Patent Application
20250019422
The present invention pertains in the fields of antibody technology, protein engineering, medicine, pharmacology, infection biology, virology, and medical diagnostics. More specifically, the present disclosure provides VHH antibodies that prevent cell entry of and infection by SARS-COV-2 and that have been selected for potent cross-reaction and cross-neutralization between the original Wuhan strain and the Alpha, Beta, Gamma, Delta, and Mu variants of concern.
The COVID-19 pandemics has plunged the world into a nearly unprecedented medical and economic crisis—mainly because only insufficient means for treatment are available. COVID-19 is caused by SARS-COV-2—a recently evolved virus belonging to the Coronaviridae. The virus infects the upper airways initially but can also cause pneumonia and more systemic infections with damages to lungs, kidneys and/or the circulatory system. Approximately 2% of the WHO-documented infections had a lethal outcome so far, whereby the probability of a severe course increases with the age of the individuals and with risk factors such as obesity, diabetes, and chronic lung or kidney diseases.
SARS-COV-2 enters cells with the help of its homotrimeric spike protein, which docks to the ACE2 receptor of the host and subsequently mediates fusion between the viral and host cell membranes to allow entry of the viral genomic RNA into the now infected host cell. The host cell is then re-programmed to produce viral proteins, to replicate the viral RNA, and package replicated RNA into the next generation of viral particles, which are then shed in large numbers off the host cell to infect further cells.
The adaptive immune system can control viral infections by a T-cell mediated cytotoxic response that eliminates infected cells and/or by B-cells producing antibodies that bind to the virus's surface and block infection (for a textbook view see: Murphy and Weaver, 2016). Such infection-blocking antibodies are referred to as neutralizing ones. Typically, however, only a small fraction of produced anti-viral antibodies also has neutralizing properties.
Immunoglobulin G (IgG) is not only the most abundant class of antibodies but also the most important one in terms of long-term immunity. IgGs are built from four polypeptide chains, two light and two heavy ones. They contain two antigen-binding sites formed each by one heavy and one light chain. Such a composite binding site can confer a virus-neutralizing activity. In addition, IgGs comprise an Fc fragment, formed only by the two heavy chains. The Fc fragment is a prototypic immune effector domain that can interact with cell surface receptors (called Fc receptors) as well as with components of the complement system.
In some cases, however, antibodies might even help viral entry by an effect called ADE (for antibody-dependent enhancement) that is mediated by Fc-receptor interactions (Kliks et al., 1989; Littaua et al., 1990). Likewise, the immune system might aggravate the condition of patients with COVID-19 by a systemic over-reaction that manifests itself as a cytokine storm (Chen et al., 2020; Mehta et al., 2020) or an inappropriate complement activation (Magro et al., 2020).
An effective immune response will either prevent a virus from establishing an infection in the first place or reduce, after an initial infection, the virus-load to non-pathogenic levels. An insufficiently controlled SARS-COV-2 infection, however, might have a lethal outcome. In survivors, it often causes permanent damage (e.g., lung dysfunction) and impairs quality of life. An efficient anti-viral therapy would be highly desirable, ideally with orthogonal lines of treatment that target distinct steps of the viral infection cycle independently.
Remdesivir represents so far one line of treatment. This nucleoside analogue inhibits the SARS-COV-2 RNA polymerase. As of May 2020, clinical studies gave mixed outcomes: from no decrease in mortality to a modest reduction in hospitalization time (Wang et al., 2020b).
Another treatment option for severely infected patients is passive immunization with polyclonal antibodies/blood plasma from individuals who have already recovered from COVD-19 (Duan et al., 2020). This approach is considered as a last resort and generally has several drawbacks, such as the risk of ADE in case of an insufficient neutralization, limited availability of sera from convalescent individuals, inevitable batch-to-batch variability of plasma samples, the risk of transmitting other infectious diseases, and all other potential adverse effects when large amounts of antibodies of mixed specificity are transferred from one person to another.
A more advanced approach is the isolation of monoclonal antibodies from SARS-COV-2 infected or convalescent patients (Wang et al., 2020a). These can be characterized in-depth and produced reproducibly in a recombinant form. This solves some of the issues raised for the plasma-therapy mentioned above. However, monoclonal antibodies are very costly to produce, making it hard to imagine that it will be feasible to scale up production to levels needed for the treatment of large numbers of patients. In addition, immunological side effects, such as aggravating cytokine storms, inappropriate complement activation, or ADE, cannot be ruled out either, mainly due to the Fc region present in human/humanized antibodies.
By now, several SARS-COV-2 variant strains with mutated RBDs, i.e., escape mutants, have emerged that are causing devastating outbreaks. The emergence of such escape mutants is presumably driven by the selective pressure of the immune system, particularly in immunocompromised, persistently infected patients (Clark et al., 2021; Starr et al., 2021). Escape mutants can bypass antibody-based immunity in previously infected or vaccinated individuals. This has the potential of sustaining infection waves in populations that have acquired herd immunity against earlier strains.
Escape mutants also pose a tremendous challenge for therapeutic antibodies, which may be rendered ineffective by a given mutation. Indeed, several monoclonal anti-SARS-COV-2 antibodies developed for treating COVID-19 are ineffective against the South African and Brazilian variants (Garcia-Beltran et al., 2021; Hoffmann et al., 2021; Li et al., 2021; Tada et al., 2021; Wibmer et al., 2021; Zhou et al., 2021).
Camelids are unusual in that they produce heavy chain-only antibodies, whose simpler antigen-binding domain can easily be cloned and recombinantly expressed in bacteria or yeast (Arbabi Ghahroudi et al., 1997). Such isolated antigen-binding domains are then referred to as nanobodies or VHH antibodies (variable domain of heavy homodimer antibodies). They are just ˜14kDa in size—about one—tenth of a traditional IgG molecule. They have found numerous useful applications, for example as crystallization chaperones (Rasmussen et al., 2011; Chug et al., 2015), as tools in affinity chromatography, for localizing proteins in cells or as an animal-friendly substitute for secondary antibodies (Pleiner et al., 2015, 2018).
VHH antibodies recognizing the SARS-COV-2 spike protein S1 domain are described in co-owned application PCT/EP2021/071309 the content of which is herein incorporated by reference.
It was an object of the present invention to provide VHH antibodies showing improved binding and neutralization characteristics with SARS-COV-2 variant strains including the Delta variant.
