WO2023148397A1

WO2023148397A1 - Engineered stabilizing aglycosylated fc-regions

Info

Publication number: WO2023148397A1
Application number: PCT/EP2023/052940
Authority: WO
Inventors: Nico Callewaert; Loes VAN SCHIE; Chiara LONIGRO; Wannes WEYTS; Bert Schepens
Original assignee: Vib Vzw; Universiteit Gent
Priority date: 2022-02-07
Filing date: 2023-02-07
Publication date: 2023-08-10

Abstract

The invention relates to the field of therapeutic antibody development. The present invention relates to a mutant Fc domain based on the human IgG1 constant domain, whereby the mutations comprise a substitution of the asparagine at position 297 with an alanine, to prevent N-glycosylation, and additionally comprise two amino acid substitutions at position R292 and V302 with a cysteine as to increase the stability of those aglycosylated Fc-containing proteins by forming an additional disulfide bridge upon expression in a host. More specifically, the introduction of this specific combination of mutations allows for aglycosylated Fc-region containing protein production in yeast with favorable biophysical characteristics, and for which the stability is at least comparable to the stability observed for conventional non-mutated Fcs. Even more specifically, immunoglobulin single variable domains (ISVDs), or VHHs, fused to said stabilizing aglycosylated Fc-regions are provided herein, for use as a medicament and in treatment of diseases, such as Covid-19.

Description

ENGINEERED STABILIZING AGLYCOSYLATED FC-REGIONS

FIELD OF THE INVENTION

The invention relates to the field of therapeutic antibody development. The present invention relates to a mutant Fc domain based on the human IgGl constant domain, whereby the mutations comprise a substitution of the asparagine at position 297 with an alanine, to prevent N-glycosylation, and additionally comprise two amino acid substitutions at position R292 and V302 with a cysteine as to increase the stability of those aglycosylated Fc-containing proteins by forming an additional disulfide bridge upon expression in a host. More specifically, the introduction of this specific combination of mutations allows for aglycosylated Fc-region containing protein production in yeast with favorable biophysical characteristics, and for which the stability is at least comparable to the stability observed for conventional non-mutated Fes. Even more specifically, immunoglobulin single variable domains (ISVDs), or VHHs, fused to said stabilizing aglycosylated Fc-regions are provided herein, for use as a medicament and in treatment of diseases, such as Covid-19.

INTRODUCTION

Monoclonal antibodies and Fc-based therapeutics are produced through large scale manufacturing processes as to use them in therapeutic treatment of various diseases such as cancer, immune disease, and viral infection. Fc-based antibodies have been engineered to address several hurdles in stability, developability and functionality for Fc-based therapeutics. Through improvement of the physicochemical properties and functions mediated by Fc fragments, their druggability is increased, and developmental hurdles may be avoided (Yang et al., 2018). Moreover, the manufacturing system or recombinant host in which such Fc-based therapeutics are produced also requires specific considerations. While mammalian expression hosts are the gold standard for monoclonal antibody production, more recently, research and optimization of alternative, cheaper and faster eukaryotic hosts, such as yeast and plants, has gained interest. Based on the choice of the production platform, different engineering strategies to obtain Fc-proteins with desired physicochemical properties may be envisaged. Bioprocess optimization efforts from the past few decades have enabled to reach a production level of approximately 10 g/L of glycosylated IgG in mammalian cells (Li et al., 2010). However, glycosylation of antibodies expressed in mammalian cells causes high heterogeneity which is often not desired for biological activity and stability of the final products. Therefore, aglycosylated antibodies have been expressed in eukaryotic hosts (mammalian cells, plant cells, or yeasts) by introducing a mutation at the N-linked glycosylation site, the Asn297 residue located within the canonical N-linked glycosylation motif (Asn- X-Ser/Thr) of Fes, or were expressed in prokaryotic hosts. Because the glycosylation status does not significantly affect the pH-dependent FcRn binding, which is critical for prolonged serum circulating half-life of IgG antibodies, the use of aglycosylated wild-type full-length IgG antibodies is a preferred choice for a range of applications such as receptor blocking and targeted delivery not requiring to activate Fc-binding ligands, while possessing the beneficial prolonged serum half-life of full-length IgG format relative to antibody fragments (Ju and Jung, 2014). The presence of a glycan at Asn297 is however indispensable for the recognition of Fc-binding ligands (FcgRs and Clq) and for the activation of a variety of therapeutically critical immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), consequently allowing the clearance of aberrant cells such as tumor cells or infected cells. Mutation of Asn297 may thus also be desired to reduce inflammation and cytotoxicity since effector functions are inhibited in this way.

A drawback of aglycosylated Fc-based therapeutics obtained by engineering the antibody Fc tail is that such biologicals often suffer from a lower stability, and/or higher aggregation potential, which leads to loss of function and increase of adverse risks. Although engineering of an additional disulfide bond has been performed in Fc or Fc domains of monoclonal antibodies, the relationships between specific engineering efforts and the introduced disulfide bond and alteration of the stability, aggregation propensity and function are not entirely clear. One example is for instance based on the evidence that Fc unfolding is first triggered by the protonation of acidic residues on CH2 domain under acidic conditions, which positions the CH2 domain as a location in the Fc tail for improvement of the stability and aggregation resistance of Fes (Zeng et al., 2018). Jacobsen et al (2017) and Liu et al. (2017) described the use of the N297G IgGl Fc mutant for production of aglycosylated monoclonals in combination with a stabilizing disulfide bridge (also see EP2970441B1). However, further exploration of combined mutations in this particular domain were not reported, although certain Fc-based therapeutics may benefit from other mutations such as the N297A for aglycosylated antibodies, which was considered less favourable for manufacturability previously, although in Jacobsen et al. (2017), both the N297A and N297G Fc mutant antibodies performed similar with regards to potency and functionality.

Notwithstanding the removal of Asn297 glycans of IgG perturbs the overall conformation and flexibility of the IgG CH2 domain, resulting in the loss of Fc-ligand interactions and therapeutically critical immune effector functions, aglycosylated full-length IgG antibodies are nearly identical to the glycosylated counterparts in terms of antigen binding, stability at physiological or low temperature conditions, pharmacokinetics, and biodistribution (Ju and Jung, 2014). This positions the research on further optimization and engineering of aglycosylated antibodies on the genetic level, expression host strain, and bioprocess into a continued benefit for the provision of aglycosylated full-length IgG antibodies or Fc-based therapeutics. SUMMARY OF THE INVENTION

The present invention is based on the finding that a particular combination of mutations in the IgGl Fc domain, in particular the CH2 domain, provide for favorable physicochemical properties for the therapeutic development of Fc-based biologicals, more specifically for Fc based fusions with single domain antibody entities such as immunoglobulin single variable domains (ISVDs), VHHs or Nanobodies.

