Lysosomal degradation

Abstract

The present invention relates to bispecific antigen-binding polypeptides that can be used to remove unwanted agents, such as viruses or toxins, from the body and target them for degradation. The bispecific antigen-binding polypeptides of the invention have a first antigen-binding domain that binds to an extracellular molecule and a second antigen-binding domain that binds to a cell surface protein, whereby the cell surface protein mediates internalization of the bound complex. The present invention also relates to pharmaceutical compositions comprising the bispecific antigen-binding polypeptides, and methods for targeting extracellular molecules for cellular internalization and degradation via the lysosomal pathway.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to bispecific antigen-binding polypeptides that can be used to remove unwanted agents, such as viruses or toxins, from the body and target them for degradation. The bispecific antigen-binding polypeptides of the invention have a first antigen-binding domain that binds to an extracellular molecule and a second antigen-binding domain that binds to a cell surface protein, whereby the cell surface protein mediates internalization of the bound complex. The present invention also relates to pharmaceutical compositions comprising the bispecific antigen-binding polypeptides, and to methods of targeting extracellular molecules for cellular internalization and degradation via the lysosomal pathway. [Background technology]

[0002] BACKGROUND OF THE INVENTION The body encounters many unwanted agents, including infectious agents, viruses, microbial pathogens and toxins, damaged proteins, and other endogenous products that have become toxic, including excess cytokines. To remove such unwanted and harmful agents, the body has evolved numerous mechanisms, many of which involve activation of the immune system. However, in pathogenic conditions, such as HIV infection, the body's removal mechanisms appear insufficient, likely because pathogenic agents have acquired an evolutionary advantage that allows them to evade the body's natural clearing mechanisms.

[0003] Worldwide, 6 in 1,000 people are infected with the human immunodeficiency virus (HIV). Since 1981, more than 25 million people have died from acquired immunodeficiency syndrome (AIDS), the disease caused by HIV. Current antiretroviral drug cocktails are effective in suppressing viral replication and reducing AIDS-related morbidity and mortality. However, antiretroviral therapy does not eliminate HIV that persists in a latent state, nor does it stop viral production from these latent reservoirs. Therefore, interruption of treatment leads to the resumption of viral replication and rapid viral regrowth from these latent reservoirs (Kreider, EF and Bar, KJ, Current HIV / AIDS Reports, 2022; doi.org / 10.1007 / s11904-022-00604-2). This requires infected individuals to adhere to lifelong daily antiretroviral therapy, which is expensive and therefore not available to all infected people.

[0004] Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoproteins can be used as an alternative to antiretroviral therapy (Caskey, M., Current Opinion in HIV and AIDS; 2020; 15: 49-55). These bNAbs bind to one of several conserved epitopes present on the envelope glycoprotein gp120 (which plays an essential role in the infection process), thereby inhibiting viral entry into target immune cells and inducing long-term suppression of viral replication. However, due to the high mutation rate of HIV, a major problem associated with the use of bNAbs is the emergence of antibody-resistant viral variants. Furthermore, bNAb binding results in the persistence of the virus in the circulation, and the actual rate of HIV viral clearance is unclear and likely reduced in HIV patients.

[0005] Targeted protein degradation (TPD) has been recognized as a promising new therapeutic modality designed to promote the clearance of unwanted factors from the body (Deshaies, RJ, Nature 2020;580:329-338). Lysosomal-targeting chimeric molecules (LYTACs), heterofunctional molecules containing a target protein recruiting molecule (e.g., an antibody) connected by a linker to a ligand for a cell surface receptor, such as the cell-type-specific lysosomal targeting receptor, the cation-independent mannose-6-phosphate receptor (CI-M6PR) or the asialoglycoprotein receptor (ASGPR), have been developed. For example, the recruiting molecule can be tagged with a tri-GalNAc motif, a ligand that can engage ASGPR for specific targeted degradation by hepatocytes (Ahn G, Banik SM, Miller CL, Riley NM, Cochran JR, Bertozzi CR, Nat Chem Biol. 2021;17(9):937-946). In this way, extracellular proteins can be degraded by cells that specifically express these lysosomal targeting receptors.

[0006] Another approach is to use so-called "sweeping antibodies." Sweeping antibodies require modifications of both the variable and constant regions, as described, for example, in U.S. Patent Publication No. US9890377B2. The variable region is modified to enable the antibody to bind to antigens in plasma and undergo pH-dependent dissociation from the antigen in endosomes, while the constant region is modified to increase cellular uptake of the antibody-antigen complex into endosomes via the FcRn receptor. This combination of modifications significantly accelerates the clearance of soluble antigens from the circulation.

[0007] Both LYTACs and sweeping antibodies rely on utilizing cells in the body that have a natural function in clearing cargo. In general, the immune system has a remarkable adaptive capacity to combat unwanted factors in the body. However, as mentioned above, in certain pathogenic conditions, such as HIV infection, the virus adapts to evade removal by these natural clearance mechanisms. Because the body's natural immune function may be impaired, there remains a need to identify novel approaches for the rapid targeting of unwanted soluble factors for degradation that are independent of a functional immune system and do not rely on specialized cell types. Summary of the Invention

[0008] (Summary of the Invention) The present inventors have developed a novel, alternative approach independent of a functional immune system that can capture and remove a wide range of unwanted soluble factors by redirecting them to unspecialized, non-immunological cells where they are internalized and degraded. The present invention utilizes a bispecific antigen-binding molecule that can bind to circulating unwanted soluble factors via one domain and to cell surface proteins, such as membrane receptors, via the other domain, such that binding results in internalization of the bispecific polypeptide containing the bound unwanted factor. These cells are therefore forced to become novel degradation units for the unwanted factors.

[0009] The present inventors have demonstrated that this novel approach can be used to eliminate HIV, for example, by forcibly targeting the virus to cells expressing the epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. By utilizing bispecific VHH antibodies designed to bind to both the HIV envelope protein and EGFR, the present inventors have demonstrated that HIV can be efficiently recruited to EGFR (present on cells in the body where the virus is not adapted), internalized, and degraded in the lysosomal pathway.

[0010] In a first aspect, the present invention provides a method for producing a composition comprising: a) a first antigen-binding domain that binds to an extracellular molecule; and b) a second antigen-binding domain that binds to a cell surface protein

[0013] The present invention provides a bispecific antigen-binding polypeptide comprising:

[0011] In some embodiments, the extracellular molecule is internalized and degraded when both the extracellular molecule and the cell surface protein bind to the bispecific antigen-binding polypeptide.

[0012] In certain embodiments, the extracellular molecule is degraded via the lysosomal pathway.

[0013] In one embodiment, the bispecific antigen-binding polypeptide is a bispecific antibody, a bispecific antibody fragment, or a bispecific VHH antibody. Preferably, the first antigen-binding domain is a VHH single domain and the second antigen-binding domain is a VHH single domain, optionally wherein the two VHH single domains are connected via a linker.

[0014] In some embodiments, the extracellular molecule is a viral antigen, a toxin, a microbial pathogen, an allergen, a damaged or deregulated protein, an autoantibody, or other pathological or infectious agent. In some preferred embodiments, the extracellular molecule is an HIV envelope glycoprotein. In some preferred embodiments, the extracellular molecule is a Sars-Cov2 spike protein.

[0015] In some embodiments, the cell surface protein is epidermal growth factor receptor (EGFR), low density lipoprotein receptor (LDLR), transferrin receptor (TfR), hepatocyte growth factor receptor (cMet), MHC class II, vascular endothelial growth factor receptor (VEGFR) or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4-1-BB, or ICOS.

[0016] In some embodiments, the cell is a fibroblast, an epithelial cell, an endothelial cell, a blood cell, or a platelet.

[0017] In some preferred embodiments of the bispecific antigen-binding polypeptide, the first antigen-binding domain binds to HIV and the second antigen-binding domain binds to EGFR. In some preferred embodiments of the bispecific antigen-binding polypeptide, the first antigen-binding domain binds to Sars-Cov2 and the second antigen-binding domain binds to EGFR.

[0018] In a second aspect, the present invention provides a pharmaceutical composition comprising a bispecific antigen-binding polypeptide disclosed herein.

[0019] In a third aspect, the present invention provides a method of targeting an extracellular molecule for cellular internalization and degradation, comprising administration of a bispecific antigen-binding polypeptide or pharmaceutical composition disclosed herein to a subject in need thereof.

[0020] In particularly preferred embodiments, there is provided a method for treating or preventing HIV, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or pharmaceutical composition disclosed herein. In particularly preferred embodiments, there is provided a method for treating or preventing Sars-Cov2, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or pharmaceutical composition disclosed herein.

[0021] In certain embodiments, the bispecific antigen-binding polypeptide is administered orally, sublingually, topically, intravenously, intramuscularly, intradermally, transdermally, intraperitoneally, subcutaneously, intranasally, intravaginally, intrarectally, or by inhalation. [Brief explanation of the drawings]

[0022] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]Figure 1: Bispecific VHH llama antibodies target HIV to the EGFR extracellular domain. (a) The VHH in the bispecific construct retains good binding properties. Wells were coated with antigen, and binding was measured by ELISA. n=2, a representative experiment is shown. (b) Bispecific VHH mediates binding of HIV-1 antigen to the EGFR extracellular domain. Schematic configuration [1 = immobilized EGFR in the well, 2 = (heterospecific) bihead VHH, and 3 = IR800-labeled HIV-1 protein] with IR800-labeled gp140 UG membrane protein specifically targeted to EGFR in the well. n>2, a representative experiment is shown. (c) Schematic of the relative efficacy of targeting by bispecific VHH. gp140 is gp140(UG37), and the virus-like particles (VLPs) are VLPHIV, except for Hep1E1, which is VLPHBV. Shown are quantitative IR800 FLISA results; for overview, IR800 scans of a representative microtiter plate (dotted circles indicate wells) are shown. n=2. (d) Efficient targeting of low and high concentrations of HIV-1 to the coated EGFR extracellular domain. Virus was preincubated with 18 nM or 3 nM bihead for 1 hour and allowed to bind to the EGFR-coated ELISA plate for 2 hours. Bound HIV was detected by p24 ELISA. Left graph: Virus concentrations in the input were 10.1, 4.9, 3.9, and 9.0 ng / 100 μl for clade A (92UG037), clade C (96ZM651), and clade B reference strains Bal and HXB2, respectively. Right graph: The same was done with 10-fold concentrated HXB2. Clade B, n = 2, mean ± sd; clades A and C, n = 1, shown in duplicate. [Figure 2]Figure 2: Binding, internalization, and degradation in EGFR-expressing cells. (a) Kinetics of binding and internalization. Labeled antigen (gp140 UG37) was preincubated with bispecific VHH for 30 min and then incubated with cells. At the indicated times, cells were washed to reveal total bound antigen (well B) or detached to remove cell surface antigen and reveal internalized antigen (well I). Bound antigen (B) and internalized antigen (I) were scanned and quantified for IR800. The top panel shows IR800 scans of several illustrative wells. The bottom panel shows the quantification graph. The difference between bound and internalized represents the amount still exposed on the cell surface. While all four show similar net binding kinetics, internalization is more variable. n = 2, representative experiment shown. (b) Immunofluorescence demonstrates that the envelope protein gp140 and virus-like particles are internalized in a similar manner. Covalently green fluorescently labeled antigen (gp140, VLPHBV) was preincubated with the bispecific VHH and added to cells for 0 min, 90 min, and 18 h. Blue indicates nuclei. Scale bar: 5 μm. n=2, a representative experiment is shown. (c) Bispecific VHH-mediated internalization leads to rapid degradation of HIV proteins in cells. Development of an IR800-based internalization and degradation assay. Top: The complex IR800-labeled gp140 + bispecific VHH H2E1 was preincubated and allowed to bind to the indicated cells in 48-well plates on ice for 2 h. After washing, chases were performed in conditioned medium at 37 °C for the indicated times (in minutes). Cells were washed and scanned for IR800. (Bottom) Next, cells in the wells were solubilized, and proteins were separated on an SDS-PAGE gel and analyzed for IR800. Note that after binding (0 min chase), only full-length HIV envelope protein is observed; after overnight chase (o / n), labeled proteolytic products are primarily detected at the front of the gel. n>2, representative experiment shown. (d) Comparison of the kinetics of degradation of IR800-labeled EGF, gp140, and VLPADW. After labeling, gp140 and VLPHBV were first preincubated with the bispecific VHHs H2E1 and Hep1E1, respectively.Next, incubation was performed on ice for 2 hours with Her14 cells, the cells were washed, and the complexes were internalized at 37°C for 0, 90, 180 minutes, or overnight. Cells were then lysed, run on SDS-page gels, and scanned for IR800. The gels were then blotted onto PVDF and Western blot analysis using α-actin was performed to confirm identical loading. IR800 scans of the gels show IR800-labeled EGF, gp140, and VLPADW, respectively. Intact and degraded proteins are part of the same gel. The actin panel is a Western blot. n=2, representative experiments are shown. Graph: IR800 quantification of intact protein and total IR800 dye in cells, n=2, mean ± SD. [Figure 3] Figure 3: Bispecific VHH-mediated recruitment leads to lysosomal degradation. (a) Colocalization of Alexa-488 fluorescently labeled EGF, gp140, and VLP (green) with Lysotracker Red in EGFR-expressing cells. EGF was bound for 10 minutes and overnight, and viral proteins were internalized overnight mediated by the bispecific VHHs H2E1 and Hep1E1, respectively. Nuclei are blue. Scale bar: 5 μm. n = 2, representative experiment shown. (b) Chloroquine inhibits EGF degradation. SDS-PAGE gel of IR800-labeled EGF in cells. After 60 minutes of pretreatment with chloroquine (shown in μM), EGF was incubated on the cells. Cells were lysed and run on an SDS-PAGE gel, and IR800 was detected. n = 2, representative experiment shown. (c) Similarly, chloroquine inhibits HBV degradation of gp140 and VLP. SDS-PAGE gel of IR800-labeled gp140 and VLPHBV internalized into cells after treatment with or without chloroquine (100 μM). Intact and degraded forms are in the same lane in the gel. n=2, representative experiment shown. [Figure 4]Figure 4: Specific binding of infectious HIV particles / virions to EGFR-expressing cells leads to internalization and lysosomal degradation. (a) Upon incubation, cell-bound HIV is internalized and degraded. Preincubated HXB2 cells were incubated with Her14 and 14C cells (both expressing EGFR) for 3 hours (loading), and unbound virus was washed away. Viral protein was determined directly (loading) and 21 hours later by measuring p24 in cell lysates by ELISA. n = 2, mean ± sd. (b) Trans-infectivity assay. Separately, after the indicated time points, MT2 indicator cells were added to establish trans-infectivity of bound HIV. Titers indicate the virus dilution at which syncytia still formed. n = 2, identical results. (c) Chloroquine inhibits degradation of internalized HIV. Her14 cells were treated with (+) or without (-) 100 μM chloroquine and incubated with virus in the presence of heparin for 3 hours, then washed and incubated for 24 hours. The presence of HIV was determined by p24 ELISA. n=2, a representative experiment is shown. (d) The graph shows the percentage of uncleaved HIV after 24 hours as a percentage of input bound after 3 hours. n=2, mean ± sd. [Figure 5]Figure 5: Targeted degradation of SARS-CoV-2 spike protein by SARS-CoV-2-specific bispecific single-chain llama antibodies. (a) shows that the bispecific biheads C1-E3 and C2-E3 specifically bind spike protein and target it to EGFR-expressing Her14 cells, whereas the E3-E3 control bihead does not. Background amounts of spike-IR800 protein bind in the absence of the bihead but produce a much lower signal. (b) PAGE gel analysis shows that spike protein targeted to EGFR-expressing cells by C1-E3 and C2-E3 is largely degraded after 4 hours, similar to the EGF-IR800 control protein. In all cases, the lower part of the gel shows labeled small degradation fragments. The top and bottom panels in (b) are from the same gel. DETAILED DESCRIPTION OF THE INVENTION