According to the present invention, SARS-COV-2-neutralizing VHH antibodies are provided, which are suitable for the effective treatment of COVID-19 patients. Specifically, the invention provides VHH antibodies that neutralize virus already at a very low concentration. Further, the invention provides stable VHH antibodies. In particular embodiments, the VHH antibodies are monomeric VHH antibodies. In further embodiments, the VHH antibodies are multimeric VHH antibodies, particularly homotrimeric VHH antibodies or heterodimeric VHH antibodies.
A first aspect of the invention relates to an VHH antibody recognizing the SARS-COV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-COV-2 S1 domain.
The VHH antibody is selected from the group of VHH antibodies comprising a CDR3 sequence as disclosed herein or a related CDR3 sequence. In certain embodiments, the VHH antibody is selected from the group of VHH antibodies comprising a combination of CDR1, CDR2 and CDR3 sequences as disclosed herein or a related combination of CDR1, CDR2 and CDR3 sequences. In certain embodiments, the VHH antibody is selected from the group of antibodies comprising a VHH sequence as disclosed herein.
In certain embodiments, the SARS-COV-2-neutralizing VHH antibody is a stable, particularly a thermostable or a hyperthermostable VHH antibody.
In certain embodiments, the VHH antibody as described above is covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a labeling group, a capture group or an effector group.
In certain embodiments, the VHH antibody as described above is fused to a heterologous polypeptide moiety, which may be selected from a multimerization module, e.g., dimerization, trimerization or tetramerization module. In particular embodiments, the multimerization module is trimerization module.
In certain embodiments, the VHH antibody is conjugated to one or several polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG), to increase the molecular weight of the antibody conjugate and, thus, delay renal clearance. The molecular weight of a polymer moiety may vary over a broad range, for example in the range of about 5 kDa to about 80 kDa. Such coupling may be performed through e.g., amino or carboxyl groups already present in the VHHs and/or through the side chains of engineered lysine, aspartic acid, glutamic acid or cysteine residues and involve known chemistries for forming amide bonds, secondary amine bonds or thioether bonds.
In certain embodiments, the VHH antibody as described above is directed against the SARS-COV-2 spike protein receptor-binding domain (RBD). In certain embodiments, the VHH antibody binds to the RBD of the SARS-COV-2 Wuhan strain and to a tetramutant RBD of a SARS-COV-2 having the immune-escape mutations K417T, L452R, E484K and N501Y.
The numbering of the indicated positions in the RBD in which mutations are present refers in each case to the spike protein of the original Wuhan strain (SEQ ID NO. 118).
In certain embodiments, the VHH antibody is capable of virus neutralization. In certain embodiments, the SARS-COV-2-neutralizing VHH antibody neutralizes a SARS-COV2 variant, e.g., a variant comprising at least one mutation in the RBD, particularly at position K417, L452, T478, E484 and/or Y501, more particularly at least one mutation from the group consisting of K417T, K417N, L452R, E484K, N501Y and T478K. In certain embodiments, the VHH antibody cross-neutralizes the SARS-COV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, the Mu variant, and the Delta variant.
A further aspect of the present invention relates to a set of two or more different VHH antibodies as described above, wherein the different VHH antibodies may recognize different epitopes, particularly different epitopes on the RBD, and more particularly non-overlapping epitopes on the RBD.
A VHH antibody described above is suitable for use in medicine, e.g., human medicine, particularly for use in therapy, e.g., in the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-COV-2, or in diagnostics, e.g., for detecting SARS-COV-2 virus or viral components in a patient sample, e.g., in a body fluid or tissue sample.
Still a further aspect of the invention relates to a nucleic acid molecule encoding a VHH antibody as described above, particularly in operative linkage with a heterologous expression control sequence, a vector comprising said nucleic acid molecule or a recombinant cell or non-human organism transformed or transfected with said nucleic acid molecule or said vector.
Still a further aspect of the invention relates to a method for recombinant production of a VHH antibody as described above, comprising cultivating a cell or an organism as described above in a suitable medium and obtaining the VHH antibody from the cell or organism or from the medium.
Still a further aspect of the invention relates to a method for the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-COV-2 including a SARS-COV2 mutant, comprising administering an effective dose of the VHH antibody as described above or the set of at least two different VHH antibodies as described above to a subject in need thereof, particularly to a human subject infected with SARS-COV-2 or at risk from being infected with SARS-COV-2.
A list of VHH antibodies of the present invention and their utility for virus neutralization is shown in the following Table 1:
The variable regions (CDRs) of each VHH antibody of Table 1 as well as assignment of SEQ ID NOs for VHHs and CDRs are listed in the following Table 2:
A list of complete sequences of VHH antibodies is shown at the end of the specification (see also
There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy chain variable regions can be made according to any method known in the art, including, but not limited to, the methods known as KABAT, Chothia, and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example. According to some embodiments of the present invention, the CDR sequences of the mAb variable regions are determined using the KABAT methods. CDRs may also be defined through a multiple alignment (with many other VHH antibodies), to identify the hot-spots of variability and relate them to a standard VHH antibody structure. It is also possible to define CDRs by analysing the structure of the VHH antibody and deciding what is a loop and what is the antibody's scaffold. In some cases, CDR-adjacent residues are also variable, and are therefore included in the CDR definition.
In the following, specific embodiments of the invention are disclosed as follows:
The primary aim of this aspect of the invention has been to generate, select, engineer and optimize SARS-COV-2-neutralising VHH antibodies for effective treatment of COVID-19 patients. Specifically, we aimed for VHH antibodies that are capable of virus neutralization of SARS-Cov-2 variants, particularly SARS-COV-2 variants of concerns and of cross-neutralization of the original SARS-COV-2 Wuhan strain and SARS-COV-2 variants already at a very low concentration. Further, we aimed for VHH antibodies that have sufficient stability for pharmaceutical applications.
According to the present invention, VHH antibodies are provided which cross-neutralize the SARS-COV-2 Wuhan strain and variants of concern at very low concentrations.
In previous work, e.g., as described in PCT/EP2021/071309, supra, VHH antibodies from three alpacas immunized with SARS-COV-2 S1 and RBD were obtained including neutralizing VHH antibodies directed against two RBD epitopes, namely epitope 1 (the main epitope defined by VHH antibody Re5D06 shown SEQ ID NO: 1) and epitope 2 (the epitope defined by the VHH antibody Re9F06 shown in SEQ ID NO: 115). Epitope 1 has a very large overlap with ACE2, epitope 2 only a small overlap. Epitope 1-binders such as Re5D06 were found to be more potent in neutralization (neutralizing down to concentrations of 50 pM) than epitope 2 binders (so far not better than 1.7 nM). However, epitope 1 was found to be mutated in all variants of concern, namely in Alpha (N501Y), Beta (N501Y, E484K, K417N), Gamma (N501Y, E484K, K417T), Delta (L452R, T478K), Epsilon (L452R) and Mu (E484K, N501Y). These escape mutations impede binding and neutralization by epitope 1-binding VHH antibodies, rendering them less suitable in therapeutic applications.