The first aspect of the invention relates to a protein which comprises an antigen-binding domain and an Fc tail which is derived from the human IgGl Fc region and which contains a mutation which modifies the Asparagine at position 297, according to EU numbering (Edelman et al., 1969), to an Alanine (N297A), and which further contains two mutations in the CH2 domain sequence at the Arginine at position292 and the Valine at position 30, both substituted with a cysteine (R292C/V302C). In a specific embodiment, said protein as described herein comprises an Fc region, tail or domain, which corresponds to the human IgGl Fc domain sequence, containing the R292C/N297A/V302C mutations, according to EU numbering, as provided in SEQ. ID NO:1, or corresponding to a homologues thereof with at least 90 % amino acid identity, wherein the R292C/N297A/V302C mutations are maintained. Said protein comprising said Fc domain is thus an Fc-fusion protein, preferably wherein said Fc domain is in the C-terminal part, and wherein the antigen-binding domain is fused to the N-terminus of the Fc domain. A further specific embodiment relates to said Fc-fusion protein comprising said Fc domain with the R292C/N297A/V302C mutations, which is an antigen-binding protein, thus contain a further antigen-binding portion, even more specifically which is an antibody. Further specific embodiments relates to said antigen-binding Fc-fusion proteins wherein the antigen-binding domain comprises an immunoglobulin single variable domain (ISVD), a single domain antibody, a VHH or a Nanobody. In a specific embodiment, said antigen-binding domain is thus fused to said Fc region.

Further aspects of the invention relates to nucleic acid molecules encoding said Fc proteins as described herein, or compositions, in particular pharmaceutical compositions comprising said proteins or nucleic acid molecules.

A further embodiment relates to host cells comprising the protein or the nucleic acid molecule as described, or and more specifically host cells which are eukaryotic organisms, such as mammals, plants, or yeast, preferably Pichia pastoris.

Further aspects of the invention relate to the use of said Fc-containing protein, nucleic acid molecule or pharmaceutical composition for treatment of disease, such as as viral or infectious disease. A specific embodiment of the present invention relates to the Fc fusion protein comprising an antigenbinding domain, which specifically binds via its antigen-binding domain to the spike protein of the SARS- CoV-2 virus. Specifically, said protein may be used for treatment of COVID-19.

A final aspect of the invention relates to production methods for obtaining a stabilizing aglycosylated Fc-fusion protein, comprising the steps of: a) Expressing the nucleic acid molecule as described herein in a host cell, or cultivating the host cell wherein the protein as described herein is present, and b) Isolating said Fc-fusion protein from the cell culture.

DESCRIPTION OF THE FIGURES

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Figure 1. Melting curves of VHH-Fc proteins as determined by DSF.

A. CHO-produced VHH-Fc construct D72-22 (see Table 1), B. P/ch/o-produced VHH-Fc construct with a N297G Fc variant (PS51; see Table 1) and C. P/ch/o-produced VHH-Fc construct with a disulfide stabilized N297G Fc variant (PS55; see Table 1) are shown.

Figure 2. Thermostability analysis of VHH-Fc proteins as determined by DSF.

For each protein encoded by the constructs as indicated in Table 1, differential scanning fluorimetry was performed and the first derivatives were plotted to identify each melting temperature (Tm) as the peaks of these first derivatives. The black line is interpolated normalized fluorescence and the grey dashed line is first derivative dF/dT. In the S-S stabilized constructs (PS55, PS61, PS63, PS64), a small shoulder of the peak is visible at around 50°C, however, it is possible that this peak is also present but hidden (not-resolvable) in the non-stabilized without additional S-S bridge (PS51, PS60, PS62, PS65) constructs because the main peak is already increasing at that point.

Figure 3. Overlay of melting curves from thermostability analysis as determined by DSF of VHH-Fc proteins.

For the VHH-Fc proteins encoded by the constructs as indicated in the figure, DSF was performed and the first derivatives were plotted to identify each melting temperature (Tm) as the peaks of these first derivatives. In the graph the normalized fluorescence and its first derivative dF/dT are shown. The addition of the extra disulphide bond in the PS64 (A/CC) increased thermal stability (ATm ) with 5.4°C as compared to the PS65 without extra disulfide bond (A/-), whereas the addition of the extra disulfide bridge in the PS55 (G/CC) increased thermal stability with 3.9°C as compared to the PS51 without extra disulfide bond (G/-). So the stabilization effect of the additional disulfide bond is higher in the N297A mutant than in the N297G mutant.

Figure 4. Neutralization of P/c/j/a-produced VHH72-Fc with N297A-CC as compared to non-mutated Fc as present in the counterpart, D72-53.

SARS-CoV-2 BetaCov/Belgium/GHB-03021/2020 plaque reduction neutralization assay by the indicated constructs (n=2 ± SD). Synagis (palivizumab) is a non-SARSCoV-2 binding control mAb. D72-53 and PS64 constructs are indicated in Table 1 and differ in the fact that the Fc is mutated in the PS64 construct to N297A and R292C/V302C, and that D72-53 has the LALA mutation as to abolish effector function (which is no longer needed in the aglycosylated Fc of PS64 which doesn't bind FcyRs).

Figure 5. Neutralization of SARS-CoV-2 Wuhan-spike VSV pseudotype virus is retained after introducing N297A instead of N297G mutations in P. pastor/s-produced VHH72-S56A_Fc.

VSV SARS-CoV-2 Wuhan-spike pseudotype GFP reporter virus was incubated with different concentrations of the indicated P/ch/o-produced VHH72-S56A_Fc constructs (Table 1), and used to inoculate confluent monolayers of VeroE6 cells in a 96-well plate. For each dilution series, the GFP signals were normalized to the lowest and highest values of that dilution series and plotted as percentage. The graph (left) shows the normalized GFP signals (N = 2), the IC50 values in pg/ml are indicated next to the graph.

DETAILED DESCRIPTION

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases 'in one embodiment' or 'in an embodiment' in various places throughout this specification are not necessarily all referring to the same embodiment but may.

Definitions

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).

"Nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and singlestranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. "Coding sequence" is a nucleotide sequence, which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances. "Gene" as used here includes both the promoter region of the gene as well as the coding sequence. It refers both to the genomic sequence (including possible introns) as well as to the cDNA derived from the spliced messenger, operably linked to a promoter sequence. "Promoter region of a gene" as used here refers to a functional DNA sequence unit that, when operably linked to a coding sequence and possibly placed in the appropriate inducing conditions, is sufficient to promote transcription of said coding sequence. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.

The terms "protein", and "polypeptide" are interchangeably used further herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. This term also includes posttranslational modifications of the polypeptide, such as glycosylation, phosphorylation, ubiquitination, sumoylation, and acetylation, among others known in the art. Based on the amino acid sequence and the modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)dalton ((k)Da). By "recombinant polypeptide" is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide. When the chimeric polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably "isolated" from or substantially free of culture medium, i.e., culture medium represents less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation. By "isolated" or "purified" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated Fc-region-containing protein" or "purified Fc- containing fusion protein" or "purified protein comprising an Fc region" refers to a protein, fusion protein, or polypeptide which has been purified from the molecules which flank it in a naturally- occurring state, or in its production host, e.g., other membrane proteins or lipids as identified and disclosed herein which has been removed from the molecules present in the sample or mixture, or bacterial or cellular environment, such as a production host, that are adjacent to said polypeptide, by using the detergents, or other agents, and/or purification means as disclosed herein, and as known in the art. An isolated protein or complex or oligomer or composition can be generated by amino acid chemical synthesis followed by further treatments or can be generated by recombinant production or by purification from a complex sample.