[0023] (Detailed explanation) (A.Definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Without limiting any term, further clarification of some of the terms used herein is provided below.

[0024] As used herein, the term "antibody" refers to a polypeptide having a combination of two heavy chains and two light chains and possessing significant, known specific immunoreactive activity against an antigen of interest. There are five distinct classes of antibodies that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention. The following discussion generally refers to immunoglobulin molecules of the IgG class. With respect to IgG, immunoglobulins contain two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 to 70,000 daltons. These four chains are connected by disulfide bonds in a "Y" configuration, where the light chains support the heavy chains, which begin at the mouth of the "Y" and continue through the variable region.

[0025] Antibody light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can associate with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within each (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody: IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art in light of the present disclosure and, accordingly, are within the scope of the present invention.

[0026] The variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site. This four-element antibody structure forms the antigen-binding site at the end of each arm of a Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) on each of the VH and VL chains. CDRs are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of Protein of Immunological Interest (1991), as well as by Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where these definitions include overlapping or subsets of amino acid residues when compared with each other.

[0027] As used herein, the term "VHH antibody" or "heavy chain-only antibody" refers to a type of antibody produced exclusively by species in the Camelidae family, which includes camels, llamas, and alpacas. These antibodies are composed of two heavy chains and lack light chains. Each heavy chain has a variable domain at its N-terminus, and these variable domains are called "VHH" domains to distinguish them from the variable domains of the heavy chains of the conventional heterotetrameric antibodies described above, i.e., VH domains. Cartilaginous fish also have heavy chain-only antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which V NAR Single domain antibodies, called fragments, can be obtained.

[0028] As used herein, the term "VHH single-domain antibody" or "nanobody" refers to the variable domain (i.e., the target-recognition portion) of a non-traditional heavy-chain-only camelid VHH antibody. VHHs are referred to as single-domain antibodies or nanobodies (registered trademarks of Ablynx) due to their small size in the nanometer range. While retaining the target specificity of a whole Ig molecule, their small size, excellent properties, and ease of manufacture make them ideal tools for diagnostic and clinical applications. Notably, nanobodies typically lack the solubility and aggregation problems inherent to the VH domains of conventional antibodies. A notable difference between camelid VHHs and human VH domains is the length and orientation of the CDR3 loops. The longer CDR3 of VHHs increases the potential interaction surface with the target antigen, enabling nanobodies to bind unique epitopes inaccessible to conventional mAbs.

[0029] As used herein, the term "bispecific antigen-binding polypeptide" can refer to any single binding molecule that has the dual function of specifically binding to at least two different epitopes. The term "specificity" refers to the ability of an antibody to specifically bind (e.g., immunoreact) with a given target antigen. An antibody molecule or fragment thereof may be monospecific and contain one or more antigen-binding domains that specifically bind to a single epitope on a single target antigen. Alternatively, an antibody molecule or fragment thereof may be "multispecific" and contain two or more antigen-binding domains that specifically bind to different epitopes, either within the same antigen or within different target antigens. For example, a bispecific antigen-binding polypeptide has two antigen-binding domains that can recognize and bind to two different target epitopes or antigens. To achieve multispecificity, multispecific antibodies or fragments thereof are artificially engineered to contain different combinations of antigen-binding domains. Thus, the bispecific antigen-binding polypeptide of the present invention may be a bispecific antibody having different VH-VL pairs for binding to two different target epitopes or antigens. Alternatively, the bispecific antigen-binding polypeptide of the present invention may be a bispecific VHH antibody. This may be a bispecific VHH heavy chain-only antibody having different VHH domains present in each of the two heavy chains. Furthermore, a bispecific VHH antibody may refer to a bispecific VHH single-chain antibody artificially created by combining at least two different VHH domains with specificities for different epitopes or antigens. In a preferred embodiment, the bispecific VHH antibody of the present invention is composed of two VHH domains connected by a flexible linker. A bispecific VHH antibody may also be referred to herein as a "bihead," meaning that the bihead contains two functional groups. This may be a bispecific antibody that recognizes different epitopes. A bispecific antibody can recognize two different epitopes on the same molecule (biparatopic antibody) or epitopes on different molecules.The bispecific antibodies we refer to herein typically use one head to bind to an extracellular unwanted factor and the other head to bind to a cell membrane molecule that mediates internalization and degradation.

[0030] As used herein, the term "antigen-binding domain" refers to a portion of an intact antibody that is responsible for selectively binding to a target antigen. In particular, an antigen-binding domain includes a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes for antigen binding with the intact antibody (i.e., with the intact antibody from which it was derived). Exemplary antigen-binding domains include antigen-binding fragments of antibodies, such as antibody light chain variable domains (VL), antibody heavy chain variable domains (VH), single-chain antibodies (scFv), F(ab')2 fragments, Fab fragments, Fd fragments, Fv fragments, mono-armed (monovalent) antibodies, diabodies, triabodies, tetrabodies, or any antigen-binding molecule formed by combining, assembling, or conjugating such antigen-binding fragments. The term antigen-binding domain as used herein is further intended to encompass antibody fragments selected from the group consisting of unibodies, domain antibodies, and nanobodies (also referred to as VHH single-domain antibodies). Preferably, an antigen-binding domain comprises one or more CDRs that form all or part of the antigen-binding site. Fragments can be obtained by chemical or enzymatic treatment of intact or complete antibodies or antibody chains, or by recombinant means. In addition to antibodies, antigen-binding agents can also include aptamers or other entities that recognize and bind to antigens.

[0031] B. Bispecific Antigen-Binding Polypeptide Formats The present invention provides bispecific antigen-binding polypeptides comprising: a) a first antigen-binding domain that binds to an extracellular molecule; and b) a second antigen-binding domain that binds to a cell surface protein. Upon binding of both the extracellular molecule and the cell surface protein to their respective antigen-binding domains, the complex is internalized by the cell, and the extracellular molecule is then targeted for degradation, preferably via the lysosomal pathway. Internalization is mediated by the cell surface protein.

[0032] A bispecific antigen-binding polypeptide is not limited to any particular format, provided that it comprises a) a first antigen-binding domain that binds to an extracellular molecule; and b) a second antigen-binding domain that binds to a cell surface protein.

[0033] The bispecific antigen-binding polypeptide may be a bispecific antibody. For example, the bispecific antibody may be a conventional heterotetrameric antibody having two variable domains that bind to different epitopes and / or different antigens. In other embodiments, the bispecific antigen-binding polypeptide may be a bispecific antibody fragment, including but not limited to, a F(ab')2 fragment, a tandem scFv, a bispecific T cell engager (BiTE), a diabody, a triabody, a tetrabody, or a bi-nanobody.

[0034] The bispecific antigen-binding polypeptide may be a bispecific VHH antibody. In one embodiment, the bispecific VHH antibody is a bispecific heavy-chain-only antibody. Preferably, the bispecific VHH antibody is a single-chain antibody composed of at least two VHH single domains connected together. Bispecific VHH single-chain antibodies, also referred to herein as "biheads" or "bi-nanobodies," can be artificially generated by combining two different VHH domains with specificities for two different epitopes or antigens. In a preferred embodiment, the bispecific VHH antibody is composed of two different VHH domains connected by a flexible linker. In a preferred embodiment, the bispecific VHH antibody is composed of two different VHH domains connected by a peptide linker. In a preferred embodiment, the first antigen-binding domain is a VHH single domain and the second antigen-binding domain is a VHH single domain, optionally wherein the two VHH single domains are connected via a linker. For example, the two different VHH domains are [ka] They may be combined via a peptide linker sequence, however, those skilled in the art will be aware of other suitable linkages that can be effectively utilized to combine two VHH domains.

[0035] Bispecific molecules, including bispecific antibodies, are known in the art, and those skilled in the art know how to produce such bispecific molecules. Briefly, a first VHH domain (or nanobody) specific for a first target antigen (e.g., an extracellular molecule) can be generated or obtained by active immunization of a host species with a polypeptide containing the antigen. A second VHH domain (or nanobody) specific for a second target antigen (e.g., a cell surface protein) can be generated or obtained by active immunization of a host species with a polypeptide containing the antigen. To produce VHH antibodies, any species from the Camelidae family, including llamas, can be immunized with polypeptides containing the respective antigens. The genes for the two VHHs are linked to each other via a linker sequence using standard recombinant techniques. Upon intracellular expression, the bispecific VHH antibody (or bihead) protein is purified. Production is possible in bacteria, yeast, eukaryotic expression cells, etc.

[0036] Further functional specificity can be added. For example, additional linkers and VHHs can be added to produce trihead antibodies with additional specific properties. For example, when injected into the blood, the residence time of VHH antibodies in the circulation is shorter than that of conventional antibodies. One approach to extending the in vivo half-life of these molecules is to introduce a third VHH domain, such as an anti-albumin-binding VHH domain, which mediates binding to albumin and prevents rapid clearance of the antibody by the kidney in vivo. Thus, the bispecific antigen-binding polypeptides of the present invention must contain at least two antigen-binding domains, but in certain embodiments may have three or more antigen-binding domains. In certain embodiments, the bispecific antigen-binding polypeptide further comprises (c) a third antigen-binding domain, optionally wherein the third antigen-binding domain binds to albumin.

[0037] The bispecific VHH antibody (bihead) format is particularly preferred due to the small size of the VHH domain. Biheads are composed of a combination of two VHH domains. VHHs are small single-chain antibody fragments derived from llamas. VHH biheads have tremendous advantages: 1) they are easily constructed by cloning in bacteria; 2) they are easily produced and purified in large quantities at low cost; 3) for human therapeutics, they can be easily produced in yeast or other organisms suitable for producing proteins used in clinical trials; 4) they are extremely stable, can withstand harsh conditions, and can be stored without refrigeration; and 5) they are non-immunogenic. Furthermore, because multiple VHHs can be easily combined within a single molecule, VHH domains are particularly suitable for constructing bivalent, bispecific binding molecules.

[0038] Homology studies between camelid germline IgV gene repertoires and their human counterparts have found 95% sequence identity between camelid IGHV family 3 and its human FR counterpart. This means that nanobodies have a low immunogenicity profile and are therefore suitable for administration to humans. Furthermore, the sequences of therapeutic nanobodies can be "humanized" at any time, if desired.