As a solution to the problem, the inventors repeatedly re-immunized the same three alpacas with wildtype and mutant spike proteins, allowing further affinity maturation of antibodies and their adaptation to RBD mutations. Immune libraries were prepared, and phage display performed, cross-selecting with alternating RBD versions as a bait, namely with a “wildtype” RBD (comprised in SEQ. ID NO: 113) corresponding to the original Wuhan strain and a “tetramutant” RBD (comprised in SEQ. ID NO: 114) combining the four key immune escape mutations K417T, L452R, E484K, and N501Y) found in the Alpha, Beta, Gamma, Delta, Epsilon and Mu SARS-COV-2 strains. The phage display also comprised a selection against non-neutralizing VHH-antibodies, using an RBD-ACE2 fusion as a competitor that blocks VHHs that do not compete with ACE2 for RBD-binding.
This combined strategy yielded new VHH antibodies (Tables 1 and 2). These include VHH variants of previously isolated classes (e.g., belonging to the Re9B09 (SEQ: ID NO: 13) class), mostly however, new VHH classes (notably the Re32D03, and the Re30H01 classes).
The present invention relates to a VHH antibody, which is a monovalent heavy chain-only antibody comprising a CDR1 domain, a CDR2 domain and a CDR3 domain linked by framework regions including, but not being limited to, whole VHH antibodies, e.g. native VHH antibodies comprising framework regions derived from camelids, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, e.g. a fusion protein with an immunoglobulin or non-immunoglobulin peptide or polypeptide, as long as it shows the properties according to the invention.
The present invention is also directed to a covalent or non-covalent conjugate of a VHH antibody molecule to a non-proteinaceous structure, for example, a labeling group, a capture group such a solid phase-binding group, or an effector group such as a toxin. For example, the heterologous moiety may be from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.
The VHH antibody of the present invention is particularly a monoclonal VHH antibody characterized by a specific amino acid sequence. The VHH antibody may be produced in a prokaryotic host cell, a yeast cell or a mammalian cell. In certain embodiments, the VHH antibody is non-glycosylated. In certain embodiments, the VHH antibody is glycosylated, wherein a carbohydrate structure may be derived from a glycosylation site introduced into the VHH sequence and/or from a fusion partner.
A VHH antibody according to the present invention is characterized by (i) a CDR3 sequence, (ii) a combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (iii) by a complete VHH sequence, or (iv) by competition with a specific reference antibody. Specific CDR and VHH sequences are provided in the Tables, Figures and the Sequence Listing. According to the present invention, sequences related to the above sequences are encompassed. These related sequences are defined by having a minimum identity to a specifically indicated amino acid sequence, e.g., a CDR or VHH sequence. This identity is indicated over the whole length of the respective reference sequence and may be determined by using well known algorithms such as BLAST.
In particular embodiments, a related CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated CDR3 sequence, e.g., a substitution of 1, 2, or 3 amino acids.
In particular embodiments, a related combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, e.g., a substitution of 1, 2, 3, 4, 5 or 6 amino acids by different amino acids.
In particular embodiments, a related VHH sequence has an identity of least 70%, at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence, e.g., a substitution of 1, 2, 3, 4, 5 or up to 20 amino acids.
Further, the invention refers to a VHH antibody, which competes with a specific VHH antibody disclosed herein for the binding to the SARS-COV-2 spike protein S1 domain. In certain embodiments, a competing VHH antibody binds the same or an overlapping epitope on the SARS-COV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-COV-2 S1 domain as a specific VHH antibody disclosed herein. For example, the invention refers to a VHH antibody, which competes either with VHH antibodies Re32D03, Re32B11, Re32E06, or Re30D01, or with VHH antibody Ma2H07.
In particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1, CDR2 or CDR3 sequence and/or an amino acid in a framework region, is replaced by another amino acid, while preserving structural integrity and epitope-binding of the VHH antibody. These exchanges can be conservative (i.e., by a similar amino acid) or non-conservative.
In further particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1, CDR2 or CDR3 sequence and/or an amino acid in a framework region, is replaced by a conservative amino acid substitution, i.e. a substitution of an amino acid by another amino acid with similar biochemical properties, for example a substitution of an aliphatic amino acid, e.g. Gly, Ala, Val, Leu, or lle, for another aliphatic amino acid; a substitution of a basic amino acid, e.g. His, Lys or Arg, against another basic amino acid or against Met; a substitution of an acidic amino acid or an amide thereof, e.g., Asp, Glu, Asn or Gin, against another acidic amino acid or an amide thereof; a substitution of an aromatic amino acid, e.g., Phe, Tyr or Trp, against another aromatic amino acid.
In further particular embodiments, the VHH antibody is selected from antibody Re32D03 comprising a VHH sequence as shown in SEQ. ID NO: 37 or a VHH antibody, which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 37 are replaced by another amino acid.
In further particular embodiments, the VHH antibody is selected from antibody Re32E06 comprising a VHH sequence as shown in SEQ. ID NO: 105 or a VHH antibody, which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 105 are replaced by another amino acid.
In further particular embodiments, the VHH antibody is selected from antibody Re32B11 comprising a VHH sequence as shown in SEQ. ID NO: 45 or a VHH antibody, which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 45 are replaced by another amino acid.
In further particular embodiments, the VHH antibody is selected from antibody Re30D01 comprising a VHH sequence as shown in SEQ. ID NO: 89 or a VHH antibody, which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 89 are replaced by another amino acid.
In further particular embodiments, the VHH antibody is selected from antibody Ma2H07 comprising a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 25 are replaced by another amino acid.