"Homologue", "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A "substitution", or "mutation", or "variant" as used herein, results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified", "mutant" or "variant" refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wildtype gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

"Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners or interactors. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. By the term "specifically binds," as used herein is meant a binding domain which recognizes a specific target protein or specific target component or molecule, but does not substantially recognize or bind other molecules in a sample. Specific binding does not mean exclusive binding. However, specific binding does mean that proteins have a certain increased affinity or preference for one or a few of their binders.

The term "antibody", "antibody fragment", or "antigen-binding domain" as used herein refer to a protein comprising an immunoglobulin (Ig) domain or an antigen binding domain capable of specifically binding the antigen. 'Antibodies' can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Among the five isotypes of naturally occurring immunoglobulins (Igs), which are IgG, IgA, IgM, IgD, and IgE, IgG comprises the majority, representing 60 % of total serum Igs in humans. The human IgG molecule is composed of two identical fragment antigen binding (Fab) domains and one fragment crystallizable (Fc) domain that make it multivalent and multifunctional. The two Fab fragments each consist of a heterodimer of a light chain and the N-terminal part of the heavy chain, whereas the C -terminal half of the two heavy chains dimerizes to form the Fc fragment of the IgG antibody. The N-terminal domains of the Fab fragment are the variable domains (Vi and VH) that are responsible for antigen recognition, whereas the C- terminal part of the heavy chains compose the Fc fragment that is responsible for humoral and cellular effector functions. The two Fabs and the Fc are connected by the hinge region, which facilitates the spatial alignment of the three moieties for binding to antigens and effector ligands. "Fc domains" or "Fc-regions" or "Fc-tails", as interchangeably used herein, and refer to the single Fc chain and/or the dimeric Fc domain of an Fc-containing proteins. Specifically in antibodies, said Fc domain is thus responsible for antibody function, and Antibody Fc engineering stands for engineering functions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life. Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post-translational modifications, dimerization behavior, effector function, serum half-life, among others. To indicate the variations present in Fc domains based on the sequence of naturally occurring IgGs, conventional antibody numbering annotations are known in the art, such as for instance IMGT numbering (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22), Kabat numbering (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication n° 91-3242, pp 662,680,689 (1991)), or preferably used herein EU numbering (Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.;63:78- 85).

The term "antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity-determining-regions (CDRs) accounting for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies, domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. An additional requirement for "activity" or "functionality" of said fragments is that said fragments are capable of binding the antigen of interest. The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "I VD") means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigenbinding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, with binding to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

An immunoglobulin single variable domain (ISVD) as used herein, refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An "immunoglobulin single variable domains" (abbreviated as "ISVD"), as used herein, is equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDR's. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079. "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (= EP 1433793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobody, including humanization and/or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent or multispecific constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08/101985 and WO 08/142164. Nanobodies form the smallest antigen binding fragment that completely retains the binding affinity and specificity of a full-length antibody. Nbs possess exceptionally long complementarity-determining region 3 (CDR3) loops and a convex paratope, which allow them to penetrate into hidden cavities of target antigens.

Determination of CDR regions may be done according to different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum et al. (J. Mol. Biol. (1996) 262, 732-745). Or alternatively the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described on http://www.bioinf.org.uk/abs/index.html), Chothia (Chothia and Lesk, 1987; Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; 5^th edition, NIH publication 91-3242), and IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22). Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any immunoglobulin protein sequences without undue burden. These annotations differ slightly, but each intend to comprise the regions of the loops involved in binding the target.

'Antigen-binding proteins' or 'antigen-binding domains' as described herein may be derived from an antibody as described herein, or may be derived from alternative antigen-binding proteins with a different fold, so non-immunoglobulin binding proteins such as but not restricted to avimers, DARPins, alphabodies, affitins, nanofitins, anticalins, monobodies and lipocalins.

The term 'antibody' or 'Fc-fusion' as used herein further refers to the genetic linking or fusion of antigen-binding fragments or antigen-binding domains with an Fc constant domain as to obtain dimers forming an antibody structure when expressed in a recombinant host. In particular, antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N-terminus of an Fc domain, preferably via a linker or hinge region. Alternatively, antibody fragments, or single domain antibodies such as ISVDs, may be fused at the N-terminus to the C-terminal end of an Fc domain, preferably via a linker or hinge region. Said single domain antibody or ISVD fused to said Fc ma comprise one or more VHHs or Nbs, as described herein.

This invention also relates to "pharmaceutical compositions" comprising one or more antibodies of the invention, in particular, the antibody composition as described herein and, optionally, a pharmaceutically acceptable carrier or diluent or excipient. These pharmaceutical compositions can be utilized to achieve the desired pharmacological effect by administration to a patient in need thereof. The present invention includes pharmaceutical compositions that are comprised of a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a protein comprising an Fc region or an antibody composition, or salt thereof, of the present invention. A pharmaceutically effective amount of compound is preferably that amount which produces a result or exerts an influence on the particular condition being treated.

A "pharmaceutically or therapeutically effective amount" of compound or protein or composition is preferably that amount which produces a result or exerts an influence on the particular condition being treated. The Fc-containing proteins or the pharmaceutical composition as described herein may also function as a "therapeutically active agent" which is used to refer to any molecule that has or may have a therapeutic effect (i.e. curative or stabilizing effect) in the context of treatment of a disease (as described further herein). Preferably, a therapeutically active agent is a disease-modifying agent, and/or an agent with a curative effect on the disease. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably a carrier that is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the carrier do not vitiate the beneficial effects of the active ingredient. Suitable carriers or adjuvantia typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, M. F. et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311), Strickley, R.G ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324- 349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171). The term "excipient", as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surface active agents, preservatives, emulsifiers, buffer substances, stabilizing agents, flavouring agents or colorants. A "diluent", in particular a "pharmaceutically acceptable vehicle", includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.

The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein. The term "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. The terms "subject", "individual" or "patient", used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, primates, avians, fish, reptiles, livestock animals (e.g., sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs) and captive wild animals (e.g., foxes, deer, dingoes). However, it will be understood that the aforementioned terms do not imply that symptoms are present.