[0039] C. First Antigen-Binding Domain As defined in the claims, the bispecific antigen-binding polypeptide comprises a first antigen-binding domain that binds to an extracellular molecule. The bispecific antigen-binding polypeptide can be modified so that the first antigen-binding domain binds to any extracellular molecule of interest. In particular, it is an object of the present invention to bind to unwanted factors, thereby targeting these factors for removal from circulation and subsequent degradation. In one embodiment, the extracellular molecule is membrane-bound. In a preferred embodiment, the extracellular molecule is soluble.

[0040] In some embodiments, the extracellular molecule is a viral antigen, a toxin, a microbial pathogen, an allergen, a damaged or dysregulated protein, an autoantibody, or other pathological or infectious agent. In some embodiments, the extracellular molecule is a viral antigen. That is, the extracellular molecule is a viral antigen present on the surface of a virus.

[0041] In certain embodiments where the extracellular molecule is a viral antigen, the extracellular molecule is a viral antigen present on HIV, hepatitis, Sars-Cov2, influenza, herpes, Epstein-Barr virus, adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, respiratory syncytial virus, pandemic mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus.

[0042] In other embodiments, the extracellular molecule is a damaged or dysregulated protein that contributes to a disease state. In certain embodiments, the extracellular molecule is a cytokine or growth factor. In certain embodiments, the extracellular molecule is an interferon, an interleukin, a tumor necrosis factor (TNF), or a transforming growth factor b (TGFb). In specific embodiments, the extracellular molecule is type 1 interferon (IFN), IL-6, PDL-1, GM-CSF, Gal-3BP, BAG3, an IL-17 family member, EGF, VEGF, NRG1, NRG2, NRG3, NRG4, HGF, RANK ligand, TNF-α, a soluble TNF-α receptor, IL-Ib, IL-5, IL-17A, IL-12, IL-23, C5, BAFF, IgE, or TGFb. In certain embodiments, the extracellular molecule is a protein that accumulates in a disease state, such as alpha-synuclein. In other embodiments, the extracellular molecule is a cholesterol carrier such as ApoB or ApoE4. In other embodiments, the extracellular molecule is a clotting factor such as factor IX. In other embodiments, the extracellular molecule is a mucin such as MUC1, MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, or GPIb. In certain embodiments, the extracellular molecule is a hormone such as insulin or ACTH.

[0043] In some embodiments, the extracellular molecule is an autoantibody. Non-limiting examples of autoantibodies include: rheumatoid factor (RF), antinuclear antibodies (ANA), antineutrophil cytoplasmic antibodies (ANCA), anti-double-stranded DNA (anti-dsDNA), anticentromere antibodies (ACA), anti-histone antibodies, cyclic citrullinated peptide antibodies (CCP), extractable nuclear antigen antibodies (e.g., anti-SS-A (Ro) and anti-SS-B (La), anti-RNP, anti-Jo-1, anti-Sm, Scl-70), cardiolipin antibodies, beta-2 glycoprotein 1 antibodies, anti-ribonucleotide ... These include phospholipid antibodies (APA), lupus anticoagulant (LA), diabetes-associated autoantibodies, anti-tissue transglutaminase (anti-tTG), anti-gliadin antibodies (AGA), intrinsic factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., anti-TPO, TSH receptor antibodies), smooth muscle antibodies (SMA), antimitochondrial antibodies (AMA), liver-kidney microsomal type 1 antibodies (anti-LKM-1), anti-glomerular basement membrane (GBM), or acetylcholine receptor (AChR) antibodies.

[0044] In other embodiments in which the extracellular molecule is a toxin, the extracellular molecule is a snake toxin such as toxic shock syndrome toxin (TSST-1), cobra toxin (Cbtx), a bacterial toxin such as a clostridial toxin, or myeloperoxidase.

[0045] Antibodies or antigen-binding domains have also been generated against small proteinaceous and non-proteinaceous toxins. Because lysosomal targeting results in the inactivation of toxins, these targets can also serve as targets for the present invention. Among such putative targets are opioids. Fatal opioid-related overdoses are common. Monoclonal antibodies are an emerging therapeutic strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier and thereby preventing or reversing opioid toxicity. Monoclonal antibodies with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine have previously been generated (Rodarte et al., 2023; Structure; 31(1): 20-32). The present invention is particularly advantageous. The small size and uncomplicated nature of VHHs are beneficial for facilitating binding to these opioid targets. Furthermore, the lysosomal targeting method of the present invention not only sequesters opioids but also rapidly clears them. Thus, in some embodiments, the extracellular molecule is an opioid, hi some embodiments, the extracellular molecule is selected from oxycodone, morphine, fentanyl, and nicotine.

[0046] In general, the extracellular molecule can be any factor to which the first antigen-binding domain can bind. In particular, the present invention provides an alternative to known blocking antibodies. While blocking antibodies specifically require that the antigen-binding domain bind to a functionally relevant epitope on the target, the bispecifics of the present invention can bind to any epitope on the target, provided that this results in rapid clearance. This is a significant improvement over blocking antibodies, which, once bound to their target, remain in the circulation for a significant period of time, increasing the risk of toxic factor release. Therefore, the high affinity binding requirement of blocking antibodies is much higher than the binding affinity required for the bispecifics of the present invention. Because binding to a random epitope on the target is sufficient, suitable antibodies are much easier to obtain, and furthermore, several bispecifics binding to different epitopes can be used in combination, thereby increasing binding strength to the target.

[0047] The present invention also contemplates that bispecific antigen-binding polypeptides can be used in conjunction with known therapies. For example, bispecific antigen-binding polypeptides can be used in conjunction with known blocking antibodies. In this way, the present invention can be used to enhance the efficacy of blocking antibody therapies that are already successful in clinical practice. As discussed above, conventional blocking antibodies bind to their targets but remain in the circulation for a significant period of time before being cleared. Advantageously, the bispecifics of the present invention are engineered such that the first antigen-binding domain binds to an antigen that has already been blocked (i.e., binds to a blocking antibody-antigen complex), either by binding to a blocking antibody or by binding to another epitope on the antigen. The inventors hypothesize that this approach may provide an additional boost because the bispecific targets the entire complex to a cell surface protein, resulting in internalization and degradation of the entire complex, including the target. In this way, the target is rapidly cleared, rather than simply blocked. Accordingly, in certain embodiments, the extracellular molecule is a blocking antibody-antigen complex. In certain embodiments, the first antigen-binding domain binds to a blocking antibody-antigen complex.

[0048] In certain embodiments exemplified herein, the extracellular molecule is HIV. That is, the extracellular molecule is a viral antigen present on HIV. In certain embodiments, the first antigen-binding domain binds to HIV. In certain embodiments, the first antigen-binding domain binds to a viral antigen on HIV. In certain embodiments, the extracellular molecule is HIV-1. In certain embodiments, the first antigen-binding domain binds to HIV-1. The extracellular molecule may be present on any HIV-1 clade, and the first antigen-binding domain may be engineered to bind to any HIV-1 clade. In certain preferred embodiments, the extracellular molecule is an HIV envelope glycoprotein. In certain preferred embodiments, the first antigen-binding domain binds to an HIV envelope glycoprotein. In certain embodiments, the extracellular molecule is gp120, gp41, and / or gp140. In certain embodiments, the first antigen-binding domain binds to gp120, gp41, and / or gp140 on the surface of HIV.

[0049] In some embodiments, a combination of different bispecific antigen-binding polypeptides can be produced, in which the first antigen-binding domain of each bispecific antigen-binding polypeptide is designed to bind to a different epitope of the HIV envelope glycoprotein. In this way, multiple bispecific antigen-binding polypeptides can be used to ensure effective targeting of a wide range of HIV subtypes (also called clades) and variants. Multiplexing also ensures that a single mutation that may limit the binding of one of these bispecifics has limited effect. Therefore, this approach can also be used to minimize the risk of escape mutants.

[0050] D. Second Antigen-Binding Domain As defined in the claims, the bispecific antigen-binding polypeptide comprises a second antigen-binding domain that binds to a cell surface protein. The bispecific antigen-binding polypeptide can be engineered so that the second antigen-binding domain binds to a specific cell surface protein, thereby directing the bound extracellular molecule to a selected cell type. Alternatively, the bispecific antigen-binding polypeptide can be engineered so that the second antigen-binding domain binds to a ubiquitously expressed cell surface protein. Binding to the cell surface protein, e.g., a membrane receptor, results in receptor-mediated endocytosis of the complex (i.e., the antigen-bispecific receptor) and subsequent degradation of the internalized extracellular molecule.

[0051] Different cell types express a vast variety of membrane proteins. Clearing functions have been assigned to some of these, with some acting as "clearing receptors." Typically, such clearing receptors are present only in certain cell types, limiting the clearing ability to only cells expressing these clearing receptors. In contrast, the present invention demonstrates that, regardless of this assigned function, other membrane proteins can be utilized with the same result (i.e., clearing of the target) simply by binding to these membrane proteins, which has been shown to result in internalization and degradation.

[0052] The cell surface protein may be any suitable cell surface protein capable of mediating endocytosis once bound by the bispecific antigen-binding polypeptide. The cellular receptor can be selected for its biological activity that induces internalization of the complex into the cell, resulting in degradation of the unwanted extracellular molecule. Importantly, the cellular receptor may not be the natural receptor for the extracellular molecule. In certain preferred embodiments, the cell surface protein is not naturally involved in mediating lysosomal degradation of the cargo. In certain embodiments, the cell surface protein is not a lysosomal targeting receptor. In certain embodiments, the cell surface protein is not a cation-dependent mannose-6-phosphate receptor (CI-M6PR). In certain embodiments, the cell surface protein is not an asialoglycoprotein receptor (ASGPR). In certain preferred embodiments, the cell surface protein is not naturally involved in mediating lysosomal degradation of the extracellular molecule. In certain embodiments, the cell surface protein does not mediate lysosomal degradation of the extracellular molecule when the extracellular molecule and the cell surface protein are not bound to the bispecific antigen-binding polypeptide. In some embodiments, the second antigen-binding domain binds directly to the cell surface protein, hi some embodiments, the second antigen-binding domain binds indirectly to the cell surface protein, for example, by binding to a ligand of the cell surface protein.

[0053] In some embodiments, the cell surface protein is epidermal growth factor receptor (EGFR), low density lipoprotein receptor (LDLR), transferrin receptor (TfR), hepatocyte growth factor receptor (cMet), MHC class II, vascular endothelial growth factor receptor (VEGFR) or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4-1-BB, or ICOS.

[0054] In one embodiment, the cell surface protein is a member of the EGFR family, such as EGFR, ErbB2, ErbB3, or ErbB4. In one embodiment, the second antigen-binding domain binds to EGFR, ErbB2, ErbB3, or ErbB4. In a preferred embodiment, the cell surface protein is EGFR. In a preferred embodiment, the second antigen-binding domain binds to EGFR. In a preferred embodiment, the second antigen-binding domain binds to an epitope in the extracellular portion of EGFR.

[0055] The cell surface protein may be present on any desired cell type, provided the cell is capable of lysosomal degradation of the extracellular molecule. A particular advantage of the present invention is that unwanted extracellular molecules can be recruited to membrane proteins on cells that have not been described to naturally participate in cargo clearance but are nonetheless capable of doing so. The examples provided herein demonstrate that unwanted extracellular molecules can be targeted to, for example, EGFR, which is ubiquitously expressed in most tissues of the body. This forced targeting leads to the cellular internalization and degradation of the extracellular molecule. Without wishing to be bound by theory, it is hypothesized that the complex of the bispecific-bound extracellular molecule piggybacks on the natural turnover of EGFR. Interestingly, the inventors have found that EGF is not required for this process. Membrane EGFR has many signaling functions. Most notably, it responds rapidly to binding to the growth factor EGF, resulting in the activation of various signaling pathways. During this process, the EGFR-EGF complex is internalized and targeted to lysosomes, where both the receptor and growth factor are degraded. Degradation of both the receptor and growth factor is initiated by EGF binding to EGFR. The present invention demonstrates that even a moiety that blocks EGF binding still results in internalization and degradation of the receptor and the bound bihead, including degradation of the protein of interest bound to the bihead, by simply binding to EGFR with an antibody. This is a novel finding that is unexpected given the function of EGFR. This differs from biheads, which recognize EGFR with both heads, induce EGFR clustering at the membrane, and then internalize and degrade the receptor.

[0056] In certain embodiments, the cell surface protein is present on a cell that is capable of lysosomal degradation of extracellular molecules. In certain embodiments, the cell surface protein is present on a cell that does not naturally undergo lysosomal degradation of extracellular molecules. In certain embodiments, the cell surface protein is present on a cell that does not undergo lysosomal degradation of extracellular molecules when the extracellular molecule and cell surface protein are not bound to the bispecific antigen-binding polypeptide. That is, the cell may be able to internalize and degrade the bound extracellular molecule, but this event does not occur in the absence of the bispecific antigen-binding polypeptide. In certain embodiments, the cell surface protein is present on a cell that (a) is capable of lysosomal degradation of extracellular molecules, and (b) does not undergo lysosomal degradation of the extracellular molecule when the extracellular molecule and cell surface protein are not bound to the bispecific antigen-binding polypeptide.