Further, the present invention relates to a nucleic acid molecule, e.g., a DNA molecule, encoding a VHH as indicated above, or a vector, comprising said nucleic acid molecule as indicated above in operative linkage with an expression control sequence, particularly with a heterologous expression control sequence. Furthermore, the invention relates to a cell comprising a nucleic acid molecule or a vector as described above. Vectors for the recombinant production of VHH antibodies are well known in the art. In certain embodiments, the vector is an extrachromosomal vector. In other embodiments, the vector is a vector for genomic integration. The cell may be a known host cell for producing antibodies or antibody fragments, e.g., a prokaryotic cell such as an E. coli or a Bacillus sp. cell, a yeast cell, particularly a Pichia yeast cell, an insect cell or a mammalian cell, e.g., a CHO cell, or a plant cell. In certain embodiments, the cell comprises the nucleic acid or the vector extrachromosomally. In other embodiments, the cell comprises the nucleic acid or the vector integrated into the genome, e.g., as a genomically integrated expression cassette.
Still a further aspect of the present invention is a method of recombinantly producing a VHH antibody by growing a cell as described above in a culture medium and obtaining the VHH antibody from the cell or the culture medium. Suitable culture media and culture conditions are well known in the art.
The VHH antibodies of the present invention bind to the S1 domain, particularly to the RBD of the S1 domain of SARS-COV-2. The inventors have identified VHH antibodies which bind with high affinity to the RBD of the SARS-COV-2 Wuhan strain and to a tetramutant RBD of a SARS-COV-2 having the mutations K417T, L452R, E484K and N501Y as shown in Table 1, supra. Some of the identified VHH antibodies, in particular Re32D03 and Re9B09 or variants thereof, additionally bind to the RBD of the SARS-CoV-2Omicron variant BA.2.75 (
In certain embodiments, the VHH antibody of the present invention binds to the RBD of the SARS-COV-2 Wuhan strain (comprised in SEQ. ID NO: 113) and to a tetramutant RBD of a SARS-COV-2 having the mutations K417T, L452R, E484K and N501Y (comprised in SEQ. ID NO: 114) with an affinity expressed as dissociation constant KD of about 1000 pM or less, of about 500 pM or less, of about 100 pM or less or of about 50 pM or less. Some of the identified VHH antibodies, in particular Re32D03 or variants thereof, additionally bind to the RBD of the SARS-COV-2Omicron variant BA.2.75 with an affinity constant KD of about 1000 pM or less, or of about 500 pM or less.
The binding affinity may be determined as described herein in detail below.
The VHH antibodies of the present invention are capable of neutralizing SARS-COV-2, e.g., the SARS-COV-2 Wuhan strain, or a SARS-COV-2 variant. The inventors have identified VHH antibodies which cross-neutralize the SARS-COV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, and the Delta variant, as shown in Table 1, supra.
In certain embodiments, the VHH antibody of the present invention neutralizes SARS-CoV-2, e.g., the SARS-COV-2 Wuhan strain, or a SARS-COV-2 variant at a concentration of about 5 nM or less, of about 1000 pM or less, of about 500 pM or less of about 170 pM or less, or of about 100 pM or less. The neutralization potency may be determined as described herein in detail below.
In particular embodiments, the VHH antibodies are capable of neutralizing a SARS-CoV2 variant comprising a spike protein RBD including at least one mutation, particularly at position K417, L452, T478, E484 and/or Y501, more particularly at least one mutation selected from the group consisting of K417T, K417N, L452R, E484K, N501Y and T478K. In even more particular embodiments, the VHH antibodies are capable of neutralizing a SARS-COV-2 variant of concern including Alpha (N501Y), Beta (N501Y, E484K, K417N), Gamma (N501Y, E484K, K417T), Delta (L452R, T478K), Epsilon (L452R) and/or Mu (E484K, N501Y).
In further particular embodiments, the VHH antibodies are capable of cross-neutralizing the SARS-COV-2 Wuhan strain, and at least one of the Alpha variant, the Beta variant, the Gamma variant, and the Delta variant. In further particular embodiments, the VHH antibodies are capable of cross-neutralizing the SARS-COV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, and the Delta variant. In even more particular embodiments, the VHH antibodies are capable of cross-neutralizing the SARS-COV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, and the Delta variant at a concentration of about 5 nM or less, of about 1000 pM or less, of about 170 pM or less, or of about 100 pM or less. The neutralization potency may be determined as described herein in detail below.
In particular embodiments, the VHH antibodies neutralize the Delta variant of SARS-CoV-2 a concentration of about 5 nM or less, of about 1000 pM or less, of about 170 pM or less, or of about 100 pM or less, or of about 50 pM or less.
Some of the identified VHH antibodies, e.g. Re32D03 or variants thereof, additionally neutralize SARS-COV-2 Omicron variants, in particular BA.2.75.
For the intended therapeutic application, the anti-SARS-COV-2 VHH antibodies should not only be highly potent in virus neutralization, but also, they should be developable as biological drugs. This includes that they are stable enough to survive a lengthy, large-scale production process as well as transportation and storage (ideally for years in liquid formulation) without aggregation or loss of activity.
A good predictor for stability is thermostability, which can be measured, e.g., by thermal shift assays or specifically by differential scanning fluorimetry. The present inventors have identified several thermostable or hyperthermostable VHH antibodies as shown in Table 1, supra.
In a particular embodiment, the invention relates to a VHH antibody, which is stable, particularly thermostable or hyperthermostable. Preferably, the VHH antibody has a melting point (melting temperature) of at least about 65° C., of at least about 80° C., of at least 90° C. or of at least about 95° C., and/or an aggregation temperature of at least about 50° C., of at least about 60° C., of at least 70° C. or of at least about 80° C. when measured under non-reducing conditions. Melting and aggregation temperatures are determined as described herein.
The hyperthermostable VHH antibodies includes some extremely strong RBD-binders and very potent neutralizers, such as Re32D03 and Re32B11 (neutralization of the Delta variant at 50 or 170 pM).
In a further aspect, the present invention relates to a set comprising at least 2, 3, 4 or more of the above VHH antibodies. In such a set, the individual VHH antibodies are present in suitable molar ratios. Typically, the molar ratios are in the range of about 2:1 to about 1:2, particularly about 1.5:1 to about 1:1.5, even more particularly about 1:1. In certain embodiments, the VHH antibody set may comprise a single composition wherein the VHH antibodies in said set consist of a predetermined number of different species of VHH antibodies as described above. The VHH antibody set may comprise a plurality of compositions each comprising a different species of VHH antibody as described above. The set of the present invention may be free from other VHH antibodies.
In certain embodiments, the VHH antibody is a heterodimeric VHH antibody, particularly a covalently linked VHH heterodimer comprising a first VHH antibody and a second VHH antibody wherein the first VHH antibody and the second VHH antibody bind to different epitopes on the RBD of the SARS-COV-2 S1 domain.