Detailed description

The present disclosure is based on research for launching a yeast production platform for therapeutic antibody production with the aim to provide aglycosylated antibody-type of proteins at high yield, and with favorable developability, such as high stability, low aggregation potential and good potency, providing for biologicals with similar inherent biophysical properties and shelf-life as antibody-based therapeutics produced in the established mammalian platforms. In view of the state of the art on monoclonal antibodies (e.g. Jacobsen et al., 2017; Liu et al., 2017), one would try to obtain aglycosylated Fc-based therapeutics by mutation of the N297 residue, which is known to prevent N- glycosylation in monoclonal antibodies, and was previously shown to allow substitution by glycine, alanine, or glutamine. Although these aglycosylated Fc-domains provide for functional and potent monoclonal antibodies, depending on the specific substitutions, their biophysical properties differ and should be analyzed in more detail as to determine which type of Fc variant is preferred in obtaining aglycosylated Fc-based therapeutics, depending on the desired Fc-format, the production host, and the indication or purpose. So far, N297 mutant Fes are mostly used in monoclonal antibody production. Whether aglycosylated antibodies in a specific format wherein ISVDs or VHHs are fused to Fes behave similar to aglycosylated monoclonal antibodies is still unknown and whether yeast-based production of such aglycosylated VHH-Fc fusions provides for Fc-based therapeutics that have similar biophysical properties and potency as their mammalian produced counterparts (glycosylated or aglycosylated) has not been reported previously. This invention relates to the use of N297A mutant Fc domains, which prevent N-glycosylation, but were also reported to have a slightly lower stability and higher aggregation potential as compared to wild type Fc, or even as compared to the known N297G Fc mutant used in monoclonal antibodies. A known method to increase stability of monoclonal antibodies is to introduce cysteine substitutions in the CH2 domain, which tends to work well for N297G Fc mutants to restore thermostability (Jacobsen et al., 2017). Though for the less favoured N297A Fc mutants, with higher aggregation potential, introduction of additional disulfide bridges may in fact increase the aggregation potential. In this study, we found that specifically the combination of N297A and R292C/V302C mutations resulted in VHH-Fc fusions with fully restored stability as compared to their wild type Fc counterparts. Especially in the context of VHH-Fc fusions, which differ from monoclonal antibodies in that no VL light chains are present, thus implicating different conformational, structural and sterical properties, the finding disclosed herein that production of VHH-Fc fusions with N297A and R292C/V302C in yeast resulted in developable biologicals, opens new engineering opportunities for Fc- based therapeutics.

In a first aspect, the invention relates thus to Fc-containing proteins wherein the Fc domain or Fc region, as used interchangeably herein, is derived from an IgGl Fc domain, comprising the mutation N297A, and R292C and V302C, using the numbering of the constant region according to EU annotation (Edelman et al., 1969), the latter two substitutions allowing the formation of an additional disulfide bridge in the Fc-containing protein when expressed in a host, and present in dimer form. In a specific embodiment said Fc-containing protein or Fc-fusion protein, as interchangeably used herein, comprises an Fc region which comprises SEQ ID NO:1, corresponding to the amino acid sequence of the human IgGl Fc domain containing the N297A/R292C/V302C mutations, or comprising a sequence with at least 80 % identity to SEQ. ID NO:1, or at least 90 % identity, or at least 95 % identity, or at least 97 % identity, or at least 99 % identity to SEQ ID NO:1, wherein the % identity is considered over the full length of the Fc domain of SEQ ID NO:1, and wherein the N297A/R292C/V302C are present.

Further embodiment disclosed herein relate to Fc-containing proteins wherein the Fc domain is present upon expression in a host in dimeric form, as to allow disulfide bridge formation and to provide for a multivalent or multispecific Fc-based protein, which may be bi-, tri-, tetra- valent or -specific, and may be comprise other protein domains such as an antigen-binding domain, as defined herein, for target binding. In a specific embodiment, said Fc-fusion protein comprises an antigen-binding domain that is fused at the N-terminus of the Fc region, directly or via a hinge and/or linker, as known in the art. Another specific embodiment provides for said Fc-fusion protein comprising an antigen-binding domain that is fused at the C-terminus of the Fc region, directly or via a hinge and/or linker, as known in the art. Another specific embodiment discloses said Fc-containing protein wherein said Fc domain is fused at its N- and C-terminus to a further protein domain, preferably one or more antigen-binding domains.

In a specific embodiment said Fc-containing protein of the present invention, comprising the N297A/R292C/V302C mutations in its Fc region is an antibody. Said antibody may be an IgGl, lgG2, lgG3 or lgG4. In a further specific embodiment said Fc region comprises further modifications as to provide for aglycosylated Fc containing proteins or antibodies with particular functionalities. Such further modifications may comprise additional amino acid mutations, such as for instance those known in the art to abolish effector functions (Wines, et al. 2000. J. Immunol. 164, 5313-5318), although aglycosylated antibodies are already reduced in effector function; additional cysteines, or additional tags or functional moieties.

In a further specific embodiment, said Fc-containing protein comprises an Fc domain comprising N297A/R292C/V302C mutations, preferably as provided in SEQ. ID NO:1, or a homologue thereof with at least 90 % identity, and comprises an antigen-binding domain comprising an antibody fragment, a single domain antibody, an ISVD, a VHH, or a Nb.

In a specific embodiment said Fc region of said protein described herein is fused to said antigen-binding domain or antigen-binding protein as described herein, such as an ISVD, wherein said fusions or connections can be direct fusions, made via peptide bonds between amino acid residues of the Fc chain and ISVD itself, or indirect fusions made by a linker or a hinge, as known in the art. Hinge regions as present in IgG antibodies are often used to connect Fc regions with alternative antigen-binding moieties such as ISVDs. Said hinge regions may also be engineered in order to optimize yield, stability and further properties of the resulting proteins upon expression in a recombinant host cell. Preferred "linker molecules", "linkers", or "short polypeptide linkers" are peptides with a length of about ten amino acids. Non-limiting examples of suitable linker sequences are described in the Example section, and are known by the skilled person. Linkers may be selected to keep a fixed distance between the Fc and antigen-binding domains.

A further aspect of the invention provides for nucleic acids encoding the protein as described herein, and expression cassettes or vectors containing said nucleic acid. In a further embodiment, host cells comprising the Fc-containing proteins as described herein or the nucleic acid molecule encoding said proteins are described. Host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors comprising said nucleic acid molecule into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction. A DNA construct capable of enabling the expression of the Fc-containing protein of the invention can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993) and Ausubel et al. (2016). Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Komagataella, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals.

It is to be understood that although particular embodiments, specific configurations, compositions, as well as materials and/or molecules, have been discussed herein for methods, compositions and products according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

EXAMPLES

Introduction

Production of aglycosylated antibodies, which is frequently desired to reduce Fc-effector functions of biologicals, often suffers from stability and developability issues. Moreover, when yeast is the production host, the presence of yeast glycans has been demonstrated to reduce circulatory half-life in Fc-containing molecules. In addition, we saw that Pichia produced VHH-Fc fusion proteins have a lower thermal stability than the mammalian CHO produced counterpart. To date a number of solutions are at hand to produce aglycosylated antibodies in yeast, such as the application of Glycodelete yeast cells, involving an enzymatic removal of glycan structures from the N-glycosylated proteins, or the application of engineered or mutated Fc-encoding sequences to prevent N-glycosylation. Substitution of the hallmark N297 residue with for instance glycine, glutamine, or alanine results in aglycosylated Fes in mammalian IgG producing cells (see for instance Jacobsen et al. 2017). In addition, the introduction of cysteine substitutions in the Fc CH2 region to improve Fc stability via an additional intradomain disulfide bridge has been previously explored. In this study, a specific combination of substitutions was analyzed with the aim to provide for aglycosylated Fc-containing proteins without loss of stability or developability, more specifically in view of producing VHH-Fc fusions in yeast production hosts. In this study, it was found that nucleic acids encoding VHH-Fc fusions, in which the IgGl-based Fc-region contains a N297 substitution to Alanine, for avoiding glycosylation, and in which the R292 and V302 are substituted to cysteine, stabilize the Fc via disulfide bridge formation in at least the same manner as for previously found alternative substitutions. This specific combination of mutations to obtain an aglycosylated Fc resulted in increased stability and moreover in further favorable biophysical properties for the Fc-containing molecules, even when produced in Pichia pastoris host cells, as to provide for improved variants for therapeutic antibody development.

