et al.,Structural basis for broad and potent neutralization of HIV-1by antibody VRC01.Science 329,811-817(2010).

71.E.O.Freed,D.J.Myers,R.Risser,Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.J.Virol.63,4670-4675(1989).

Sequence listing

Underlined within the sequence indicates the linker sequence; bold within the sequence indicates ferritin or ferritin subunit sequences; boxes and bold residues represent residues that are mutated with respect to a reference molecule (e.g., with respect to IgG1 Fc).

/>

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 this application is intended to cover 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 self-assembled polypeptide complex comprising

(A) A plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein said Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, and

2. The self-assembled polypeptide complex of claim 1,

Wherein (1) if the Fc polypeptide is an IgG1 Fc polypeptide, then the antigen binding fragment is not a Fab fragment which binds SARS-CoV-2; and/or (2) if the nanocage monomer is a mouse ferritin monomer and the Fc polypeptide is a mouse IgG2a Fc polypeptide, the antigen-binding antibody fragment is not a Fab fragment that binds CD 19.

3. The self-assembled polypeptide complex of claim 1 or 2, wherein the nanocage monomers are ferritin monomers.

4. The self-assembled polypeptide complex of claim 3 wherein the ferritin monomer is a ferritin light chain.

5. The self-assembled polypeptide complex of claim 4 which does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

6. The self-assembled polypeptide complex of claim 3, 4 or 5, wherein the ferritin monomer is human ferritin.

7. The self-assembled polypeptide complex of any one of claims 1-6, wherein the Fc polypeptide is an IgG1 Fc polypeptide.

8. The self-assembled polypeptide complex of any one of claims 1-6, wherein the Fc polypeptide is an IgG2 Fc polypeptide.

9. The self-assembled polypeptide complex of any one of claims 1-8, wherein the Fc polypeptide is a single chain Fc (scFc).

10. The self-assembled polypeptide complex of any one of claims 1-8, wherein the Fc polypeptide is an Fc monomer.

11. The self-assembled polypeptide complex of any one of claims 1-10, wherein the antigen-binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

12. The self-assembled polypeptide complex of claim 11 wherein the antigen-binding antibody fragment is a Fab fragment.

13. The self-assembled polypeptide complex of any one of claims 1-12, wherein each second fusion polypeptide does not comprise any CH2 or CH3 domain.

14. The self-assembled polypeptide complex of any one of claims 1-13, wherein the one or more mutations comprise a mutation or a set of mutations associated with altered binding to FcRn.

15. The self-assembled polypeptide complex of claim 14 wherein the mutation or set of mutations comprises mutations at one or more of the following residues: m252, I253, S254, T256, K288, M428, and N434, or combinations thereof, wherein numbering is according to EU index.

16. The self-assembled polypeptide complex of claim 15, wherein the mutation or set of mutations comprises mutations at M428 and N434, wherein numbering is according to the EU index.

17. The self-assembled polypeptide complex of claim 16, wherein the mutation or set of mutations comprises M428L and N434S mutations, wherein numbering is according to the EU index.

18. The self-assembling polypeptide complex of claim 14 or 15, wherein the altered binding to FcRn is reduced binding to FcRn.

19. The self-assembling polypeptide complex of claim 15, wherein the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I253V and K288A, and combinations thereof, wherein numbering is according to the EU index.

20. The self-assembled polypeptide complex of any one of claims 1-19, wherein the one or more mutations comprises a mutation or a set of mutations associated with altered effector function.

21. The self-assembled polypeptide complex of claim 20, wherein the Fc polypeptide is an IgG1 Fc polypeptide, and wherein the mutation or set of mutations comprises mutations at one or more of the following residues: l234, L235, G236, G237, P329 and a330 or combinations thereof, wherein numbering is according to EU index.

22. The self-assembled polypeptide complex as claimed in claim, wherein the altered effector function is a reduced effector function.

23. The self-assembled polypeptide complex of claim 22, wherein the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A/L235A), LALAP (L234A/L235A/P329G), G236R, G237A and a330L, wherein numbering is according to the EU index.

24. The self-assembled polypeptide complex of any one of claims 1-23, wherein the nanocage monomer or subunit thereof is a ferritin monomer subunit, and

25. The self-assembling polypeptide complex of any one of claims 1-24, wherein the self-assembling polypeptide complex is characterized by a 1:1 ratio of a first fusion polypeptide to a second fusion polypeptide.

26. The self-assembled polypeptide complex of any one of claims 1-25, wherein within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof via an amino acid linker.

27. The self-assembled polypeptide complex of any one of claims 1-26, wherein within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof.

28. The self-assembling polypeptide complex of any one of claims 1-27, wherein within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the nanocage monomer or subunit thereof via an amino acid linker.

29. The self-assembling polypeptide complex of any one of claims 1-28, wherein within each second fusion polypeptide, the antigen-binding antibody fragment is linked to the nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof.