[0057] In some embodiments, the cells are non-immunological cells, i.e., the cells are not involved in an immune response. In some embodiments, the cells are fibroblasts, epithelial cells, endothelial cells, blood cells, or platelets. This can be beneficial when the cells are particularly abundant in the body. 2 Endothelial cells, which are present on the surface of the body's blood vessels, provide a significant capacity for degradation of unwanted factors that appear in the circulation. Therefore, in certain embodiments, the cell surface protein is present on endothelial cells. Alternatively, it may be advantageous to target cells in a specific location. For many unwanted extracellular molecules, cells that are appropriately localized at the site of toxicity can be utilized. Furthermore, local administration of bispecific antigen-binding polypeptides is possible. For example, epithelial cells lining the vaginal and anal walls can be targeted using microbicides containing the bispecific antigen-binding polypeptides of the present invention. This approach can be used, for example, to capture HIV and reduce post-sexual transmission. Therefore, in certain embodiments, the cell surface protein is present on epithelial cells.

[0058] Forced internalization of unwanted extracellular molecules, such as viruses, into cells can have the detrimental effect of allowing some infection to occur via this route. However, it is worth noting that not all cell types provide the gene products required to support viral growth. Therefore, the present invention can be tailored depending on the pathogen to be removed. For example, some cell types, such as platelets, do not contain genetic material. This has the advantage that internalization of infectious agents, such as viruses, by platelets cannot result in unwanted replication of these materials. Thus, the present invention allows for targeting of cells that are completely unable to support viral growth. Thus, in some embodiments, the cell surface protein is present on platelets. In other embodiments, where the extracellular molecule is a viral antigen, the cell is not the natural target of the viral antigen. In other embodiments, where the extracellular molecule is a viral antigen, the cell is unable to support viral replication and / or growth.

[0059] Degradation of extracellular molecules preferably occurs via the lysosomal pathway. Thus, in a preferred embodiment, extracellular molecules are degraded via the lysosomal pathway. Lysosomes are ubiquitous organelles that can degrade proteins, nucleic acids, polysaccharides, and other biological substances. Extracellular substances and cytoplasmic membrane proteins (e.g., receptors or channels) enter cells through endocytosis, which is trafficking through endosomal compartments, and can undergo various sorting steps, after which they are either recycled to the plasma membrane or delivered to lysosomes for degradation. In one embodiment of the present invention, cell surface proteins are recycled to the plasma membrane. In another embodiment of the present invention, cell surface proteins are degraded together with soluble factors.

[0060] E. Targeting HIV Degradation As described in the Examples below, the inventors have shown that bispecific antigen-binding polypeptides of the invention can be used for the targeted elimination and degradation of HIV. In this study, the inventors obtained proof-of-principle that the invention is suitable for targeting HIV to EGFR-expressing fibroblasts and EGFR-expressing epithelial-derived (cancer) cells for degradation.

[0061] Thus, in one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to HIV; and b) a second antigen-binding domain that binds to a cell surface protein Contains:

[0062] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to HIV; and b) a second antigen-binding domain that binds to EGFR Contains:

[0063] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to HIV; and b) a second antigen-binding domain that binds to a cell surface protein on a non-immunological cell, a fibroblast, an epithelial cell, an endothelial cell, or a platelet. Contains:

[0064] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to HIV; and b) a second antigen-binding domain that binds to EGFR on a non-immunological cell, a fibroblast, an epithelial cell, an endothelial cell, or a platelet; Contains:

[0065] In each of the above embodiments, the extracellular molecule is preferably internalized and degraded when both the extracellular molecule and the cell surface protein bind to the bispecific antigen-binding polypeptide.

[0066] In each of the above embodiments, the extracellular molecule is preferably degraded via the lysosomal pathway.

[0067] The present invention also provides a method of targeting HIV for cellular internalization and degradation, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same.

[0068] The present invention further provides a method for treating or preventing HIV, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same.

[0069] The present invention further provides bispecific antigen-binding polypeptides for use in a method of treating or preventing HIV, the method comprising administering the bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same to a subject in need thereof.

[0070] The present invention further provides the use of bispecific antigen-binding polypeptides to target HIV for cellular internalization and degradation.

[0071] In each of the above methods or uses, the bispecific antigen-binding polypeptide may be any of the bispecific antigen-binding polypeptides disclosed herein that bind to HIV. In particularly preferred embodiments of the methods, the bispecific antigen-binding polypeptide comprises a) a first antigen-binding domain that binds to HIV; and b) a second antigen-binding domain that binds to EGFR.

[0072] In each of the above embodiments directed to targeting HIV for degradation, those skilled in the art will recognize that the first antigen-binding domain can be engineered to bind to any epitope present on the surface of HIV. For example, in certain embodiments, the first antigen-binding domain can bind to gp120, gp41, and / or gp140 on the surface of HIV. Similarly, those skilled in the art will recognize that the second antigen-binding domain can be engineered to bind to other cell surface proteins other than EGFR with the same result. For example, in other embodiments, the second antigen-binding domain can bind to low-density lipoprotein receptor (LDLR), transferrin receptor (TfR), hepatocyte growth factor receptor (cMet), MHC class II, vascular endothelial growth factor receptor (VEGFR) or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4-1-BB, or ICOS.

[0073] Furthermore, it should be understood that the orientation of the two antigen-binding domains is not fixed. The above embodiments can be modified so that a designated first antigen-binding domain binds to a cell surface protein such as EGFR, and a designated second antigen-binding domain binds to an extracellular molecule such as HIV.

[0074] In one preferred embodiment for targeting HIV for degradation, the bispecific antigen-binding polypeptide is a bispecific VHH antibody.

[0075] As used herein, a method of "preventing" a disease or condition means preventing the onset of the disease, preventing the worsening of symptoms, preventing the progression of the disease or condition, or reducing a subject's risk of developing the disease or condition. As used herein, a method of "treating" a disease or condition means curing the disease or condition and / or alleviating or eliminating symptoms associated with the disease or condition so that the patient's suffering is reduced. In the case of HIV, a method of preventing HIV can mean preventing or reducing HIV infection, preventing or reducing HIV entry into target cells, preventing or reducing HIV replication, preventing the onset of AIDS, preventing the worsening of AIDS-related symptoms, preventing the progression of AIDS, or reducing a subject's risk of developing AIDS. A method of treating HIV can mean alleviating or eliminating HIV infection or alleviating or eliminating symptoms associated with AIDS.

[0076] F. Targeting Sars-Cov-2 Degradation Another related target is Sars-Cov2. Neutralizing antibodies have been generated in an attempt to block Sars-Cov2 infection. The present invention, utilizing a targeted degradation approach, has the advantage that blocking all envelope spike proteins is not necessary, since binding of only a small amount of the envelope spike protein should be sufficient to rapidly remove and degrade the virus. This requires a lower concentration of antibody than neutralizing all spike proteins. This allows the use of a mixture of different antibodies, each targeting a different epitope, which can enhance efficacy and reduce the chance of mutational escape.

[0077] As described in the Examples below, the inventors have demonstrated that the bispecific antigen-binding polypeptides of the present invention can be used for targeted elimination and degradation of Sars-Cov2. Therefore, the present invention also provides a method for treating or preventing Sars-Cov2. In this scenario, a bispecific antigen-binding polypeptide can be artificially engineered in which an anti-Sars-Cov2 VHH is fused to an anti-EGFR VHH so that the Sars-Cov2 virus is internalized and degraded in EGFR-expressing fibroblasts and epithelial cells. It should be noted that the damaging effects of COVID-19 are largely due to the body's secondary immunological response. Antibody binding to the virus triggers a cascade of immunological reactions that have deleterious effects in a certain percentage of cases. On the other hand, the present invention provides rapid clearance and degradation of the virus and antibody without the need for further immunological activation (due to the Fc tail present on natural antibodies), thereby potentially circumventing these problems. Furthermore, bispecific VHH single-domain antibodies can be delivered intravenously and (due to their small size) by aerosol delivery. Delivery of inhaled therapeutics to the respiratory system results in lower dosage requirements and direct engagement of the virus at the site of initial infection. Bispecific VHH-mediated direct clearance of virus by epithelial cells is likely to be much more efficient than immunological clearance of virus that is merely coated with neutralizing VHHs.

[0078] Thus, in one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to Sars-Cov2; and b) a second antigen-binding domain that binds to a cell surface protein Contains:

[0079] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to Sars-Cov2; and b) a second antigen-binding domain that binds to EGFR Contains:

[0080] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to Sars-Cov2; and b) a second antigen-binding domain that binds to a cell surface protein on a non-immunological cell, a fibroblast, an epithelial cell, an endothelial cell, or a platelet. Contains:

[0081] In one embodiment, the bispecific antigen-binding polypeptide comprises: a) a first antigen-binding domain that binds to Sars-Cov2; and b) a second antigen-binding domain that binds to EGFR on a non-immunological cell, a fibroblast, an epithelial cell, an endothelial cell, or a platelet; Contains:

[0082] In each of the above embodiments, the extracellular molecule is preferably internalized and degraded when both the extracellular molecule and the cell surface protein are bound to the bispecific antigen-binding polypeptide.

[0083] In each of the above embodiments, the extracellular molecule is preferably degraded via the lysosomal pathway.

[0084] The present invention also provides a method for targeting Sars-Cov2 for cellular internalization and degradation, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same.

[0085] The present invention further provides a method for treating or preventing Sars-Cov2, comprising administering to a subject in need thereof the bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same.

[0086] The present invention further provides a bispecific antigen-binding polypeptide for use in a method of treating or preventing Sars-Cov2, the method comprising administering the bispecific antigen-binding polypeptide or a pharmaceutical composition comprising the same to a subject in need thereof.

[0087] The present invention further provides the use of bispecific antigen-binding polypeptides to target Sars-Cov2 for cellular internalization and degradation.

[0088] In each of the above methods or uses, the bispecific antigen-binding polypeptide may be any of the bispecific antigen-binding polypeptides disclosed herein that bind to Sars-Cov2. In particularly preferred embodiments of the methods, the bispecific antigen-binding polypeptide comprises a) a first antigen-binding domain that binds to Sars-Cov2; and b) a second antigen-binding domain that binds to EGFR.

[0089] In each of the above embodiments relating to targeting Sars-Cov2 for degradation, those skilled in the art will recognize that the first antigen-binding domain can be engineered to bind to any epitope present on the surface of Sars-Cov2. For example, in some embodiments, the first antigen-binding domain can bind to the spike protein on the surface of Sars-Cov2. Similarly, those skilled in the art will recognize that the second antigen-binding domain can be engineered to bind to other cell surface proteins other than EGFR with the same results. For example, in other embodiments, the second antigen-binding domain can bind to low-density lipoprotein receptor (LDLR), transferrin receptor (TfR), hepatocyte growth factor receptor (cMet), MHC class II, vascular endothelial growth factor receptor (VEGFR) or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4-1-BB, or ICOS.

[0090] Furthermore, it should be understood that the orientation of the two antigen-binding domains is not fixed. The above embodiments can be modified so that a designated first antigen-binding domain binds to a cell surface protein such as EGFR, and a designated second antigen-binding domain binds to an extracellular molecule such as Sars-Cov2.

[0091] In one preferred embodiment of targeting Sars-Cov2 for degradation, the bispecific antigen-binding polypeptide is a bispecific VHH antibody.

[0092] In the case of Sars-Cov2, a method of preventing Sars-Cov2 can mean preventing or reducing Sars-Cov2 infection, preventing or reducing Sars-Cov2 entry into target cells, preventing or reducing Sars-Cov2 replication, preventing the onset of COVID-19, preventing the worsening of COVID-19 associated symptoms, preventing the progression of COVID-19, reducing a subject's risk of developing COVID-19, preventing the onset or progression of post-COVID syndrome (long covid), preventing the worsening of post-COVID syndrome associated symptoms, or reducing a subject's risk of developing post-COVID syndrome. A method of treating Sars-Cov2 can mean alleviating or eradicating Sars-Cov2 infection, or alleviating or eradicating symptoms associated with COVID-19.

[0093] G. Other Therapeutic Uses Although the present invention has been demonstrated for the targeted elimination of HIV and SARS-CoV-2, it is clear that the concept can be applied to the elimination of a wide range of extracellular molecules and therefore to treat or prevent a variety of diseases and disorders.

[0094] Thus, the present invention provides a method of targeting an extracellular molecule for cellular internalization and degradation, comprising administering to a subject in need thereof a bispecific antigen-binding polypeptide or a pharmaceutical composition comprising same.

[0095] The present invention further provides the use of bispecific antigen-binding polypeptides to target extracellular molecules for cellular internalization and degradation.

[0096] In one embodiment the method or use comprises administering a combination of bispecific antigen-binding polypeptides, wherein the combination of bispecific antigen-binding polypeptides binds to multiple extracellular molecules and / or multiple epitopes on extracellular molecules.

[0097] In certain embodiments, the bispecific antigen-binding polypeptide is administered orally, sublingually, topically, intravenously, subcutaneously, intranasally, intravaginally, intrarectally, or by inhalation.

[0098] In certain embodiments, degradation is via the lysosomal pathway.