The examples as described herein demonstrate that engineering of Fc-regions of anti-SARS-CoV-2- specific VHH-Fc fusions provides for developable biologicals to combat COVID-19. As disclosed in Schepens et al. (2021, Sci. Transl. Med. 13, eabi7826), VHH72-Fc constructs have undergone a protein engineering campaign for their production in CHO cells previously. Herein, we further established the optimal developability properties for production of these VHH-Fcs in Pichia pastoris. If Pichia as a host is usable for large-scale manufacturing to provide for biologicals of similar quality, this comes with a number of advantages, such as fewer competition for the capacity of large-scale manufacturing plants, and a faster and cheaper manufacturing process. The monoclonal antibody Vyepti, containing a N297A Fc, was the first ever manufactured in Pichia and has been approved by the FDA for the treatment of migraine, thereby providing a good benchmark for future regulatory Pichia-manufactured antibody studies.

Herein, we investigated production of aglycosylated VHH-Fc molecules in Pichia in terms of protein stability and quality, especially in view of the Fc sequence variants discussed herein. Although it has previously been demonstrated that the glycine substitution is more stable than the N297A, so the N297A substitution in an IgGl-Fc conventional antibody is more destabilizing than the N297G IgGl-Fc mutant (as reported by Jacobsen et al., 2017), as compared to the wild type IgGl-Fcs, surprisingly we found that the additional modification of the R292C/V302C induced disulfide bridge in the N297A VHH- Fcs which drive the stability to a similar level as compared to a N297G mutant containing said R292C/V302C induced disulfide bridge. So the addition of the disulfide bridge via R292/V302 cysteine modification in the N297A VHH-Fc increases its thermostability with a larger extend as when the same modification is introduced in the N297G Fc, more specifically within a range of 5°C - 10°C increase as compared to the N297A Fc its thermostability. Moreover, further beneficial properties were identified for said Fes such as the fact that this N297A/R292C/V302C mutated VHH-Fc also has reduced aggregate/oligomer induction in stress conditions, as shown in Example 3, which encourages that these modified VHH-Fcs are suitable in view of their stability and developability.

Example 1. Aglycosylated VHH-Fc proteins are less stable than the CHO-produced glycosylated counterpart but addition of an extra disulfide bond in the Fc' CH2 domain restores thermal stability.

Production of aglycosylated Fc-containing proteins is often required, although, as compared to the production of glycosylated Fc-containing proteins, these aglycosylated forms are often less stable. In this study, not only the impact of the Fc glycosylation on protein stability and developability was investigated, but also the production of such aglycosylated Fc protein forms in yeast as alternative production system was optimized, providing for faster and cheaper manufacturing options. Production of wild-type, glycosylated forms of VHH-Fc fusion proteins in mammalian CHO cells resulted in Fc- containing proteins with melting temperature of around 65°C (Figure 1 A). However, when aglycosylated forms were produced in yeast, such as the N297G mutant Fc, these were found to have a melting temperature that is about 5°C lower as compared to CHO-produced glycosylated VHH-Fc, with a melting temperature of 60°C on differential scanning fluorimetry (as determined by a thermofluor assay; Figure IB). To counteract on this effect, addition of certain disulfide bonds in the Fc' CH2 domain has been reported to restore protein thermal stability in aglycosylated monoclonal antibodies (Gong et al., 2009; Jacobsen et al., 2017). Based on that observation, we investigated whether VHH-Fc stability could also be restored in the yeast produced Fc fusions by cysteine replacements to result in specific disulfide bridges. For the known N297G aglycosylated Fes, the substitution of R292C/V302C indeed restored the melting temperature, which is a measure for thermostability, back to the 'wild-type' level of 65° C (Figure 1C).

In an effort to optimize aglycosylated Fc protein production in yeast, the stability of the N297A aglycosylated Fc constructs was investigated herein, and as shown in Table 2 and Figure 2 and 3, the melting temperature, as a parameter for thermal stability of the antibodies, was compared for a set of different VHH-Fc fusions, as listed in Table 1.

To evaluate the biophysical properties of different VHH-Fc variants, the constructs as listed in Table 1 were expressed in the NCYC2543 Pichia pastoris host cells and the Fc fusion proteins were purified using Protein A affinity chromatography followed by a polishing step by gel filtration with SD200 16/600pg in PBS pH7.2 (see Methods). For the constructs with Cys substitutions, after expression in Pichia pastoris the correct formation of the new S-S bridge at the expected location was confirmed by mass spec analysis.

Table 1. Different VHH-Fc fusion constructs used in this study.

Thermal stability was assessed by differential scanning fluorimetry (DSF) which was performed as described in the Methods.

Table 2. Melting temperatures as calculated from the DSF plots.

The results shown in Table 2 indicate that for two different VHH-Fc fusion constructs wherein the hinge region between the VHH and the Fc part differs in its EPKS-sequence (Table 1), the melting curves for each of those constructs (Figure 2) provides for a melting temperature ™ value for the VHH fusions with the N297A Fc mutant starting the unfolding at about 1°C lower as compared to the N297G Fc mutant, while the melting temperatures for the cysteine-substituted stabilized mutants of both the N297G and N297A Fes are similar. This indicates that introduction of an extra disulfide bond in the CH2 domain (R292C and V302C) restores the thermal stability with a larger degree in the N297A mutant as compared to the restoration in the N297G mutant (ATm due to CC in Table 2), with at least 0.3-1.7° C. Alternatively, Figure 3 provides an overlay of melting curves from thermostability measurements as determined by DSF of the listed VHH-Fc proteins, confirming that addition of the extra disulphide bond in the PS64 (A/CC), as well as in the PS55 (G/CC) increased thermal stability back to levels which were also observed for CHO produced glycosylated Fc constructs, around 66°C, though with a larger increase for the addition of the disulfide bridge in the N297A mutant (ATm = 5.4°C), as compared to the increase for the addition of the disulfide bridge in the N297G mutant (ATm = 3.9°C).

Finally, the thermal stability of the aglycosylated VHH-Fc CC-stabilized forms produced in Pichia is comparable as compared to a wild type Fc produced in CHO cells, independent from other particular modifications, such as a truncated hinge, which was the difference in the construct 1 & 2 indicated in Table 2 (AEPKSC; also see Table 1).

Example 2. Characterization of purified SARS-CoV-2 VHH-Fc proteins expressed in Pichia.

The production yields from the Pichia expressed VHH-Fc construct (Table 1) are shown in table 3 and expressed in mg/L based on total purified protein obtained after gel filtration. Expression volume varied between 150-200mL.