30. The self-assembled polypeptide complex of any one of claims 1-29, further comprising a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) an antigen-binding antibody fragment linked to (2) a nanocage monomer or subunit thereof, wherein the third fusion polypeptide is different from the second fusion polypeptide.

31. The self-assembling polypeptide complex of claim 30, wherein the antigen-binding antibody fragment within the third fusion polypeptide comprises a light chain variable domain and a heavy chain variable domain.

32. The self-assembling polypeptide complex of claim 31, wherein the antigen-binding antibody fragment within the third fusion polypeptide is a Fab fragment.

33. The self-assembling polypeptide complex of any one of claims 30-32, wherein each third fusion polypeptide does not comprise any CH2 or CH3 domains.

34. The self-assembled polypeptide complex of any one of claims 31-33, wherein the antigen-binding antibody fragment of the second fusion polypeptide is capable of binding a first epitope, the antigen-binding fragment of the third fusion polypeptide is capable of binding a second epitope, and the first epitope and the second epitope are different and non-overlapping.

35. The self-assembled polypeptide complex of claim 34 wherein the first epitope and the second epitope are from the same protein.

36. The self-assembling polypeptide complex of any one of claims 35, comprising a total of 24 to 48 fusion polypeptides.

37. The self-assembling polypeptide complex of any one of claims 1-36, comprising a total of at least 24 fusion polypeptides.

38. The self-assembling polypeptide complex of claim 37, comprising a total of at least 32 fusion polypeptides.

39. The self-assembling polypeptide complex of claim 38, having a total of about 32 fusion polypeptides.

40. The self-assembling polypeptide complex of any one of claims 1-39, wherein the self-assembling polypeptide complex has a half-life of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days when administered to a subject in need thereof.

41. The self-assembling polypeptide complex of any one of claims 1-40, wherein upon administration of a composition comprising the self-assembling polypeptide complex, the self-assembling polypeptide complex has a half-life substantially similar to a reference IgG molecule administered by the same route of administration and in a similar composition.

42. The self-assembled polypeptide complex of claim 41 wherein the reference IgG molecule is an antibody from which the antigen-binding antibody fragment within the second fusion polypeptide is derived or an antibody from which the antigen-binding antibody fragment within the third fusion polypeptide is derived.

43. The self-assembling polypeptide complex of any one of claims 40-42, wherein the self-assembling polypeptide complex has a half-life of about 3 days to about 35 days when administered to a subject in need thereof.

44. The self-assembling polypeptide complex of any one of claims 1-43, wherein the self-assembling polypeptide complex is capable of being detected in serum for at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration to a subject in need thereof.

45. The self-assembling polypeptide complex of any one of claims 1-44, wherein the area under the curve (AUC) of the self-assembling polypeptide complex is at least 10 days μg/mL, at least 25 days μg/mL, at least 50 days μg/mL, at least 100 days μg/mL, at least 200 days μg/mL, at least 300 days μg/mL, at least 400 days μg/mL, at least 500 days μg/mL, at least 750 days μg/mL, at least 1000 days μg/mL, at least 1500 days μg/mL, at least 2000 days μg/mL, at least 2500 days μg/mL, at least 3000 days μg/mL, at least 4000 days μg/mL, at least 5000 days μg/mL, at least 6000 days μg/mL, at least 7000 days μg/mL, or at least 8000 days μg/mL when administered to a subject in need thereof.

46. The self-assembled polypeptide complex of any one of claims 1-45, wherein the area under the curve (AUC) of the self-assembled polypeptide complex is from about 10 days · μg/mL to about 8000 days · μg/mL when administered to a subject in need thereof.

47. The self-assembling polypeptide complex of any one of claims 1-46, wherein the maximum concentration of the self-assembling polypeptide complex (C _max) is at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 250 μg/mL, at least 500 μg/mL, at least 750 μg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 75mg/mL, at least 100mg/mL, at least 250mg/mL, at least 500mg/mL, or at least 750mg/mL when administered to a subject in need thereof.

48. The self-assembling polypeptide complex of any one of claims 1-47, wherein the maximum concentration of the self-assembling polypeptide complex (C _max) is about 10 μg/mL to about 750mg/mL when administered to a subject in need thereof.

49. The self-assembled polypeptide complex of any one of claims 40-48, wherein the subject is a human.

50. The self-assembled polypeptide complex of any one of claims 40-49, wherein administering to the subject is by parenteral administration.

51. The self-assembled polypeptide complex of any one of claims 40-49, wherein administration to the subject is by subcutaneous administration, intravenous administration, intramuscular administration, intranasal administration, or by inhalation.

52. The self-assembled polypeptide complex of any one of claims 1-51, wherein the self-assembled polypeptide complex induces ADCP in an Antibody Dependent Cell Phagocytosis (ADCP) in vitro model.

53. The self-assembled polypeptide complex of claim 52, wherein the ADCP is induced at a level of target internalization of at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

54. A method comprising administering to a mammalian subject a composition comprising the self-assembled polypeptide complex of any one of claims 1-53.