[0099] The present invention further provides a method of treating or preventing an inflammatory pathology, optionally wherein the inflammatory pathology is selected from inflammatory bowel disease; psoriasis; rheumatic inflammatory pathologies such as rheumatoid arthritis, ankylosing spondylitis, etc.; multiple sclerosis; autoimmune pathologies such as systemic lupus erythematosus, neuromyelitis optica, etc.; asthma; and allergies.

[0100] Also provided is a bispecific antigen-binding polypeptide for use in a method for treating or preventing an inflammatory pathology, the method comprising administering the bispecific antigen-binding polypeptide or a pharmaceutical composition comprising the same to a subject in need thereof. In certain embodiments, the inflammatory pathology is selected from inflammatory bowel disease; psoriasis; rheumatic inflammatory pathologies such as rheumatoid arthritis and ankylosing spondylitis; multiple sclerosis; autoimmune pathologies such as systemic lupus erythematosus and neuromyelitis optica; asthma; and allergies.

[0101] As disclosed elsewhere herein, the extracellular molecule may be a viral antigen, and thus the present invention contemplates methods of treating or preventing diseases and disorders characterized by viral infection.

[0102] The present inventors have demonstrated that hepatitis B virus-like particles can be efficiently targeted to EGFR on cells in vitro. Upon targeting to cells, the virus-like particles are internalized and degraded in lysosomes. This demonstrates the versatility of the present invention and demonstrates that various viruses can be addressed in this manner. Thus, the present invention also provides a method for treating or preventing hepatitis B.

[0103] Influenza viruses cause annual epidemics and occasional pandemics of respiratory tract infections in humans, resulting in a wide spectrum of clinical disease severity. With regard to SARS-CoV-2, both viral and host factors determine the degree and severity of virus-induced lung injury. The host response to viral infection, while necessary for viral clearance, can be detrimental and contribute to severe disease phenotypes. Influenza possesses hemagglutinin (HA) and neuraminidase (NA) spike proteins. HA plays a key role in binding to sialic acid within the cell membrane, leading to internalization. NA activity is then required for proteolytic processing of the HA spike, resulting in virus release into the cytoplasm of target cells. The present invention contemplates internalization via different pathways, followed by lysosomal degradation. Accordingly, the present invention also provides methods for treating or preventing influenza.

[0104] As disclosed elsewhere herein, the extracellular molecule may be a toxin, and thus the present invention contemplates methods of treating or preventing diseases and disorders characterized by toxicity.

[0105] Circulating bacterial toxins are also suitable for treatment with the present invention. Staphylococcus aureus is a major human pathogen that produces numerous toxins. Among the secreted toxins, staphylococcal superantigens (SAgs) play a key role in host debilitation. They exert superantigenic activity, resulting in the activation of large populations of T cells and the release of large amounts of proinflammatory cytokines. This overstimulation of the immune system can ultimately lead to a systemic, life-threatening response known as toxic shock syndrome (TSS). Besides the administration of antibiotics, current therapies are primarily supportive, using fluid resuscitation and vasopressors. Current therapies are not sufficiently effective, so new treatments are needed. Because the toxic shock syndrome toxin TSST-1 is considered the primary etiologic factor in TSS, specific removal of TSST-1 from the blood may be beneficial for patients suffering from TSS. Indeed, there is some evidence supporting the possibility that plasmapheresis in severe cases of sepsis can improve the course of this syndrome. The present inventors have conducted in vitro studies showing that TSST-1 induces upregulation of MHC class II molecules on endothelial cells. TSST-1 itself binds to these MHC class II molecules and is rapidly endocytosed and degraded (unpublished data). The same results are likely to be achieved more quickly and completely by applying bispecific antibodies. Using bispecific antigen-binding polypeptides that target TSST-1 to EGFR or a suitable receptor on endothelial cells, the toxin is targeted for rapid degradation, which is thought to be fast enough to prevent the toxic effects of TSST-1 in the body. The present inventors have generated VHH antibodies against TSST-1 that can be combined with anti-EGFR antibodies in bispecific antibodies to remove TSST-1. Thus, the present invention also provides methods for treating or preventing toxic shock syndrome.

[0106] Since their initial development and use by Calmette in the late 1800s, conventional antivenoms have remained the only specific treatment for envenomation. Most antivenoms consist of either whole IgG (150 kDa), F(ab')2 antibody fragments (100 kDa), or occasionally Fab antibody fragments (50 kDa) derived from horses or sheep immunized with a single venom (monospecific) or a combination of venoms (polyspecific). While intravenous administration of antivenom is usually effective in treating systemic envenomation, conventional antivenoms are usually ineffective in treating localized effects on tissues near the snakebite due to the rapid development of localized lesions and the inability of antivenom antibodies to penetrate affected tissues, often resulting in permanent disability. Furthermore, conventional antivenoms often induce life-threatening adverse reactions in patients, such as anaphylaxis or serum sickness. Recently, construction of a VHH library from a llama immunized with crude venom of the N. kaouthia (monoclus cobra) led to the isolation of high affinity VHHs that conferred complete protection (100% mouse survival) against α-Cbtx (α-cobra venom). Bispecific VHHs can also more rapidly clear and degrade the venom. Thus, the present invention also provides methods for treating or preventing envenomation.

[0107] As disclosed elsewhere herein, the extracellular molecule may be a cytokine, and thus the present invention contemplates methods of treating or preventing diseases and disorders characterized by excessive cytokine production.

[0108] Excessive induction of proinflammatory cytokines, also known as cytokine storm or cytokine release syndrome (CRS), is one of the key aspects of the ongoing SARS-CoV-2 pandemic. This has also been described for other major human coronaviruses and influenza A subtypes (H5N1, SARS-CoV, MERS-CoV, and H7N9). Recent studies of various viruses have highlighted SARS-CoV-2-specific dysregulation of the type I interferon (IFN) response and its downstream cytokine signature. Therefore, targeted degradation of type I IFN may have a beneficial effect in preventing CRS in COVID-19 disease pathogenesis. Bispecific VHHs that remove type I IFN can be administered at appropriate doses. Many other diseases result in cytokine imbalance. This balance can be restored by administering a combination of bispecific VHHs against abundantly expressed cytokines. Because the bispecific VHHs themselves are relatively rapidly cleared from the body through the kidneys, this allows for quantitative regulation of cytokine removal. Therefore, the present invention also provides methods for treating or preventing cytokine release syndrome.

[0109] H. Pharmaceutical Compositions and Routes of Administration The present invention includes pharmaceutical compositions containing one or a combination of the bispecific antigen-binding polypeptides described herein, which may be formulated with one or more pharmaceutically acceptable carriers or excipients. Techniques for formulating antibodies for therapeutic use in humans are well known in the art and are reviewed, for example, in Wang et al., Journal of Pharmaceutical Sciences, Vol. 96, pp. 1-26, 2007, the contents of which are incorporated herein in their entirety.

[0110] In certain embodiments, the composition comprises a combination of bispecific antigen-binding polypeptides that bind to multiple extracellular molecules and / or multiple epitopes on extracellular molecules.

[0111] Pharmaceutical compositions according to the invention can be administered alone or in combination with other treatments, either simultaneously or sequentially. For example, in certain embodiments where the pharmaceutical composition is for use in the treatment or prevention of HIV, the composition can be administered in combination with antiretroviral therapy, broadly neutralizing antibodies, or other suitable treatments.

[0112] Pharmaceutically acceptable excipients that can be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0113] Bispecific antigen-binding polypeptides are typically formulated as pharmaceutical compositions and administered to a subject in a "therapeutically effective amount." As used herein, the term "therapeutically effective amount" is intended to mean an amount or dose of a bispecific antigen-binding polypeptide that is sufficient to produce a therapeutic effect, e.g., an amount or dose required to affect the internalization and degradation of unwanted extracellular molecules and / or to eradicate or at least alleviate symptoms associated with a disease or disorder. An appropriate amount or dose can be determined by a physician, as appropriate. For example, the dose can be adjusted based on factors such as the size or weight of the subject being treated, the age of the subject being treated, the general physical condition of the subject being treated, the disease being treated, and the route of administration.

[0114] The experimental results described herein demonstrate that even at low concentrations of bispecific antigen-binding polypeptides, a sufficiently high proportion of HIV-1 particles are coated with bispecific VHHs and therefore recruited to cells displaying EGFR on their surface. Binding to a single HIV-1 envelope protein is sufficient to target the virus to cells and degrade it. This contrasts with current approaches that attempt to obtain antibodies that block virus attachment to target cells. To be effective, these current approaches require antibodies to occupy all 14 spike surface proteins. Thus, the present invention requires fewer antibodies than traditional blocking methods. As a result, the present invention readily enables the use of combinations of, for example, five or more different bispecific antigen-binding polypeptides, each with its own unique clade specificity. This dramatically limits the virus's evasion potential.

[0115] For clinical use, in certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject in one or more doses of about 0.05 mg / kg body weight to about 20 mg / kg body weight. In certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject at a dose of about 0.05 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject at a dose of about 0.1 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject at a dose of about 0.5 mg / kg body weight to about 10 mg / kg body weight. In certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject at a dose of about 0.05 mg / kg body weight to about 5 mg / kg body weight. In certain embodiments, a bispecific antigen-binding polypeptide described elsewhere herein is administered to a subject at a dose of about 0.05 mg / kg body weight to about 2 mg / kg body weight.

[0116] In certain embodiments, the composition is formulated for administration to a subject via any suitable route of administration, including, but not limited to, oral, sublingual, topical, intravenous, intramuscular, intradermal, transdermal, intraperitoneal, subcutaneous, intranasal, intravaginal, rectal, or by inhalation.

[0117] Given their small size and robust thermostability, the bispecific VHH antibodies of the present invention can be aerosolized for the direct treatment of respiratory diseases.

[0118] HIV-1 can be transmitted during sexual intercourse. In addition to the use of condoms to reduce transmission, microbicides that neutralize the virus have been developed. Therefore, microbicides containing the bispecific antigen-binding polypeptides described herein can be administered directly to the vagina or anus as an ointment, which can locally release the bispecific antigen-binding molecules. Furthermore, localized production by Lactobacilli can be utilized: for certain microbicides, Lactobacilli can be used to steadily produce the microbicides. Certain species of Lactobacillus are vaginal commensals, and these species have been modified to produce specific microbicides. Thus, using lactobacilli, which are natural commensals in the healthy vaginal microbiota, bispecific antigen-binding polypeptides against HIV-1 can be expressed in soluble or cell wall-anchored form, for example, in Lactobacillus rhamnosus DSM14870 (Pant, Neha et al., 2006; The Journal of Infectious Diseases; vol. 194;11:1580-8. doi:10.1086 / 508747).

[0119] The invention will now be further understood with reference to the following non-limiting examples. [Example]

[0120] (Example) Example 1: Induction of HIV-1 Degradation by Non-Target Cells Using Bispecific Single-Chain Llama Antibodies (Rationale) Described herein is a highly efficient method for removing HIV from the circulation and targeting it for degradation using bispecific antibodies. These antibodies bind to HIV with one head and target the virus with the other head to cells where the virus cannot replicate but is instead degraded. The concept is that HIV is captured and destroyed by unspecialized, non-immune cells in the body. Removal of the virus from the circulation, alone or in combination with a cocktail of antiretroviral drugs, can completely control and limit HIV. To realize this concept, we created a series of bihead constructs in which four different anti-HIV antibodies are coupled to two epidermal growth factor receptor (EGFR) antibodies that bind with high affinity to EGFR. We used small, single-chain llama antibody fragments (approximately 15 kD in size), known as VHHs or nanobodies. These bispecific bihead antibodies were generated by cloning. The anti-HIV VHHs were selected based on their broad binding ability to different clades of HIV, including clades A, B, and C. EGFR is widely expressed in the body on cells that do not express the HIV receptor. Because the HIV receptor CD4 and co-receptors CCR5 and CXCR4 are required for productive HIV replication, retargeting HIV to these non-immunological target cells does not result in viral replication. We have shown in vitro that bispecific VHHs can bind to HIV-1 envelope glycoproteins and deliver them to EGFR-expressing cells, where viral antigen-antibody complexes are internalized and degraded by the lysosomal degradation pathway. This approach can be used to actively clear the virus from the body of infected patients.