The % of the peak corresponding to multimers in the SEC chromatogram is indicated for the different constructs in Table 3, providing for a first indication on their aggregation potential.

In Jacobsen et al. 2017, a stronger aggregation propensity of the N297A variant had been observed, which is confirmed here in that the % multimeric fraction for both VHH-Fc constructs (PS65 and PS62) is higher as compared to the N297G counterpart. The introduction of the stabilizing extra S-S bond however reduces the % multimeric fraction, and while this was only performed in the N297G molecule by Jacobsen et al, the additional disulfide bond in the N297A variant also reduces the multimeric fraction, but even to a larger extent as compared for the N297G+CC counterpart (A % for addition of the S-S bridge to N297A is more than 2-fold as compared to N297G).

Table 3. Purification yield from NCYC2543 produced cells and aggregation potential of VHH-Fc proteins.

To further explore the aggregation potential of the different proteins, Dynamic Light scattering (DLS) and Static Light Scattering (SLS) measurements were performed on the purified samples by defining the aggregation temperature (T_agg) at 266 nm and 243nm (Uncle instrument; two last columns in Table 3), as to determine the amount of particles or aggregates. From these measurements, we confirmed that for the VHH-Fc constructs a reduction in aggregation is obtained by adding the additional disulfide bridge, as well in the N297G as N297A Fc mutants, however the reduction in the latter is more significant.

Example 3. Accelerated aging stress test of Pichia-made N297A-CC-stabilized VHH-Fcs.

An "accelerated aging" stress test was performed on the VHH-Fc construct with the preferred N297A- CC mutations produced in Pichia (PS64) as compared to the non-stabilized N297A (PS65 construct) and as compared to the CHO produced D72-53 with non-mutated Fc, to analyze the effects of the particular stabilizing disulfide bridge on protein (storage) stability.

Table 4 shows the results of SEC-MALS analysis of purified VHH-Fc samples before (to) and after (tlO) 10 days at 40 °C incubation. The peak corresponding to the molar weight of an assembled VHH-Fc protein was indicated as 'monomer peak'. Peak quantitation (%) is based on the refraction signal. Qualitative analysis of the monomer peak was performed on the 200 pL peak elution fraction. Values reported are from a single stress tested sample.

Table 4. SEC MALS results.

HMW = high molecular weight species; LMW = low molecular weight species; aggreg, = aggregates.

Before accelerated aging, all three N297A variants occur as > 97% fully assembled monoparatopic bivalent VHH-Fc monomers, both with and without Cys-Cys Fc stabilization.

The monomer mass fraction after stress is retained to 97.8% upon Cys-Cys stabilization and decreases to 91.8% without Cys-cys stabilization. Furthermore, Cys-Cys stabilization limited the increase of LMW fractions (degradation products) during accelerated aging.

For the R292C-V302C stabilized VHH-FC-N297A variants 'PS64', monomer recovery after 10 days of incubation at 40 degrees is consistently higher than for the non-stabilized variants 'PS65'. In parallel, after 10 days, the increase in multimer/HMW/aggregate species as well as LMW species is limited in stabilized variants and consistently higher for non-stabilized N297A variants.

To conclude, the Fc molecules stabilized by introducing the additional disulfide bridge showed 4-6 °C higher melting temperature, in the range typical for CHO-glycosylated Fes. In addition, it seems that the introduction of the disulfide bridge also provides for a lower tendency of forming soluble and/or insoluble protein aggregates in accelerated aging temperature stress experiments (10 days at 40°C, Table 4), at least equal as compared to CHO-made glycosylated D72-53. Although Jacobsen et al (2017) reported that the N297A was less suited for developability due to the stronger aggregation potential, here we unexpectedly observed that the N297A and N297G are both restored to near-native stability, and this both usable in development of VHH-Fc fusions for therapeutic use.

Example 4. Neutralization potential of aglycosylated SARS-CoV-2 specific VHH-Fc with N297 and R292C/V302C mutations.

As to confirm that the potency of the P/ch/o-produced constructs is retained to the expected level, the neutralization potential was analyzed in comparison to the D72-53 construct, which has an Fc that is not mutated in N297 or R292/V302. Since the interest of this study lays in the application of the N297A R292C/V302C Fc variant, the PS64 purified protein construct was analyzed in a plaque reduction assay, and as shown in Figure 4, the in vitro neutralization potency was not affected by the Fc engineering, since similar potency in neutralizing real SARS-CoV-2 virus in vitro and reducing plaque formation, was observed as compared to non-mutated Fc (D72-53) protein.

Example 5. Neutralization of SARS-CoV-2 Wuhan-spike VSV pseudotype virus is retained after introducing N297A instead of N297G mutations in P. pastor/s-produced VHH72-S56A_Fc.

To analyze the effects of the triple mutations N297A/R292C/V302C substitutions on the viral neutralization potential, as compared to the N297G/R292C/V302C and as compared to the single mutants N297 lacking the stabilizing disulfide bridge, a neutralization assay of SARS-CoV-2 Wuhan-spike VSV pseudotype virus was performed (Figure 5) with several P/ch/o-produced VHH72-S56A_Fc constructs (see Table 1). The resulting IC₅o values below 0.1 pg/ml show a better or equal performance of the preferred N297A and N297A-CC-stabilized mutants as compared to the N297G and N297G-CC mutants. This effect was independent from other particular modifications, such as a truncated (AEPKSC) or single amino acid substituted (EPKSS) hinge region (Table 1). This observation confirmed our hypothesis that the application of aglycosylated VHH-Fc with this particular triple mutation N297A/R292C/V302C is beneficial and even preferred for in vivo prophylactic or therapeutic use of antibodies, in particular VHH-Fc type of antibodies. Material and methods.

Strains, media, and reagents

Escherichia coli (E. coli) MC1061 was used for standard molecular biology manipulations. For plasmid propagation, E. coli were cultured in LB broth (0.5% yeast extract, 1% tryptone, and 0.5% NaCI) supplemented with 25 pg/mL chloramphenicol (MP Biomedicals), 50 pg/mL carbenicillin (Duchefa Biochemie) and 50 pg/mL Zeocin® (Life Technologies). P. pastoris NCYC-2543 strain was provided by the National Collection of Yeast Culture. Yeast cultures were grown in liquid YPD (1% yeast extract, 2% peptone, 2% D-glucose) or on solid YPD-agar (1% yeast extract, 2% peptone, 2% D-glucose, 2% agar) at pH 7.5 and selected with 100 pg/mL Zeocin. For protein expression, cultures were grown in a shaking incubator (28°C, 225 rpm) in BMGY (same composition but with 1% glycerol replacing the 2% D-glucose) or BMMY (same composition but with 1% methanol replacing the 2% D-glucose).