A heterodimeric VHH antibody comprises two different VHH antibodies optionally connected by linker. In particular embodiments, at least one of the VHH antibodies is a VHH antibody of the present invention as described herein. A VHH antibody of a heterodimeric VHH antibody is characterized by (i) a CDR3 sequence, (ii) a combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (iii) by a complete VHH sequence, or (iv) by competition with a specific reference antibody.
In certain embodiments, the first and the second VHH antibody do not compete with each. Competition can be measured either as an at least 90% loss of fluorescence staining of the SARS-COV-2 spike protein by a fluorophore-labelled candidate VHH antibody in the presence of a 100-fold molar excess of an unlabelled competitor VHH antibody, e.g., selected from Re32D03, Re32B11, Re32E06, Re30D01, or Ma2H07, or conversely as an at least 90% loss of spike protein-staining by a fluorophore-labelled VHH antibody in the presence of a 100-fold molar excess of the unlabelled candidate VHH antibody as a competitor.
Preferably, competition can be measured by label-free biolayer interferometry-performed as a cross-competition or epitope binning assay using a label-free detection system, e.g., an Octet® system from Sartorius, according to the manufacturer's instructions.
The first and the second VHH antibody may be directly fused to each other or fused through a linker sequence. In particular embodiments, the first and the second VHH antibody are genetically fused by a peptidic linker sequence.
In certain embodiments, the linker is a peptidic linker, particularly having a length of 5 to 50 amino acid residues, particularly of 10 to 40 amino acid residues and more particularly of 12 to 30 amino acid residues, particularly as described in PCT/EP2021/071309 supra.
In certain embodiments, the VHH antibody is a homotrimeric antibody.
Homotrimerization of a VHH antibody requires an appropriate polypeptide fusion partner as trimerization module. According to the present invention, a fusion partner is preferred, comprising at least one of the following features:
Preferred trimerization modules include domains derived from collagen, the most abundant class of proteins of the human body, including the trimerization (NC1) domains of collagen XV (PDB 3N3F, 54 residues) (Wirz et al., 2011), collagen XVIII (PDB 3HSH; 54 residues) (Boudko et al., 2009) and collagen X (PDB 1GR3; 160 amino acids) (Bogin et al., 2002). Another very appropriate homotrimeric fusion partner is the lung surfactant protein D (LSFPD) that recognizes sugars on viral and bacterial pathogens (see e.g., PDB 1B08, 3IKN, 3IKQ, 3IKR). As the SARS-COV-2 spike protein is heavily glycosylated, a VHH-LSPD fusion could confer a bimodal inhibition of spike function.
According to the present invention, it is preferred that the VHH antibody is linked to the trimerization domain via a spacer having a length of at least about 5 amino acids, particularly about 5 amino acids to about 100 amino acids, and more particularly of about 10 to about 50 amino acids, particularly as described in PCT/EP2021/071309 supra.
It should be note that different arrangements for the subunits of homotrimeric VHH antibodies are possible. As just described, the trimerization module might be located N-terminally, followed by the spacer and the VHH antibody. Alternatively, the VHH antibody might be located N-terminally, followed by spacer and trimerization module. The best arrangement will depend on the geometry of the target to be neutralized.
In particular embodiments, the present invention relates to a neutralizing homotrimeric VHH antibody, e.g., a VHH antibody which has a lowest SARS-COV-2 neutralizing concentration of about 100 pM or less, of about 25 pM or less, of about 17 pM or less, of about 10 pM or less, of about 5 pM or less or of about 1.7 pM or less.
VHH antibodies including monomeric, heterodimeric and homomultimeric, particularly homotrimeric VHH antibodies may be produced as described in PCT/EP2021/071309 supra.
A VHH antibody may be recombinantly produced in a suitable host cell, e.g., in a prokaryotic or eukaryotic host cell or host organism. For this purpose, a nucleic acid molecule encoding the VHH antibody is introduced into the host cell or host organism and expressed in the host cell or host organism. The nucleic acid molecule may encode a monomeric VHH antibody or a subunit of a homomultimeric VHH antibody, particularly of a homotrimeric VHH antibody.
Any therapeutic application of neutralizing anti-SARS-COV-2 VHH antibodies requires their production at a large scale. In this aspect, production in bacteria or yeast, e.g., Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, may be preferred. Production in yeast, e.g., Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, is particularly preferred because it ensures a quantitative formation of disulfide bonds, allows for excellent yields (in the range of grams per liter culture), while the secretion into the medium allows the subsequent purification to begin with only few contaminating proteins.
In a particular embodiment, the VHH antibody is recombinantly produced in a bacterium, e.g., E. coli or Bacillus. For example, expression in a bacterium may involve cytoplasmic and/or periplasmic expression and purification of the VHH antibody from the host cell, or secretory expression and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression to the periplasm and/or into the culture medium.
In a further particular embodiment, the VHH antibody is recombinantly produced in a eukaryotic host cell or host organism, preferably in yeast, e.g., in Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha. For example, expression in a eukaryotic host cell or host organism, e.g., yeast may involve cytoplasmic and/or periplasmic expression and purification of the VHH antibody from the host cell, or preferably secretion from the host cell and purification of the VHH antibody from the culture medium. In certain embodiments, the nucleic acid sequence encoding the VHH antibody is fused to at least one sequence directing the expression into the culture medium.
The VHH antibody encoding nucleic acid molecule may comprise a sequence encoding a monomeric VHH antibody, a sequence encoding a heterodimeric VHH antibody or a sequence encoding a subunit of a multimeric VHH antibody, particularly of a trimeric VHH antibody. The sequence encoding a heterodimeric VHH antibody may comprise two sequences each encoding a monomeric VHH antibody arranged in tandem fused to each other optionally through a linker. The subunit sequence may comprise (from N-terminus to C-terminus) the VHH antibody sequence, optionally a spacer, and a multimerization domain, particularly a trimerization domain, e.g., the Collagen XVIII NC1 domain. Alternatively, the subunit sequence may comprise (from N-terminus to C-terminus) a multimerization domain, particularly a trimerization domain, optionally a spacer and the VHH antibody sequence.
Still a further aspect of the present invention is the use of an active agent as described above selected from a monomeric VHH antibody, a heterodimeric VHH antibody, a set of at least 2 different VHH antibodies, or a multimeric, particularly a trimeric VHH antibody in medicine, particularly for therapeutic and/or in vitro or vivo diagnostic use. In certain embodiments, the monomeric VHH antibody, the heterodimeric VHH antibody, the set of VHH antibodies or the multimeric, particularly trimeric VHH antibody is used in human medicine.