[0121] (Construction and functionality of bispecific biheaded VHHs) Previously, anti-HIV-1 VHHs (small llama antibody fragments) were generated, yielding a substantial number of VHHs with broad binding capabilities, including binders with blocking / neutralizing activity. In this study, we investigated a novel approach by constructing bispecific VHH antibodies capable of direct HIV-1 degradation by non-target cells. We utilized previously generated VHHs that bind to the extracellular domain of EGFR, including VHHs that are internalized after binding to EGFR. We combined four different anti-HIV-1 VHHs (1F10, H3, 2H10, and 2E7), designated here as H2, H3, H4, and H5, with two different anti-EGFR VHHs (EgA1 and EgB4), designated as E1 and E2. The anti-HIV-1 VHHs were selected based on their broad binding capabilities to different clades of HIV-1, including clades A, B, and C, specific for different envelope glycoprotein epitopes. The E1 and E2 VHHs bind to two distinct epitopes on the EGFR extracellular domain with high affinity. Table 1 lists the bispecific VHH domains used and summarizes their characteristics. The bispecific VHHs (biheads) were generated by PCR cloning and contain a flexible linker sequence of 10 amino acids [(G4S)2]. Table 1: VHH llama antibodies combined with a [G4S]2 sequence between VHHs in bispecific VHHs. H is anti-HIV-1, E is anti-EGFR, and Hep is anti-Hepatitis B VHH. Bispecific VHHs were generated in various combinations. For example, H2E2 represents a bispecific VHH antibody in which the N-terminal VHH 1F10 is linked to the C-terminal EGb4. E1 (EgA1) is a competitive anti-EGFR VHH that blocks EGF binding, while E2 (EgB4) is a non-competitive antibody that binds to domain I of EGFR. Anti-HIV antibodies H2 and H3 were generated against the gp120 envelope protein, H4 against gp4140, and H5 against the gp140 protein. Hep1 binds to the hepatitis B virus (HBV) surface antigen (HBV). This HB is expressed in HBV virus-like particles (ADW and ADY). The bispecific VHHs generated in this study were H2E2, E2H2, H2E1, H3E1, H4E2, E2H4, H5E2, Hep1E1, and the control biheads E2E1, H2H2, and H5H4. [Table 1]

[0122] After fusion to the bihead, both anti-HIV-1 and anti-EGFR VHHs retained excellent binding properties regardless of the orientation of the VHH antibody moiety within these molecules. This was demonstrated by ELISA analysis showing binding to purified EGFR extracellular domain proteins and purified HIV-1 envelope proteins (clade A, 92UG037 and clade C, gp140ZM96) (Fig. 1a). For these monospecific biheads, no binding to other targets was observed, so the control proteins E2E1 and H2H2 demonstrated the specificity of the VHHs. Next, we investigated whether the bispecific VHHs could recruit HIV-1 proteins to immobilized EGFR extracellular domains. Instead of using a multistep ELISA method, we covalently labeled the HIV-1 proteins with the near-infrared dye IR800CW, which allowed us to obtain quantitative binding results using IR800 FLISA (fluorescence-linked immunosorbent assay). Bound IR800-labeled HIV-1 proteins were detected directly in the wells using a scanner. Clearly, unlike the H2H4 and E2E1 controls, the bispecific VHHs mediated recruitment of IR800-labeled envelope proteins to coated EGFR (Fig. 1b). We next expanded our panel of bispecific VHHs to include VHHs that bind gp41. Functional envelope glycoproteins on the viral surface consist of a trimer of covalently linked gp41 and gp120 proteins, of which gp41 possesses a transmembrane domain. In this study, we used a trimeric gp140 molecule containing the extracellular domain of gp41, stabilized by the addition of a heterologous trimerization motif to the C-terminus of the gp41 sequence, in addition to gp120. H2 and H3 bind only the gp120 epitope and therefore do not recognize the gp41 trimer. H4 (α-gp41) binds to the gp41 portion of gp140, and H5 binds to gp140 37 binds to (Figure 1c).

[0123] The recombinant glycoproteins gp41 and gp140 exhibit their native conformation and form trimers. However, because actual HIV-1 viral particles are much larger and have complex membrane structures, we generated noninfectious HIV-1 virus-like particles (VLPs) that are the size of virions and express the envelope protein of the ZM96 strain (clade C) in their membranes. Furthermore, to demonstrate that directed VHH-mediated recruitment is not limited to HIV-1 but may be more generally applicable, we also tested hepatitis B virus-like particles using Hep, a VHH specific for this virus type. HIV and hepatitis B VLPs were labeled with IR800 dye. They were also shown to specifically target EGFR depending on the bispecific VHH used (Figure 1c). The exceptions are H4E2 and E2H4, which target gp41 to EGFR but not to HIV-1 VLP, presumably because they are too short to be linked or due to other steric hindrances. Under the labeling conditions used, there was never any indication that the label interfered with binding. In conclusion, we have generated bispecific VHHs that can specifically target viral envelope glycoproteins and HIV-1 and HBV virus-like particles to EGFR.

[0124] Note that at moderate to low antibody concentrations (nM), VLPs (pM) bind well to EGFR. Trimers contain three binding sites, which contribute to higher avidity due to the cooperative action of multiple binding. This has been demonstrated for gp140 trimers in quantitative FLISA experiments. Due to the higher number of viral membrane proteins per particle, VLPs contain many more binding sites, resulting in higher avidity. As a result, in competition experiments, pre-incubated VLPs containing many binding groups on the particle surface were only inhibited two-fold when a 20-fold higher concentration of competing free antibody was added. This illustrates the increased avidity when multiple epitopes per particle are involved.

[0125] To test infectious HIV-1, two common HIV-1 reference strains, HxB2 (clade B) and BaL (clade B), and two patient-derived HIV-1 strains, 92UG037 (clade A) and 96ZM651 (clade C), were cultured and tested for bispecific VHH-mediated targeting of the EGFR extracellular domain (Fig. 1d). The bispecific VHH was shown to be effective by its binding efficiency for different clades; a concentration of 3 nM was sufficient to bind to the envelope protein and target all four infectious HIV-1 strains to immobilized EGFR. Because 3 nM of the bispecific VHH already provided a molar excess over the HIV-1 envelope protein, increasing the concentration to 18 nM did not significantly increase the amount of HIV-1 bound to EGFR. A 10-fold increase in added HIV-1 targeted, on average, 8-fold more HIV-1 to EGFR (Fig. 1d), demonstrating the efficacy of the bispecific VHH. In conclusion, bispecific VHHs can efficiently recruit HIV-1 reference strains and clinical isolates to the EGFR extracellular domain in vitro.

[0126] (Targeted internalization and degradation) Next, we aimed to investigate the binding of HIV-1 antigens to EGFR-expressing cells mediated by bispecific VHHs, their internalization, and the intracellular fate of internalized viral proteins. The viral antigen gp140 and VLPs were labeled with IR800 and preincubated with bispecific VHHs for 30 minutes. The resulting preformed antigen-antibody complexes were added to Her14 mouse fibroblasts, which express the EGFR receptor, and NIH3T3 2.2 cells, which lack EGFR expression. As a positive control for the experiment, we employed IR800-labeled EGF. After binding for the indicated time points (Fig. 2a), cells were washed and gp140-IR800 was determined (Panel B, binding), or cells were stripped with acid and internalized HIV envelope proteins were determined (Panel I, internalization). As shown in Figure 2a, we found that the antigen-antibody complex specifically bound to EGFR in Her14 mouse fibroblast cells, but not to the negative control cells, NIH3T3 2.2. Binding and internalization of HIV proteins continuously increased over 18 hours. As expected, the positive control EGF-IR800 showed rapid internalization compared to the internalization of HIV-1 envelope proteins (compare the difference between internalization and binding). This is due to the fact that EGF-IR800 activates the EGFR receptor, resulting in rapid internalization. In contrast, our bispecific VHH antibody does not activate EGFR. Nevertheless, we still observed continuous uptake of bound envelope proteins, increasing from 35% to 80% and 95% after 90 minutes, 6 hours, and 18 hours, respectively. We also found that binding of hepatitis B VLPs (VLPs) to HIV-1 envelope proteins (VLPs) was significantly increased. HBVWe also found that the VHH-mediated targeting of EGFR-expressing cells results in clear uptake of viral envelope proteins and VLPs in tissue culture. Gp140 and hepatitis B VLPs were covalently labeled with AlexaFluor488, and internalization was demonstrated by immunofluorescence microscopy (Fig. 2b). After 90 min of incubation, labeled proteins were observed within endocytosed vesicles for both antigens. After overnight incubation, all signals were present in endocytosed, mostly perinuclear vesicles. We therefore conclude that bispecific VHH-mediated targeting results in clear uptake of viral envelope proteins and VLPs by EGFR-expressing cells, even in the absence of EGFR activation.

[0127] Bispecific VHH-mediated uptake leads to lysosomal degradation Binding of the natural ligand EGF to its receptor EGFR activates a negative feedback loop, leading to internalization and degradation of the receptor-growth factor complex. We evaluated whether bispecific VHH-mediated uptake of virus leads to subsequent degradation of the complex components, even in the absence of EGF growth factor activation. Degradation was observed with covalently IR800-labeled EGF, gp140, and VLPs. HBVWe analyzed the IR800 dye using denatured proteins. Remarkably, we found that it remained inside cells even when the carrier protein to which it was conjugated was degraded. This allows for simple quantification of protein-linked and protein-free dye in cell lysates. Cells were incubated with IR800-labeled proteins for various periods of time. After IR800 quantification in FLISA wells, we solubilized the cells and analyzed the protein quantity and integrity by denaturing protein gel analysis using quantitative IR800 analysis. Figure 2c shows that when recruited HIV-1 envelope protein is internalized (Figure 2a), it becomes rapidly degraded (a decrease in the full-length gp140 signal). After 100 minutes of chase, most of the dye was still present inside (or on) the cells, but more than 40% of the (internalized) envelope protein was degraded, and some IR800-labeled degradation products were visible on the gel. Similar results were obtained with the other bispecific VHH. The degradation kinetics of recruited gp140 was compared with those of VLPs and the EGFR ligand EGF (Fig. 2d). As expected, EGF was degraded faster, and degradation of both HIV-1 proteins and VLPs was slower. However, degradation was nearly complete within a day. Western blot analysis measuring actin confirms that equal amounts of cell lysate were loaded in these experiments. Binding and degradation were similar when other EGFR-expressing cell types were tested, including HeLa, A431, and 14C cells.

[0128] Next, we investigated the observed bispecific VHH-mediated degradation of viral proteins in EGFR-expressing cells. Upon EGF binding to EGFR, the complex is internalized, sorted into early endosomes, late endosomes, and finally degraded in the lysosomal compartment. Therefore, mouse fibroblast Her14 cells were labeled with LysoTracker dye, which stains acidic compartments such as lysosomes. After internalization of the labeled proteins (fluorescently labeled EGF, gp140, and VLPs) mediated by bispecific VHHs, the colocalization of the proteins with LysoTracker was analyzed with fluorescently labeled EGF, gp140, and VLPs. Figure 2a shows that the majority of the bound EGF is internalized after 30 minutes of incubation, whereas Figure 3a shows that it resides in small endosomal vesicles without colocalization with lysosomes. After 30 min, colocalization gradually occurred, was complete by 3 h (data not shown), and persisted for at least 18 h (Fig. 3a, overnight panel [O / N]). After overnight incubation, the fluorescent (degradation) products of EGF, recruited HIV-1 envelope proteins, and VLPs showed similar lysosomal localization. This result suggests that targeting of EGFR-expressing cells mediated by bispecific anti-HIV-1 VHHs against viral antigens leads to internalization and degradation via the lysosomal degradation pathway.

[0129] To further confirm these observations, mouse fibroblast Her14 cells expressing the EGFR receptor and negative control NIH3T3 2.2 cells were incubated with the lysosomal inhibitor chloroquine. The optimal functional range of chloroquine concentrations inhibiting lysosomal degradation in these cells was established using EGF-IR800 at chloroquine concentrations of 0, 25, 100, and 200 μM (Figure 3b). Chloroquine inhibited EGF degradation in a dose-dependent manner, with the optimal concentration already reaching 100 μM. In cells not treated with chloroquine, internalized EGF was still intact after 15 minutes but was completely degraded after 60 minutes. Similarly, Figure 3c shows the results of IR800-labeled envelope protein (gp140) and IR800-labeled hepatitis virus-like particles (VLPs) preincubated with the corresponding bispecific antibodies and incubated overnight on cells. HBV ) without chloroquine (--), these are largely degraded, but 100 μM chloroquine prevents degradation, indicating that after uptake, these proteins are targeted for lysosomal degradation. Further analysis of the EGFR signaling pathway indicates that internalization does not, per se, require EGFR activation and does not involve activation of signaling pathways associated with oncogenic signaling.

[0130] Bispecific VHH-mediated lysosomal degradation of infectious HIV in EGFR-expressing cells. Next, we analyzed the infectious HIV-1 reference strain HxB2 for targeted degradation in cells. Like many viruses, HIV binds to heparin sulfate proteoglycans and therefore also binds to fibroblasts. In vivo, this "nonspecific binding" is thought to play a physiological role in infection by pre-concentrating virion particles on the cell surface as an intermediate step toward specific binding to CD4. To facilitate the determination of the specific effect of the bihead in our studies, we first investigated methods of blocking nonspecific binding using IR800-labeled HIV-VLPs. While none of these provided satisfactory results, bispecific VHHs containing anti-HIV and anti-EGFR moieties appeared to mediate binding that was 30% greater on average than control biheads containing two anti-HIV-1 moieties. Thus, for VLPs, bispecific VHHs provide slightly higher binding than background binding alone.