55. The method of claim 54, wherein the subject is a human.

56. The method of claim 54 or 55, comprising administering by a systemic route.

57. The method of claim 56, wherein said systemic route comprises subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

58. The method of any one of claims 54-57, wherein the self-assembling polypeptide complex has a half-life in the mammalian subject of at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, or at least 28 days after administration.

59. The method of any one of claims 54-58, wherein the self-assembling polypeptide complex has a half-life in the mammalian subject of 3 days to 35 days after administration.

60. The method of any one of claims 54-59, wherein the area under the curve (AUC) of the self-assembled polypeptide complex in the mammalian subject after administration is at least 10 days, μg/mL, at least 25 days, μg/mL, at least 50 days, μg/mL, at least 100 days, μg/mL, at least 200 days, μg/mL, at least 300 days, μg/mL, at least 400 days, μg/mL, at least 500 days, μg/mL, at least 750 days, μg/mL, at least 1000 days, μg/mL, at least 1500 days, μg/mL, at least 2000 days, μg/mL, at least 2500 days, μg/mL, at least 3000 days, μg/mL, at least 4000 days, μg/mL, at least 5000 days, μg/mL, at least 7000, μg/mL, or at least 8000 days, μg/mL.

61. The method of any one of claims 54-60, wherein the area under the curve (AUC) of the self-assembled polypeptide complex in the mammalian subject after administration is from about 10 days μg/mL to about 8000 days μg/mL.

62. The method of any one of claims 54-61, wherein the maximum concentration (Cmax) of the self-assembling polypeptide complex in the mammalian subject after administration is at least 10 μg/mL, at least 25 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 250 μg/mL, at least 500 μg/mL, at least 750 μg/mL, at least 1mg/mL, at least 10mg/mL, at least 25mg/mL, at least 50mg/mL, at least 75mg/mL, at least 100mg/mL, at least 250mg/mL, at least 500mg/mL, or at least 750mg/mL.

63. The method of any one of claims 54-62, wherein the maximum concentration (Cmax) of the self-assembling polypeptide complex in the mammalian subject after administration is about 10 μg/mL to about 750mg/mL.

64. A fusion polypeptide comprising (1) an Fc polypeptide linked to (2) a nanocage monomer or subunit thereof, wherein the Fc polypeptide comprises an Fc chain having one or more mutations relative to a reference Fc chain of the same Ig class, wherein the one or more mutations comprise a mutation or set of mutations associated with altered binding to FcRn and/or altered effector function.

65. The fusion polypeptide of claim 64, wherein the nanocage monomers are ferritin monomers.

66. The fusion polypeptide of claim 65, wherein said ferritin monomer is a ferritin light chain.

67. The fusion polypeptide of claim 66, which does not comprise any ferritin heavy chain or a subunit of ferritin heavy chain.

68. The fusion polypeptide of any one of claims 65-67, wherein the ferritin monomer is human ferritin.

69. The fusion polypeptide of any one of claims 64-68, wherein the Fc polypeptide is an IgG1 Fc polypeptide.

70. The fusion polypeptide of any one of claims 64-68, wherein the Fc polypeptide is an IgG2 Fc polypeptide.

71. The fusion polypeptide of any one of claims 64-70, wherein the Fc polypeptide is a single chain Fc (scFc).

72. The fusion polypeptide of any one of claims 64-71, wherein the mutation or set of mutations comprises a mutation at one or more of the following residues: m252, I253, S254, T256, K288, M428, and N434, or combinations thereof, wherein numbering is according to EU index.

73. The fusion polypeptide of any one of claims 64-72, wherein the altered binding to FcRn is reduced binding to FcRn.

74. The fusion polypeptide of claim 73, wherein the mutation or set of mutations associated with reduced binding to FcRn is selected from the group consisting of I253A, I V and K288A, and combinations thereof, wherein numbering is according to the EU index.

75. The fusion polypeptide of any one of claims 64-71, wherein the Fc polypeptide is an IgG1 Fc polypeptide, and wherein the mutation or set of mutations comprises mutations at one or more of the following residues: l234, L235, G236, G237, P329 and a330 or combinations thereof, wherein numbering is according to EU index.

76. The fusion polypeptide of any one of claims 64-71 and 75, wherein the altered effector function is a reduced effector function.

77. The fusion polypeptide of claim 76, wherein the mutation or set of mutations associated with reduced effector function is selected from the group consisting of LALA (L234A/L235A), LALAP (L234A/L235A/P329G), G236R, G237A and a330L, wherein numbering is according to the EU index.

78. The fusion polypeptide of any one of claims 64-77, wherein the nanocage monomer or subunit thereof is a ferritin monomer subunit, and

79. The fusion polypeptide of any one of claims 64-78, wherein within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomers or subunits thereof via an amino acid linker.

80. The fusion polypeptide of any one of claims 64-79, wherein within each first fusion polypeptide, the Fc polypeptide is linked to the nanocage monomer or subunit thereof at the N-terminus of the nanocage monomer or subunit thereof.