In particular embodiments, a therapeutic VHH antibody is selected from (i) a VHH antibody comprising the CDR3 sequence of the hyperthermostable VHH antibody Re32D03 or a related sequence, (ii) a VHH antibody comprising the CDR1, CDR2 and CDR3 sequences of the hyperthermostable VHH antibody Re32D03 or related sequences, or (iii) a VHH antibody comprising the VHH sequence of the hyperthermostable VHH antibody Re32D03 or a related sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the VHH sequence of the reference antibody are replaced by another amino acid. Specific variants of the VHH antibody Re32D03 are described above as embodiments 3.1-3.3.
In particular embodiments, a therapeutic VHH antibody is selected from (i) a VHH antibody comprising the CDR3 sequence of the hyperthermostable VHH antibody Re32E06 or a related sequence, (ii) a VHH antibody comprising the CDR1, CDR2 and CDR3 sequences of the hyperthermostable VHH antibody Re32E06 or related sequences, or (iii) a VHH antibody comprising the VHH sequence of the hyperthermostable VHH antibody Re32E06 or a related sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the VHH sequence of the reference antibody are replaced by another amino acid.
In particular embodiments, a therapeutic VHH antibody is selected from (i) a VHH antibody comprising the CDR3 sequence of the VHH antibody Re30D01 or a related sequence, (ii) a VHH antibody comprising the CDR1, CDR2 and CDR3 sequences of the VHH antibody Re30D01 or related sequences, or (iii) a VHH antibody comprising the VHH sequence of the VHH antibody ReRe30D01 or a related sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the VHH sequence of the reference antibody are replaced by another amino acid.
In particular embodiments, a therapeutic VHH antibody is selected from (i) a VHH antibody comprising the CDR3 sequence of the hyperthermostable VHH antibody Re32B11 or a related sequence, (ii) a VHH antibody comprising the CDR1, CDR2 and CDR3 sequences of the hyperthermostable VHH antibody Re32B11 or related sequences, or (iii) a VHH antibody comprising the VHH sequence of the hyperthermostable VHH antibody Re32B11 or a related sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the VHH sequence of the reference antibody are replaced by another amino acid.
In particular embodiments, a therapeutic VHH antibody is selected from (i) a VHH antibody comprising the CDR3 sequence of the VHH antibody Ma2H07 or a related sequence, (ii) a VHH antibody comprising the CDR1, CDR2 and CDR3 sequences of the thermostable VHH antibody Ma2H07 or related sequences, or (iii) a VHH antibody comprising the VHH sequence of the thermostable VHH antibody Ma2H07 or a related sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the VHH sequence of the reference antibody are replaced by another amino acid.
Therapeutic applications of an active agent as described above, e.g., a monomeric VHH antibody, a heterodimeric VHH antibody, a set of at least 2 different VHH antibodies, or a multimeric, particularly trimeric VHH antibody include methods for preventing or treating a disorder caused by and/or associated with a Coronavirus infection, particularly a disorder caused by and/or associated with a SARS-COV-2 infection and more particularly of preventing or treating COVID-19. The therapeutic applications also include methods for preventing or treating a disorder caused by and/or associated with an infection by a SARS-COV-2 variant including the Alpha variant, the Beta variant, the Gamma variant, the Delta variant and the Epsilon variant as well as variants comprising at least one of the amino acid substitutions in the RBD domain in any one of the above variants.
In therapeutic applications, the active agent is administered in an effective amount to a subject in need thereof, particularly to a human subject. The dose will depend on the specific type of agent, e.g. monovalent VHH antibody or multimeric VHH antibody, and the type of disease.
In the prevention or treatment of COVID-19, a daily dose of a monomeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally, or systemically, particularly from about 5 μg to about 5000 mg per day.
In the prevention or treatment of COVID-19, a daily dose of a heterodimeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally, or systemically, particularly from about 5 μg to about 5000 mg per day.
In the prevention or the treatment of COVID-19, a daily dose of a trimeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally or systemically, particularly from about 1 μg to about 2500 mg per day.
In the prevention or treatment of COVID-19, a unit dose of a monomeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally, or systemically, particularly from about 5 μg to about 5000 mg per unit.
In the prevention or treatment of COVID-19, a unit dose of a heterodimeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally, or systemically, particularly from about 5 μg to about 5000 mg per unit.
In the prevention or the treatment of COVID-19, a unit dose of a trimeric VHH antibody for a human subject is any suitable dose based on the severity of the disease, the specific type of agent, and the mode of administration, e.g., locally, or systemically, particularly from about 1 μg to about 2500 mg per unit.
Typically, the VHH antibody is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art.
Depending on the stage and the severity of the disorder, the pharmaceutical composition may be administered once or several times during the course of the disorder. For example, it may be administered once or several times daily, each second day, two times weekly or weekly for a suitable period of time.
In certain embodiments, the pharmaceutical composition is administered parenterally, e.g., by subcutaneous, intramuscular or intravenous injection or by infusion. In further embodiments, the pharmaceutical composition may be administered locally, e.g., orally, nasally or intrapulmonary, for example by inhalation as an aerosol.
In certain embodiments, the pharmaceutical composition is administered in an early infection stage, e.g., in an infection stage where the upper airways such as the oral cavity, the nasal cavity, the paranasal sinus, the pharynx and/or the throat are infected, but the lower airways such as the bronchi and/or the lung are not infected. In further embodiments, the pharmaceutical composition is administered in a late infection stage wherein the lower airways such as the bronchi and/or the lung are infected. Further, the pharmaceutical composition may be administered in an infection stage where the subject suffers from a respiratory dysfunction and optionally is ventilated. In still further embodiments, the pharmaceutical composition may be administered prophylactically to a subject being at risk of a Coronavirus infection, e.g., a subject, which has previously, for example, within 1, 2, 3, 4, or 5 days or even longer, been in close contact with an infected subject.
The VHH antibody may be administered alone or together with a further active agent, which may be selected from anti-viral agents such as remdesivir, methotrexate, Molnupiravir, or Paxlovid (Wang et al., 2020b; Malone and Campbell, 2021; Stegmann et al., 2021).