[0131] Infectious HIV-1 HxB2 viral particles were then incubated with the bispecific VHH for 1 hour to allow the formation of HIV-antibody complexes. These complexes were then added to wells containing either Her14 or 14C cells (both expressing EGFR) for 3 hours (loading). Unbound virus was then removed by washing, and the cells were allowed to rest for 21 hours (chase). Viral proteins were detected in lysates from treated cells using an HIV-1 p24 ELISA. The amount of HIV-1 present on or within cells after 3 hours of binding (loading) was reduced by 82–96% after 21 hours of chase, with both the bispecific VHH and negative control VHH antibodies, either due to release of bound virus during the chase or due to internalization and degradation (Figure 4a). While this was not a discernible difference, a parallel transinfectivity assay showed that binding by the bispecific VHH was four times more effective than the control at presenting HIV-1 to MT2 indicator cells (Figure 4b). Virus was allowed to bind for 3 hours, washed, and co-cultured with MT2 indicator cells for 7 days, after which HIV-1-mediated syncytia were scored. This was performed using subsequent dilutions of the treated virus; the maximum dilution at which syncytia were observed is shown (Figure 4b). The effectiveness of H5E2 was most clearly demonstrated with Her14 cells. 14C cells bound HIV just as efficiently (Figure 4a), but either presented the virus in a manner that was less functional for infection of the indicator cells, or the virus was rapidly internalized and no longer able to infect the indicator cells. After a 21-hour chase, infectious virus was no longer present on the surface of Her14 cells, suggesting that after initial loading / binding, the virus was internalized and degraded. To examine whether degradation occurred in lysosomes, cells were treated with chloroquine, which inhibits lysosomal degradation but does not affect internalization (Figure 4c). We show that HIV-1 binds when bound for 3 hours with or without chloroquine and washed, and that binding to H5E2 is approximately 20% greater than binding to the control bihead H5H4.Approximately 90% of the nonspecifically bound virus (H5H4-treated) was subsequently lost during the 21-hour chase, both in the presence and absence of chloroquine. When H5E2 was used in the presence of chloroquine, 70% of the virus remained intact within the cells. This means that despite the high level of nonspecific binding, 70% of the H5E2-treated virus was internalized and degraded in lysosomes, which could be inhibited by chloroquine. Despite nonspecific binding, H5E2 clearly targets the virus to the lysosomal degradation pathway. The graph in Figure 4d shows the percentage of HIV present at 24 hours as a percentage of the input bound at 3 hours. Thus, most of the bound HIV-1 in the presence of the bispecific VHH is mediated to lysosomal degradation, which is inhibited by chloroquine. Meanwhile, the nonspecifically bound HIV could be released or hypothetically follow a non-EGFR-dependent internalization and degradation pathway. In conclusion, bispecific VHHs mediate targeting of infectious HIV to the lysosomal degradation pathway, leading to efficient degradation.

[0132] (Conclusion) In conclusion, we have generated many different bispecific antibodies to target HIV proteins to EGFR. We tested HIV spike proteins, HIV virus-like particles, and infectious HIV particles, and showed that all efficiently targeted EGFR. We also investigated whether biheads could target HIV spike proteins to cells. Direct covalent labeling of HIV proteins and VLPs with near-infrared dyes enabled rapid quantitative assessment. Her14 mouse fibroblasts expressing human EGFR were compared with control NIH3T3 2.2 fibroblasts lacking EGFR expression. In addition to binding, the level of internalization was also determined by acid wash, which removes bound proteins on the outside of the cells. Already after 3 hours of incubation, most of the bound spike protein had been internalized into the cells, and by 18 hours, more than 95% appeared to be internalized. Most importantly, using a novel method, we show that at that time, internalized HIV proteins and VLPs are nearly completely degraded (>95%).

[0133] Materials and Methods (Construction and production of biheaded VHHs) Anti-HIV VHHs 1F10 (H2), 2E7 (H5), 2H10 (H4), and H3 (this study, sequence = [ka] ), and anti-EGFR VHHs EGa1 (E1) and EGb4 (E2) (Hofman). [ka] The N-terminal VHHs were combined in various combinations in a bihead using standard PCR methods with a linker encoding [ka] and a 3' primer encoding a portion of a 10 amino acid (AA) linker including a BamHI digestion site. [ka] The C-terminal VHH was cloned using the 5' primer containing the second part of the linker, which contains a BamHI digestion site. [ka] while the 3' primer [ka] The fragments contained a NotI digestion site. The PCR fragments were digested with PstI, BamHI, and NotI (Fermentas), agarose gel purified, and co-cloned into a phagemid vector for display on a filamentous bacteriophage digested with PstI and NotI. The resulting clones contained Myc- and His-tags. Expression was carried out in Escherichia coli (E. coli) TG1, DH10, or DH5α. Colonies were screened for inserts by colony PCR using M13 primers. Expression of the recombinant VHH proteins in E. coli and purification by immobilized metal ion affinity chromatography (IMAC) was carried out using His-tag-conjugated Talon-beads (Clontech). 34 The isolated products were checked for purity on a Coomassie blue-stained 15% SDS-polyacrylamide gel. All clones were confirmed by DNA sequencing.

[0134] Covalent labeling of proteins and VLPs with fluorescent dyes HIV envelope protein gp140 (UG37) subtype A was kindly provided. Recombinant HIV-1 envelope protein gp140CN54 subtype C was obtained from the Centre for AIDS Reagents, NIBSC HPA UK, supported by the EC FP6 / 7 Europrize Network of Excellence, the NGIN consortium, and the Bill and Melinda Gates GHRC-CAVD Project, and was a gift from Polymun, Immunodiagnostics, Immune Technology. gp41 (GCN) contains extracellular domains that are linked together to form trimers. HIV protein-based VLPs (VLPs) displaying envelope protein ZM96 gp145 subtype C were also prepared. HIV ) was produced in 293T cells after large-scale transient cotransfection with two plasmids encoding HXB2 Gag and ZM96 gp145, which contains the entire external gp120 portion, the extracellular domain of gp41, and its transmembrane domain. VLPs carrying HIV envelope proteins were sucrose gradient purified essentially as described. Hepatitis B virus-like particles (recombinant surface antigen ADW subtype HC87-2) were obtained from HyTest Ltd. For labeling, protein concentrations were ≥1 mg / ml. Where necessary, proteins were concentrated using Amicon® Ultra centrifugal filters or Microcon YM-3 spin columns (Millipore).

[0135] VHHs and other proteins were labeled with IRdye800 (IRdye 800CW NHS ester infrared dye from Licor, product 92970020) at a ratio of 20 μg:0.67 μg in PBS for 30 minutes with shaking at room temperature. For VLPs, this was 20 μg containing 2 μg of dye and 1 / 10 volume of 0.5 M NaHCO3 pH 9. The reaction was quenched with 10% 2-(methyl-amino)ethanol (1 M, pH 9.0, Sigma), and unbound dye was removed from the labeled proteins by size-exclusion separation on a homemade 1 ml G-25 Sephadex (GE Healthcare) column. Protein concentration and labeling efficiency were determined with a Nanodrop 1000 spectrophotometer (Thermo Scientific). For immunofluorescence, proteins were similarly labeled with Alexa 488-NHS (Invitrogen).

[0136] (ELISA) 96-well MaxiSorp plates were coated overnight with gp140 subtype A (=UG37) and subtype C (=CN54) or BSA control (250 ng / 50 μl PBS). After blocking with 200 μl of 4% w / v skim milk (Marvel, in PBS), VHHs were added for 1 hour (200 ng / 100 μl). Incubations were performed with 1% Marvel in PBS, and all washes were with PBS containing 0.05% Tween-20. Detection was performed with αMyc (Roche Diagnostics, 1 / 2000) or αHis (Amersham, 1 / 5000), peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, 1:5,000), and o-phenylenediamine (OPD). The secondary antibodies used were donkey anti-mouse and donkey anti-rabbit IgG.

[0137] (Bihead EGFR binding, FLISA and ELISA) Coating was performed with polyclonal rabbit anti-human IgG (DakoCytomation, 1 / 2000 in 50 μl PBS). After blocking with skim milk (4% w / v Marvel in PBS), incubation was performed with EGFR extracellular domain containing the Fc tail (EGFR-ect) (85 ng in 50 μl 2% Marvel) followed by incubation with the indicated concentrations of bihead in 1% Marvel. For FLISA, results were analyzed using an Odyssey infrared imaging system (Li-Cor Biosciences). For ELISA, α-Myc (1 / 2000), α-mouse-Ig-peroxidase (1 / 5000), and o-phenylenediamine (OPD) were used.

[0138] (Pre-incubated complex) IR800-labeled protein and VLP were preincubated with a 2-3-fold molar excess of bihead, and the preincubated complex was allowed to form for 30 min at room temperature (typically 50 ng of gp140-IR800 or VLP-IR800 + 37 ng of bihead VHH in 20 μl). For ELISA and FLISA, preincubations were performed in 2% Marvel, and for cell application, in 1% BSA (filtered). Binding of the preincubated complex in the assay was typically performed using 5 ng of labeled protein in 200 μl, i.e., 2 nM preincubated complex.

[0139] (Bihead-mediated HIV-1 binding) The HIV-1 strains used were grown and purified by standard procedures, and the resulting concentrations were determined by HIV-1 p24Ag ELISA (Aalto Bioreagents): HXB2, 940 ng / ml p24Ag; Bal, 109 ng / ml; 92UG037 (NIH Aids Reagent Program), 37 ng / ml; and 96ZM651 (NIH Aids Reagent Program), 24 ng / ml p24Ag. The required amount of virus was spun at 17,000 rpm for 1 hour at 4°C, and the viral pellet was resuspended in PBS + 2% BSA. 100 μl (typically 10 ng of viral p24) was incubated with 60 ng or 10 ng of bispecific bihead (18 nM and 3 nM bihead, respectively) for 1 hour at 37°C. Virus-bihead complexes were incubated in EGFR-coated wells for 2 h at 37°C, washed four times with PBS, and bound virus was dissolved in 100 μl of 0.1% empigen (Sigma) in TBS and transferred for analysis by p24 ELISA.

[0140] (cell culture) The mouse fibroblast cell line NIH 3T3 clone 2.2 (designated 3T3) lacks EGFR expression, and HER14 was derived from it as a stable transfectant expressing human EGFR. The tumor cell line UM-SCC-14C was kindly provided by GAMS van Dongen (Department of Otolaryngology, VU University Medical Center, Amsterdam, The Netherlands). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% fetal bovine serum (v / v), 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine (all Gibco, Invitrogen) at 37°C in a 5% CO2 humidified atmosphere. For incubations outside the incubator, the medium was replaced with CO2-independent medium (Gibco, Invitrogen).

[0141] (Internalization assay) Corning plate wells were coated with 0.25% gelatin (Merck, autoclaved) and washed with PBS. Her14 and 3T3 2.2 cells were plated at 4 × 10 in a 48-well plate. 4 Cells were seeded at 1000 kJ / well and grown overnight in DMEM medium at 37°C in a CO2-containing atmosphere. EGF-IR800 (LiCor) or pre-incubated complexes were added to the medium and incubated for 30, 90, 6, or 18 hours. Cells were washed twice with CO2-independent medium, chilled on ice, and detached once with strip buffer (250 mM NaCl, 100 mM glycine, pH 2.5) for 5 minutes. Cells were washed twice with CO2-independent medium, and IR800 was measured on an Odyssey.

[0142] (fluorescence microscopy) Coverslips (Menzel-Glazer) in 24-well plates were coated with 0.25% gelatin and washed with PBS. 2 × 10 cells per well were added. 5 Her14 or 3T3 2.2 cells were seeded onto glass coverslips and grown overnight. The cells were pre-incubated with Alexa488-labeled EGF (Life Technologies, 8 nM) or homemade Alexa488-labeled VLPs. HBV Cells were incubated with gp140UG37 (2 nM) and EGF-Alexa488 (200 ng) in PBS. After washing with PBS, cells were fixed with 4% paraformaldehyde (PFA) (Sigma) for 30 minutes at room temperature in the dark. Cells were then washed, treated with 20 ng / ml 4',6-diamidino-2-phenylindole (DAPI, Roche Diagnostics Corporation), and embedded in Mowiol (Sigma) containing PPD on microscope slides. For colocalization with Lysotracker, cells were incubated with EGF-Alexa488 for 10 minutes or with complexes of the bispecific VHH H2E1 with gp140-Alexa488 and Hep1E1 with VLPs. HBVCells were preincubated with α-Alexa488 complexes for 6 hours, after which they were washed three times with PBS and grown in fresh DMEM (8% FBS). After overnight growth, Lysotracker Red (Life Technologies, 75 nM) was added to the cells 90 minutes before washing and paraformaldehyde fixation. Images were acquired using a Zeiss Axiovert 200M confocal microscope (Carl Zeiss Microscopy GmbH, Germany) equipped with a 63x water-immersion objective (NA 1.2).