Generation of expression plasmids

Most of the expression vectors for the VHH-Fc variants were generated using an adapted version of the Yeast Modular Cloning toolkit based on Golden Gate assembly (Lee et al., 2015. ACS Synth. Biol. 2015, 4, 9, 975-986). Briefly, coding sequences for the VHH and hlgG-Fc variants, were codon optimized for expression in P. pastoris using GeneArt (Thermo) proprietary algorithm and ordered as gblocks at IDT. Each coding sequence was flanked by unique part-specific upstream and downstream Bsal-generated overhangs. The gblocks were inserted in a universal entry vector via BsmBI assembly which resulted in different "part" plasmids (entry vectors), containing chloramphenicol resistance cassette. Part plasmids were assembled to form expression plasmids (pX-VHH-Fc) via a Golden Gate Bsal assembly. Each expression plasmid consists of the assembly of 9 parts: Pl_ConLS, P2_pGAP (or P2_pAOXl), p3-0stl- VHH, P4a-hlgG-Fc, P4b_AOXltt, P5_ConRl, P6-7 Lox71-Zeo, P8 AmpR-ColEl-Lox66. Selection of correctly assembled expression plasmids was made in LB supplemented with 50 pg/mL carbenicillin and 50 pg/mL Zeocin®. All the entry vectors and expression plasmids were sequence verified. Point mutations N297G, N297A, R292C and V302C were introduced by site directed mutagenesis using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent) according to the manufacture's protocol.

Generation of Pichia pastoris expression strains

Prior to transformation, all expression constructs were linearized in the promoter region (Avril for pGAP and Pmel for AOX1) to facilitate single homologous recombination and targeted integration in the P. pastoris genome. Transformations were performed using the lithium acetate electroporation protocol as described by Wu and Letchworth (2004. Biotechniques, 36(l):152-4.). Briefly, a culture of P. pastoris was grown overnight in YPD to obtain an OD600 of 1.5. The cells were harvested and were left to incubate in 100 mM LiAc, 10 mM DTT, 0.6 M sorbitol and 10 mM Tris-HCI, pH 7.5. The cells were thoroughly washed with 1 M ice-cold sorbitol. Cells were transformed by electroporation (1.5 kV, 25 pF, 200 O) with 100-1000 ng of linearized vector. Positive transformants were selected on YPD-agar supplemented with 100 pg/mL of Zeocin®.

Protein expression and purification

For protein purification, an overnight culture of P. pastoris transformed with the expression plasmid was diluted in 125 ml of BMGY to 0.1 OD600 in 2-liter baffled shake flasks. For methanol-based expression with pAOXl promoter, after 48 hours of growth in glycerol, expression was induced by switching to media containing 1% MeOH. To maintain induction and to compensate for evaporation, cultures were spiked with 1% MeOH every 8-12 hours. For expression with the constitutive GAP promoter, expression was done in BMGY for 50-60 hours, at 28 °C, 250 rpm. Medium was collected by centrifugation at 1,500 g, 4°C for 10 minutes and filtered over a 0.22 pm bottle top filter (Millipore) before loading on a HiTrap MabSelect SuRe 5 ml column (GE Healthcare), equilibrated with Mcllvaine buffer pH 7.2 (174 mM Na2HPO4, 13mM citric acid). The column was eluted with Mcllvaine buffer pH 3 (40 mM Na2HPO4 ,79mM citric acid). Collected fractions were neutralized to pH 6.5 with 0.4M Na3PO4. The elution fractions were analyzed on SDS-PAGE. Target protein-containing fractions were pooled and finally, the protein (in a maximal volume of 10 ml) was injected on a HiLoad® 16/600 Superdex® 200 pg column (GE-Healthcare), eluted with PBS. The obtained fractions were analyzed by SDS-PAGE and the fractions containing monomeric VHH-Fc were pooled together. Protein concentration was measured by 280nm absorbance vs. a buffer blank and concentrated with Amicon 30 kDa MWCO spin columns. Purified protein was snapfrozen in liquid nitrogen and stored at -80°C.

Mass spectrometry

Intact VHH72-Fc protein (10 pg) was first reduced with tris(2-carboxyethyl)phosphine (TCEP; 10 mM) for 30 min at 37°C, after which the reduced protein was separated on an Ultimate 3000 HPLC system (Thermo Fisher Scientific, Bremen, Germany) connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fischer Scientific). Briefly, approximately8 pg of protein was injected on a Zorbax 300SB-C18 column (5 pm, 300A, 1x250mm IDxL; Agilent Technologies) or a Zorbax Poroshell 300SB-C8 column (5 pm, 300A, 1x75mm IDxL; Agilent Technologies), and separated using a 30 min gradient from 5% to 80% solvent B at a flow rate of 100 pl/min (solvent A: 0.1% formic acid and 0.05% trifluoroacetic acid in water; solvent B: 0.1% formic acid and 0.05% trifluoroacetic acid in acetonitrile). The column temperature was maintained at 60°C. Eluting proteins were directly sprayed in the mass spectrometer with an ESI source using the following parameters: spray voltage of 4.2kV, surface-induced dissociation of 30V, capillary temperature of 325°C, capillary voltage of 35V and a sheath gas flow rate of 7 (arbitrary units). The mass spectrometer was operated in MSI mode using the orbitrap analyzer at a resolution of 100,000 (at m/z 400) and a mass range of 600-4000 m/z, in profile mode. The resulting MS spectra were deconvoluted with the BioPharma FinderTM3.0 software (Thermo Fischer Scientific) using the Xtract deconvolution algorithm (isotopically resolved spectra). The deconvoluted spectra were manually annotated.

Thermal stability

To evaluate thermal stability of VHH-Fc variants, differential scanning fluorimetry (a thermofluor assay) was performed. Briefly, a final concentration of 0.1 pg/ml of VHH72-Fc in PBS was mixed with 10X SYPRO Orange dye (Life Technologies). Dye binding to molten globule unfolding protein was measured over a 0.01 °C/s temperature gradient from 20 °C to 98 °C in a Roche LightCycler 480 qPCR machine. After cubic spline interpolation of the melting curves, first derivatives were plotted to identify each melting temperature (Tm) as the peaks of these first derivatives.

SEC MALS

To determine the molecular mass and aggregation behavior of VHH-Fc variants, the protein was analyzed by size exclusion chromatography multi-angle laser light scattering (SEC-MALS). For each analysis, 100 pl sample filtered through 0.1 pm Ultrafree-MC centrifugal filters (Merck) was injected onto a Superdex200 10/300 GL Increase SEC column (GE Healthcare) equilibrated with sample buffer, coupled to an online UV detector (Shimadzu), a mini DAWN TREOS (Wyatt) multi-angle laser light scattering detector and an Optilab T-rEX refractometer (Wyatt) at 298 K. The refractive index (Rl) increment value (dn/dc value) at 298 K and 658 nm was calculated using SEDFIT V16.175 (Schuck, 2000. Biophysical Journal. 78, 1606-1619) and used for the determination of the protein concentration and molecular mass.

The peaks were defined as follows: annotation of 'LMW/HMW species' are low or high molecular weight species, resp., that do not form very discrete peaks, e.g. degradation products and aggregation products thereof; 'monomer' are completely assembled monoparatopic bivalent VHH-Fc.