A further aspect that needs to be considered is the plasma half-life of a VHH antibody. Proteins smaller than 40 kDa are subject to considerable renal clearance, and an isolated VHH antibody (˜15 kDa) shows, therefore, a plasma half-life of only 4 hours (Hoefman et al., 2015). Thus, the administration might comprise repeated daily injections or continuous perfusion during anti-viral therapy that might last for days to weeks. One solution to this problem is to increase the size of the protein. Fusion to spacer and trimerization module increases the size to >70 kDa, and such fusion should then have a far longer plasma half-life that is no longer limited by renal clearance. One can, however, also imagine alternative application routes. In fact, inhalation of an anti-SARS-COV-2 VHH antibody formulation would bring the therapeutic agent directly to the site of the highest and most problematic virus load, namely the airway epithelia.
The here outlined antiviral clinical strategy will become more robust if not just one but an entire set of several VHH antibodies is deployed. First, one should expect synergistic effects for neutralizing antibodies that bind to non-overlapping sites. Second, patients might develop anti-idiotypic antibodies against a given therapeutic VHH antibody and render it ineffective in treatment. A switch to another VHH would solve the problem. Third, and perhaps most importantly there is the clear risk that SARS-COV2 evolves further, that escape mutations in its RBD might destroy the epitope of a given VHH antibody and render such VHH inefficient in therapy. This risk can be minimized by utilizing a panel of neutralizing antibodies that recognize non-identical and possibly even non-overlapping epitopes, or by at least switching to another VHH that still neutralizes a given escape mutant.
Diagnostic applications include in vitro methods wherein a VHH antibody is used for detecting Coronavirus spike protein in a sample, e.g., in a body fluid such as saliva, blood, serum or plasma, stool samples or in a tissue or biopsy sample. Diagnostic applications further include in vivo methods wherein a VHH antibody is used for detecting Coronavirus spike protein in a subject, particularly in a human patient. For diagnostic applications the VHH antibody carry a label for direct detection or used in combination with secondary detection reagents, e.g., antibodies, including conventional antibodies or VHH antibodies, for indirect detection according to established techniques in the art.
Further, the present invention is explained in more detail by the following Figures and Examples.
An alignment of VHH sequences from Table 2 is shown. Residues that deviate from the consensus are shown in colour. Re5D06, Re5D06R25, Re6H06 and Re9B09 have been reported before (cf. PCT/EP2021/071309) and are shown as references. Sequences are sorted according to classes. The three variable CDR regions are indicated. These comprise predicted CDR loop regions plus flanking variable residues.
Bio-layer interferometry (BLI) experiments were performed for the indicated VHH antibodies with a biotinylated tetramutant (K417T, L452R, E484K and N501Y) RBD. Curves (black) were fitted with a mass transport model. Fitted curves and calculated dissociation constants KDs are shown in red. red). Re6H06 (a previously isolated mutation-tolerant anti-RBD VHH) bound with a KD of 3 nM. Ma2H07 showed with a Kp of 150 pM a far better affinity. Re32D03 and Re32B11 had a low picomolar affinity with a non-detectable dissociation rate.
Vero E6 cells were grown in 96 well plates and infected with the indicated SARS-COV-2 variants. Cells were fixed 48 h later, permeabilized and stained with DAPI (to locate cell nuclei) and a cocktail of fluorescently labelled VHH antibodies that recognize three non-overlapping epitopes on the SARS-COV-2 Spike protein. Analysis was by automated spinning disc fluorescence microscopy. Panels depict maximum intensity projections. Infected cells show bright intracellular signals in the green (anti-RBD) and red (anti-S1ARBD) channels. Where indicated, virus was pre-incubated with indicated concentrations of VHH antibodies.
The indicated VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposes aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Two replicates of each sample were placed onto a Thermal Cycler. The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step.
The indicated VHH antibodies were diluted to a concentration of 1 mg/ml. One set of samples was supplemented with Tween 80. Then, samples were heated for 10minutes to 95° C. and centrifuged. Soluble (Sup) and precipitated fractions (Pellet) were analyzed by SDS-PAGE followed by Coomassie-staining.
Vero E6 cells were grown in 96 well plates and infected with the indicated SARS-COV-2 variants. Cells were fixed 48 h later, permeabilized and stained with DAPI (to locate cell nuclei) and a cocktail of fluorescently labelled VHH antibodies that recognize three non-overlapping epitopes on the SARS-COV-2 Spike protein. Analysis was by automated spinning disc fluorescence microscopy. Panels depict maximum intensity projections. Infected cells show bright intracellular signals in the green (anti-RBD) and red (anti-S1ΔRBD) channels. Where indicated, virus was pre-incubated with indicated concentrations of VHH antibodies.
Samples of the indicated anti-SARS-COV-2 nanobody were nebulized and the impact of nebulization on the activity against the virus (Wuhan strain) was determined.
BLI experiments were performed for the indicated VHH antibodies with biotinylated wild type RBD, Delta RBD, or Omicron BA.2.75 RBD. Curves (black) were fitted with a mass transport model. Fitted curves and calculated dissociation constants (KDs) are shown in red. Where curve fitting failed due to low or no binding signal, sensograms are denoted as “no binding” (n.b.) in red.
VHH antibody sequences (shown in Table 2)
RBD Sequences
Additional VHH sequences
Monomeric VHH antibodies were expressed with an N-terminal DsbA signal sequence and a C-terminal His12 tag from a Kan-ColE1 plasmid harboring a T5/lac promoter in E. coli NEBExpress (New England Biolabs). A 125-ml pre-culture in Terrific Broth (TB) containing 50 μg/ml Kanamycin was grown overnight at 28° C. to early stationary phase. The culture was then diluted with fresh medium (500 ml, pre-warmed to 37° C.). After 30 minutes of growth at 37° C., protein expression was induced with 0.1 mM IPTG and growth was continued for 2 hours at 37° C., whereby the culture reached a final OD600 of ˜8. Bacteria were harvested by centrifugation and lysed by osmotic shock lysis: cell pellets were resuspended in 14ml 130 mM Tris/HCl PH 8.0, 10 mM EDTA, and sucrose was immediately added to 20% (w/v). After gentle mixing at 23° C. for 30 min, four volumes of ice-cold water were added and mixing was continued at 4° C. for 30 min. 20 mM Tris/HCl PH 7.5, 50 mM NaCl and 20 mM imidazole were added to the cell suspension. Periplasmic extract was then recovered as the supernatant of two consecutive centrifugation steps at 4° C.: a low-speed spin at 4000× g (20 min, F13 rotor, Thermo Fisher Scientific) and a high-speed spin at 38000 rpm (˜1 hour, T647.5 rotor). The VHH antibody was purified at 4° C. via Ni2+ EDTA-amide chelate affinity chromatography (1 ml matrix). Beads were washed with ten column volumes of 50mM Tris/HCl PH 7.5, 300 mM NaCl, 20 mM imidazole, 0.2% (w/v) Triton X-100 and ten column volumes of buffer lacking detergent. After elution with 50 mM Tris/HCl PH 7.5, 300 mM NaCl, 500 mM imidazole, the buffer was exchanged to 50 mM Tris/HCl pH 7.5, 150 mM NaCl, 250 mM sucrose via a PD 10 desalting column (GE Healthcare). Aliquots were frozen in liquid nitrogen and stored at −80° C. This expression/purification strategy was used for all VHH antibodies containing four cysteines and thus two disulfide bonds.