[0143] (Degradation assay) Wells were coated with 0.25% gelatin. Her14 and 3T3 2.2 cells were plated at 4 × 10 in 48-well plates. 4 Cells were seeded at 400 μl per well and grown overnight. The cells were cooled on ice, and the medium was collected as conditioned medium and replaced with ice-cold CO2-independent medium (300 μl per well) containing 0.5% BSA (Sigma) and 0.4% FBS. After one wash, EGF-IR800, biheaded VHH, or preincubated complexes (usually in 50 μl for rapid mixing) were added to the cells and incubated on ice in the dark for 2 hours. The cells were washed three times with CO2-independent medium, and the wells were scanned for IR800. From another plate containing the "0-minute chase" wells, the medium was aspirated and replaced with 20 μl of 2x Laemmli sample buffer, and the lysed cells were transferred to a PCR microtiter plate (stored on ice). For the other wells, the medium was replaced with conditioned medium. The cells were transferred to a 37°C and 5% CO2 environment for 0, 90, or 180 minutes, or overnight. At these time points, cells were washed once and scanned for IR800 using an Odyssey, then lysed in 16 μl of 2× Laemmli sample buffer and transferred to a PCR microtiter plate. The collected samples were boiled at 95°C for 4 minutes in a sealed PCR microtiter plate to prevent evaporation. The lysates were analyzed for EGF, VHH, and VLPs. ADWThe proteins were separated on 15% SDS-PAGE gels for gp140 and 10% for gp140. Samples were Western blotted onto PVDF membranes. Actin was detected using mouse anti-actin (MP Biomedicals, 1 / 10000) and donkey anti-mouse peroxidase (DAMPO, 1 / 5000) followed by ECL using standard procedures.

[0144] (chloroquine treatment) Coat a 48-well plate with 0.25% gelatin and incubate at 6 × 10 cells per well. 4 Her14 or 3T3 2.2 cells were seeded. Cells were incubated with the indicated concentrations of chloroquine for 60 minutes. 3T3 2.2 cells were incubated with 200 μM chloroquine. Next, cells were incubated with 13 nM EGF-IR800 for 15, 60, or 120 minutes. After washing, cells were scanned with an Odyssey. Then, the medium was removed, and cells were lysed with 2x Laemmli sample buffer, boiled, and analyzed on a 15% SDS page gel. IR800 was detected with an Odyssey. After 60 minutes of 100 μM chloroquine pretreatment, the pre-incubated complexes (gp140-IR800 complex with H2E1 and VLP, respectively) were analyzed. ADW The treatment with Hep1E1 (a complex of Hep1E1 and Hep1E1) was carried out overnight.

[0145] (HIV-1 binding assay to cells) For HIV-1 binding, cells were seeded as described above. HIV strains were preincubated with biheads as described above, but using 30 ng virus + 60 ng biheads / 200 μl conditioned medium. Where indicated, heparin (Sigma) was added to 40 μg / ml for the last 30 minutes of preincubation. The mixture was then added to the wells, the plates were centrifuged at 3000 rpm for 10 minutes, and incubated with shaking (to ensure proper mixing) for 3 or 24 hours. Cells were washed four times with PBS, and then lysed in 0.1% empigen in TBS for p24 ELISA. For transinfectivity assays after 3 or 24 h of incubation, cells were washed four times with PBS, and MT2 indicator cells (NIH Aids Reagent Program) were added at 40,000 cells / 200 μl / well in RPMI 1640 + L-glutamine (Lonza) supplemented with 10% fetal bovine serum (v / v) (Sigma) and 10 μg / ml gentamicin (Life Technologies). Cytopathic effect (CPE) was scored after 24 and 48 h. For pulse-chase experiments, cells were loaded with virus for 3 h, washed, and then conditioned medium was added for 24 h of chase (or, in some experiments, the same results were obtained in DMEM medium containing 4% FBS). Treatment of cells with 100 μM chloroquine (Sigma) began 2 h before virus addition, and the chloroquine was present throughout the 3-h loading period. Chloroquine did not need to be readded during the 24-hour chase to inhibit degradation (results not shown).

[0146] Example 2: Targeted Degradation of SARS-CoV-2 Spike Protein by a SARS-CoV-2-Specific Bispecific Single Chain Llama Antibody In addition to HIV and Hepatitis B protein target degradation, we also performed similar experiments on another target, SARS-CoV-2. VHHs C1 and C2 bind to the SARS-CoV-2 spike protein with <1 nM affinity and neutralize at 2.5 nM for C1 and 5.9 nM for C2, respectively. A third anti-EGFR VHH, 7D12, designated E3, was used to generate a bispecific antibody. SARS-CoV-2 spike protein was labeled with IRdye800. The labeled protein and bihead were preincubated and then incubated with EGFR-expressing Her14 cells. Binding was assessed in tissue culture wells where IR800 was quantified. At the indicated time points, cells in the wells were solubilized and analyzed by PAGE. All procedures were essentially as described above, including control experiments (not shown) to verify the reagents.

[0147] Figure 5a shows that the bispecific biheads C1-E3 and C2-E3 specifically bind and target spike protein to EGFR-expressing Her14 cells, whereas the E3-E3 control bihead does not. Background amounts of spike-IR800 protein bind in the absence of the bihead but produce a much lower signal. Figure 5b shows that spike protein targeted to EGFR-expressing cells by C1-E3 and C2-E3 is largely degraded after 4 hours, similar to the EGF-IR800 control protein.

[0148] These results demonstrate that the present invention can be easily applied to any extracellular target for which an antibody can be generated. Furthermore, it demonstrates the versatility of using antibodies that have different second antigen-binding domains and can therefore bind to different epitopes on cell surface proteins, i.e., EGFR (three out of three were successful). In the case of the present invention, a specific epitope is not required for functionality.

[0149] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable, as appropriate, with any and all other consistent embodiments, including those taken from other aspects of the invention (including alone).

[0150] Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. a) A primary antigen-binding domain that binds to extracellular molecules; and b) A second antigen-binding domain that binds to a cell surface protein, wherein the cell surface protein is the epidermal growth factor receptor (EGFR), A bispecific antigen-binding polypeptide comprising, Here, when both the extracellular molecule and EGFR are bound to the bispecific antigen-binding polypeptide, the extracellular molecule is internalized and degraded via the lysosomal pathway, and The bispecific antigen-binding polypeptide wherein the antigen-binding domain is selected from an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a VHH single domain, a single-chain variable fragment (scFv), a Fab fragment, an Fd fragment, and an Fv fragment.

2. The bispecific antigen-binding polypeptide according to claim 1, wherein the first antigen-binding domain is VHH, and the second antigen-binding domain is VHH, and optionally the two VHH domains are linked by a linker, and optionally the linker is a GGGGSGGGGS peptide linker sequence.

3. The bispecific antigen-binding polypeptide according to claim 1, wherein the extracellular molecule is a viral antigen, toxin, microbial pathogen, allergen, damaged or dysregulated protein contributing to a disease state, cytokine, growth factor, hormone, autoantibody, or other pathological or infectious factor.

4. The bispecific antigen-binding polypeptide according to claim 3, wherein the extracellular molecule is a viral antigen present on the surface of the virus, and optionally the virus is selected from HIV, hepatitis, SarsCoV2, influenza, herpes, Epstein-Barr virus, adenovirus, flavivirus, echovirus, rhinovirus, coxsackievirus, respiratory syncytial virus, pandemic mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arbovirus encephalitis virus.

5. The bispecific antigen-binding polypeptide according to any one of claims 1 to 4, wherein the extracellular molecule is HIV-1 or HIV envelope glycoprotein.

6. The bispecific antigen-binding polypeptide according to claim 5, wherein the first antigen-binding domain binds to HIV, and optionally, the first antigen-binding domain binds to gp120, gp41, and / or gp140 on the surface of HIV.

7. The bispecific antigen-binding polypeptide according to any one of claims 1 to 4, wherein the first antigen-binding domain binds to Sars-CoV2, or the extracellular molecule is a Sars-CoV2 spike protein.

8. (i) The extracellular molecule is a toxin arbitrarily selected from toxic shock syndrome toxin (TSST-1), snake toxin, cobra toxin (Cbtx), bacterial toxin, Clostridium toxin, myeloperoxidase, and opioids; (ii) The extracellular molecule is a growth factor or cytokine, arbitrarily selected from interferon, interleukin, tumor necrosis factor (TNF), and transforming growth factor b (TGFb); (iii) The extracellular molecule is a damaged or disregulated protein selected from type 1 interferon (IFN), IL-6, PDL-1, GM-CSF, Gal-3BP, BAG3, IL-17 family, EGF, NRG1, NRG2, NRG3, NRG4, HGF, RANK ligand, TNF-α, soluble TNF-α receptor, IL-1b, IL-5, IL-17A, IL-12, IL-23, C5, BAFF, IgE, and TGFb; (iv) The extracellular molecule is α-synuclein; (v) The extracellular molecule is a cholesterol carrier such as ApoB and ApoE4; (vi) The extracellular molecule is a coagulation factor such as factor IX; (vii) The extracellular molecule is a mucin, which is arbitrarily selected from MUC1, MUC16, MUC2, MUC5AC, MUC4, CD43, CD45, and GPIb; (viii) The extracellular molecule is a hormone, which is arbitrarily selected from insulin and ACTH; (ix) The extracellular molecule is an autoantibody, and includes rheumatoid factor (RF), antinuclear antibody (ANA), anti-neutrophil cytoplasmic antibody (ANCA), anti-double-stranded DNA (anti-dsDNA), anti-centromere antibody (ACA), anti-histone antibody, cyclic citrullinated peptide antibody (CCP), extractable nuclear antigen antibody (e.g., anti-SS-A(Ro) and anti-SS-B(La), anti-RNP, anti-Jo-1, anti-Sm, Scl-70), cardiolipin antibody, β-2 glycoprotein 1 antibody, anti Any of the following antibodies may be selected: phospholipid antibodies (APA), lupus anticoagulant (LA), diabetes-related autoantibodies, anti-tissue transglutaminase (anti-tTG), anti-gliadin antibodies (AGA), intrinsic factor antibodies, parietal cell antibodies, thyroid autoantibodies (e.g., anti-TPO, TSH receptor antibodies), smooth muscle antibodies (SMA), anti-mitochondrial antibodies (AMA), hepatorenal microsome type 1 antibodies (anti-LKM-1), anti-glomerular basement membrane (GBM), and acetylcholine receptor (AChR) antibodies. A bispecific antigen-binding polypeptide according to any one of claims 1 to 3.

9. a) A first antigen-binding domain that binds to an extracellular molecule, wherein the extracellular molecule is a viral antigen; and b) A second antigen-binding domain that binds to a cell surface protein, wherein the cell surface protein is the epidermal growth factor receptor (EGFR), A bispecific antigen-binding polypeptide containing [the specified component].

10. A pharmaceutical composition comprising the bispecific antigen-binding polypeptide described in claim 1.

11. The pharmaceutical composition according to claim 10, wherein the composition comprises a combination of bispecific antigen-binding polypeptides that bind to a plurality of extracellular molecules and / or a plurality of epitopes on extracellular molecules.

12. The pharmaceutical composition according to claim 10, wherein the composition is formulated for administration by oral, sublingual, topical, intravenous, intramuscular, intradermal, transdermal, intraperitoneal, subcutaneous, intranasal, vaginal, rectal, or inhalation.

13. A pharmaceutical composition according to any one of claims 10 to 12 for use in a therapy, wherein the therapy targets the extracellular molecule for intracellularization and degradation via the lysosomal pathway.

14. A pharmaceutical composition according to any one of claims 10 to 12 for treating or preventing an inflammatory pathology, wherein the inflammatory pathology is optionally selected from inflammatory bowel disease; psoriasis; rheumatic inflammatory pathologies such as rheumatoid arthritis and ankylosing spondylitis; multiple sclerosis; autoimmune pathologies such as systemic lupus erythematosus and neuromyelitis optica; asthma; and allergies.

15. A pharmaceutical composition according to any one of claims 10 to 12 for treating or preventing a disease or disorder characterized by a viral infection.

16. A pharmaceutical composition comprising a bispecific antigen-binding polypeptide for the treatment or prevention of HIV, wherein the bispecific antigen-binding polypeptide is a) A first antigen-binding domain that binds to an extracellular molecule, wherein the extracellular molecule is a viral antigen present on the surface of HIV; and b) A second antigen-binding domain that binds to a cell surface protein, wherein the cell surface protein is the epidermal growth factor receptor (EGFR), The pharmaceutical composition comprising the above.

17. A pharmaceutical composition comprising a bispecific antigen-binding polypeptide for treating or preventing SarsCoV2, wherein the bispecific antigen-binding polypeptide is a) A first antigen-binding domain that binds to an extracellular molecule, wherein the extracellular molecule is a viral antigen present on the surface of SarsCoV2; and b) A second antigen-binding domain that binds to a cell surface protein, wherein the cell surface protein is the epidermal growth factor receptor (EGFR), The pharmaceutical composition comprising the above.

18. The antigen-binding domain is selected from the antibody light chain variable domain (VL), antibody heavy chain variable domain (VH), VHH single domain, single chain variable fragment (scFv), Fab fragment, Fd fragment, and Fv fragment. The pharmaceutical composition according to claim 15, wherein optionally, the first antigen-binding domain is VHH and the second antigen-binding domain is VHH.

19. The pharmaceutical composition according to claim 15, wherein the binding of the second antigen-binding domain to EGFR results in EGFR-mediated endocytosis, and / or when both the extracellular molecule and EGFR are bound to the bispecific antigen-binding polypeptide, the extracellular molecule is internalized and degraded via the lysosomal pathway.

20. The pharmaceutical composition according to claim 13, wherein EGFR is present on non-immunological cells, fibroblasts, epithelial cells, endothelial cells, blood cells, or platelets.

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