Plaque reduction neutralization test (PRNT)

The PRNT was performed by the group of Dirk Jochmans. We used SARS-CoV-2 strain BetaCov/Belgium/GHB-03021/2020 (EPI ISL 407976; 2020-02-03) used from passage P6 grown on Vero E6 cells Dose-dependent neutralization of distinct VHH-Fc constructs was assessed by mixing the VHH- Fc constructs at different concentrations (three-fold serial dilutions starting from a concentration of 10 pg/ml), with 100 PFU SARS-CoV-2 in DMEM supplemented with 2% FBS and incubating the mixture at 37°C for lh. VHH-Fc-virus complexes were then added to Vero E6 cell monolayers in 12-well plates and incubated at 37°C for lh. Subsequently, the inoculum mixture was replaced with 0.8% (w/v) methylcellulose in DMEM supplemented with 2% FBS. After 3 days incubation at 37°C, the overlays were removed, the cells were fixed with 3.7% PFA, and stained with 0.5% crystal violet. Half-maximum neutralization titers (PRNT50) were defined as the VHH-Fc concentration that resulted in a plaque reduction of 50% across 2 or 3 independent plates.

SARS-CoV pseudovirus neutralization assay

To generate replication-deficient VSV pseudotyped viruses, HEK293T cells, transfected with SARS-CoV-2 Wuhan S, were inoculated with a replication deficient VSV vector containing enhanced green fluorescent protein (GFP) and firefly luciferase expression cassettes (Rentsch & Zimmer, 2011; Hoffmann et al, 2020). After a 1-hour incubation at 37°C, the inoculum was removed, and cells were washed with PBS and incubated in medium supplemented with an anti-VSV G mAb (American Type Culture Collection) for 16 hours. Pseudotyped particles were then harvested and clarified by centrifugation (Wrapp, 2020). For the VSV pseudotype neutralization experiments, the pseudoviruses were incubated for 30 min at 37°C with different dilutions of VHH-Fc fusions. The incubated pseudoviruses were subsequently added to subconfluent monolayers of Vero E6 cells. Sixteen hours later, the cells were lysed using passive lysis buffer (Promega). The transduction efficiency was quantified by measuring the GFP fluorescence in the prepared cell lysates using a Tecan Infinite 200 Pro plate reader. GFP fluorescence was normalized using the lowest and highest GFP fluorescence value of each dilution series. The IC50 was calculated by nonlinear regression curve fitting, log(inhibitor) versus response (log-normalized).

Sequence listing

>SEQ ID NO:1: human IgGl Fc with N297A R292C/V302C mutations

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYA STYRCVSVLTVLHQ.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSD IAVEWESNGQ.PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ.Q.GNVFSCSVIVIHEALHNHYTQ.KSLSLSPGK

>SEQ ID NO:2: human IgGl Fc amino acid sequence

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQ.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYP SDIAVEWESNGQ.PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ.Q.GNVFSCSVIVIHEALHNHYTQ.KSLSLSPGK >SEQ ID NO:3: PS51 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSdel- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQ.M NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQ.GTLVTVSSGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLH Q.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWESNGQ.P ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ.Q.GNVFSCSVIVI HEALHNHYTQ.KSLSLSPGK >SEQ ID N0:4: PS55 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSdel- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLH

Q.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWESNGQ.P ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQ.KSLSLSPGK

>SEQ ID NO:5: PS60 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSS- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQMNSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQ.KSLSLSPGK

>SEQ ID NO:6: PS61 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSS- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSV

LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ.KSLSLSPGK

>SEQ ID NO:7: PS65 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSdel- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLH

>SEQ ID NO:8: PS64 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSdel- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYASTYRCVSVLTVLH Q.DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWESNGQ.P ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

>SEQ ID N0:9: PS62 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSS- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSEPKSSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ.KSLSLSPGK

>SEQ ID NO:10: PS63 construct amino acid sequence (VHH72_hl_ElD_S56A-GS-hlgGlhingeEPKSS- hlgGlFc)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSGGGGSGGGGSEPKSSDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYASTYRCVSV

>SEQ ID NO:11: D72-22 construct amino acid sequence (VHH72-hl-S56A-GS-hlgGlhinge-hlgGlFc)

EVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQGTLVTVSSGSEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ.YNSTYRVVSVLTVLHQ.DWLN

GKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWESNGQ.PENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ.KSLSLSPGK

>SEQ ID NO: 12: D72-53 construct amino acid sequence (VHH72_hl_ElD_S56A-(G4S)2-hlgGlhinge- EPKSCdel-hlgGlFc_LALA_Kdel)

DVQLVESGGGLVQPGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGATYYTDSVKGRFTISRDNAK

NTVYLQM NSLRPEDTAVYYCAAAGLGTVVSEWDYDYDYWGQ.GTLVTVSSGGGGSGGGGSDKTHTCPPCPAPEAA

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQ

DWLNGKEYKCKVSNKALPAPIEKTISKAKGQ.PREPQ.VYTLPPSRDELTKNQ.VSLTCLVKGFYPSDIAVEWESNGQ.PE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQ.KSLSLSPG REFERENCES

Edelman et al. (1969). The covalent structure of an entire gammaG immunoglobulin molecule. Proc Natl Acad Sci USA.;63:78-85. doi: 10.1073/pnas.63.1.78.

Jacobsen, et al. (2017). Engineering an IgG Scaffold Lacking Effector Function with Optimized Developability. J Biol Chem. 292 1865-1875.

Ju and Jung, (2014). Aglycosylated full-length IgG antibodies: steps toward next-generation immunotherapeutics. Current Opinion in Biotechnology, 30:128-139.

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Claims

1. A protein comprising an antigen-binding protein and an Fc region, wherein said Fc region is an IgGl Fc region wherein the amino acid at position 297 is an Alanine, and the amino acid residues at positions 292 and 302 are a cysteine, according to EU numbering.

2. The protein of claim 1, wherein said Fc region comprises SEQ. ID NO:1 or a homologue with at least 90 % identity thereof.

3. The protein of claims 1 or 2, which is an antigen-binding protein, or specifically an antibody.

4. The protein of claim 3, wherein the antigen-binding portion comprises a single domain antibody, an immunoglobulin single variable domain (ISVD), a VHH or a Nanobody.

5. The protein of claim 4, wherein said antigen-binding portion is fused to said Fc region.

6. The protein of claim 5, wherein said antigen-binding portion specifically binds the SARS-CoV-2 viral spike protein.

7. A nucleic acid molecule encoding the protein of any one of claims 1 to 6.

8. A host cell comprising the protein of claims 1 to 6, or the nucleic acid molecule of claim 7.

9. The host cell of claim 8, which is a mammalian or a yeast cell, preferably a Pichia pastoris yeast cell.

10. A method to produce a stabilized aglycosylated Fc-region-containing protein, comprising the steps of: a) Expressing the nucleic acid molecule of claim 7 in a host cell, or cultivating the host cell of claims 8 or 9, and b) Isolating said Fc-region-containing protein from the cell culture.

11. A pharmaceutical composition comprising the protein of any one of claims 1 to 6, or the nucleic acid molecule of claim 7.

12. The protein of claims 1 to 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 11, for use as a medicament.

13. The protein of claims 1 to 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 11, for use in treatment of a viral or infectious disease.

14. The protein of claims 1 to 6, the nucleic acid molecule of claim 7, or the pharmaceutical composition of claim 11, for use in treatment of COVID-19.