VHH antibodies Re9B09, Ma2H07, Ma2F02 und Ma2E06 comprising just two cysteines and thus a single disulfide bond were produced as His14-ScSUMO fusions by cytoplasmic expression in E. coli NEBExpress Shuffle, which allows forming disulfide bonds in the bacterial cytoplasm. In brief, 200ml pre-cultures were grown overnight at 37° C. in TB+50 μg/ml kanamycin in 5-liter flasks to early stationary phase. They were then diluted with 700ml fresh medium, shifted to 21° C. and induced for 5 hours with 0.08 mM IPTG. 5 mM EDTA was added, bacteria were pelleted, resuspended in 50 mM Tris/HCl PH 7.5, 20 mM imidazole/HCl PH 7.5, 300 mM NaCl, frozen in liquid nitrogen and lysed by thawing plus sonication. Insoluble material was removed by ultracentrifugation at 38000 rpm (˜1 hour, T647.5 rotor).
The supernatant was applied to a 2 ml Ni2+ EDTA-amide chelate column. The matrix was sequentially washed in resuspension buffer, resuspension buffer+0.2% TritonX100, resuspension buffer +700 mM NaCl, low salt buffer (resuspension buffer minus NaCl), and protease buffer (resuspension buffer with an imidazole concentration lowered to 10 mM and supplemented with 250 mM sucrose). The VHH antibody was finally eluted by cleaving the His14-SUMO-tag using 100 nM S. cerevisiae Ulp1 for 2 hours at room temperature. The eluted VHH antibodies were frozen in liquid nitrogen and stored an −80° C. until further use.
Bio-layer interferometry (BLI) experiments were performed using High Precision Streptavidin biosensors and an Octet RED96e instrument (ForteBio/Sartorius) at 25° C. with Phosphate-Buffered Saline (PBS) pH 7.4, 0.002% (w/v) Tween 20 and 0.1% (w/v) bovine serum albumin (BSA as assay buffer. For this, RBDs had been expressed with a C-terminal Avi-tag in mammalian cells and biotinylated enzymatically with BirA.
Biotinylated RBDs, namely wildtype RBD and a tetramutant (K417T, L452R, E484K and N501Y) RBD were immobilized on biosensors until a wavelength shift/binding signal of 1 nm was reached. The biosensors were dipped into wells containing 100, 20 or 4 nM VHH for 450 seconds association, and then incubated for one hour with assay buffer for dissociation. Data were reference-subtracted, and curves (black) were fitted with a mass transport model (Octet Data Analysis HT 12.0 software).
The results are shown in Table 1 and
Re6H06 (a previously isolated mutation-tolerant anti-RBD VHH) bound with a KD of 3 nM to the tetramutant RBD. Ma2H07 showed with a KD of 150 pM a far better affinity. Re32D03 and Re32B11 had a low picomolar affinity with a non-detectable dissociation rate.
Virus stocks were prepared as supernatants from Vero E6 cells infected with SARS-CoV-2 (Wuhan strain (WT with D), Alpha variant, Beta variant, Gamma variant, or Delta variant).
The virus stocks contained between 1011 and1012 copies of the virus genome per ml, as determined by reverse transcription and quantitative PCR (qRT-PCR) according to a standard protocol (Corman et al., 2020). Approximately 107 virus copies were diluted in 100 ul of cell culture media, DMEM/2% FBS, in the presence or absence of varying concentrations of the VHH antibodies under study. After 60 min incubation at 37° C., this was added to a monolayer of Vero E6 cells, i.e., 5000 cells in a well of a standard 96 well cell culture plate. Note that the inoculated amount of virus was approximately 50 times higher than needed for a complete infection of a well.
Cells were fixed 48 h later with 4% paraformaldehyde, permeabilized with 0.15% Triton X100, and stained with 2 μg/ml DAPI (to locate cell nuclei) and a cocktail of fluorescently labelled VHH antibodies that recognize three non-overlapping epitopes at the SARS-COV-2 Spike protein (5 nM of Alexa Fluor 488-labeld Re7E02, Re9C07, Re9G12, and Re9H03 and 15 nM Re8H11-Atto 565 (cf. PCT/EP2021/071309) in PBS+1% BSA). Imaging was by spinning disc fluorescence microscopy. Neutralization was considered as successful if no infected cells were detectable within the well.
The results are shown in Table 1 and
VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposes aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Assays were performed in a volume of 20 ul, at 1 mg/ml VHH concentration in 50 mM Tris/HCl, 150 mM NaCl (pH 8.0 at 20° C.) and 1× dye (diluted from a 5000× stock; Life Technologies). Two replicates of each sample were pipetted in a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed onto a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak.
The results are shown in Table 1 and
VHH antibody Re9B09 that was produced in reducing cytosol and hence lacks disulphide bonds melts already at 35° C., while VHH antibodies Re30H02, Re32D03 and Re32B11 showed only a negligible melting amplitude and thus remained stable throughout 95° C.
VHH antibodies were diluted to a concentration of 1 mg/ml in 50 mM Tris/HCl PH 8.0, 150 mM NaCl. One set of samples was supplemented with 0.2% Tween 80 (polysorbate 80). 40ul were heated for 10 minutes to 95° C. Samples were then centrifuged at 21 000 g for 15 minutes, and soluble in precipitated fractions were analyzed by SDS-PAGE followed by Coomassie-staining.
The results are shown in Table 1 and
VHH antibody Re32B11 was fully aggregation resistant. The small extent of heat-induced aggregation of Re30H02 and Re32D03 could be fully suppressed by the addition of Tween 80.
VHH antibody was diluted to a concentration of 30 mg/ml (“Solution A”). The solution was put in a nebulizer, and the aerosol was collected into a fluid again (“Solution I”).
A dilution series of both was prepared, and a virus neutralization assay was performed with the respective solutions. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 21209549.1 | Nov 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/082808 | 11/22/2022 | WO |
