Patent Application
20240287172
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 2, 2022, is named 51656-003001_Sequence_Listing_9_2_22_ST25 and is 213,140 bytes in size.
The present application relates to protein binders against iRhom2.
ADAM metallopeptidase domain 17 (ADAM17) (NCBI reference of human ADAM17: NP_003174), also called TACE (tumor necrosis factor-α-converting enzyme), is an enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases. It is an 824-amino acid polypeptide.
ADAM17 is understood to be involved in the processing of tumor necrosis factor alpha (TNF-α) at the surface of the cell, and from within the intracellular membranes of the trans-Golgi network. This process, which is also known as ‘shedding’, involves the cleavage and release of α soluble ectodomain from membrane-bound pro-proteins (such as pro-TNF-α), and is of known physiological importance. ADAM17 was the first ‘sheddase’ to be identified, and is also understood to play a role in the release of a variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes.
Cloning of the TNF-α gene revealed it to encode a 26 kDa type II transmembrane pro-polypeptide that becomes inserted into the cell membrane during its translocation in the endoplasmic reticulum. At the cell surface, pro-TNF-α is biologically active and is able to induce immune responses via juxtacrine intercellular signaling. However, pro-TNF-α can undergo proteolytic cleavage at its Ala76-Val77 amide bond, which releases α soluble 17 kDa extracellular domain (ectodomain) from the pro-TNF-α molecule. This soluble ectodomain is the cytokine commonly known as TNF-α, which is of pivotal importance in paracrine signaling of this molecule. This proteolytic liberation of soluble TNF-α is catalyzed by ADAM17.
ADAM17 also modulates the MAP kinase signaling pathway by regulating the cleavage of the EGFR ligand amphiregulin in the mammary gland. ADAM17 is important for activating several ligands of the EGFR, TGFα, AREG, EREG, HB-EGF, Epigen. Moreover, ADAM17 has a role in shedding of L-selectin, a cellular adhesion molecule.
Recently, ADAM17 was discovered as a crucial mediator of resistance formation to radiotherapy. It was also shown that radiotherapy activates ADAM17 in non-small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and radiotherapy-induced treatment resistance.
Since ADAM17 seems to be a crucial factor for the release of different pathogenic and non-pathogenic factors, including TNFα, it has come into the focus as therapeutic target molecule. For that reason, different attempts have been made to develop inhibitors of ADAM17.
However, so far, no such inhibitor has proven clinically successful.
It is hence one object of the present invention to provide a new approach which allows the control, regulation, reduction or inhibition of ADAM17 activity.
It is another object of the present invention to provide a new approach that allows the treatment of inflammatory diseases.
These and other objects are solved by the features of the independent claims. The dependent claims disclose embodiments of the invention which may be preferred under particular circumstances. Likewise, the specification discloses further embodiments of the invention which may be preferred under particular circumstances.
The present invention provides, among others, a protein binder that binds to human iRhom2, and inhibits and/or reduces TACE/ADAM17 activity when bound to human iRhom2.
According to one aspect of the invention, a protein binder is provided which, when bound to human iRhom2, binds at least within a region of Loop 1 thereof.
According to another aspect of the invention, a protein binder is provided which binds to the extracellular domain of human iRhom2, which protein binder is an IgG antibody.
Inactive Rhomboid family member 2 (iRhom2) is a protein that in humans is encoded by the RHBDF2 gene. It is a transmembrane protein consisting of about 850 amino acids, having seven transmembrane domains. The inventors of the present invention have for the first time demonstrated that iRhom2 can act as a target for protein binders to inhibit TACE/ADAM17 activity. iRhom2 comes in different isoforms. The experiments made herein have been established with the isoform defined as NCBI reference NP_078875.4. However, the teachings are transferable, without limitation, to other isoforms of iRhom2, as shown in the following table:
Loop 1 of Rhom2 comprises amino acid residues 474-660 of SEQ ID NO 181. See item “B” in
In one embodiment, of the invention, the protein binder binds also to one or more other regions of human iRhom2, like e.g. the juxtamembrane domain (JMD) located N-terminally of Loop 1 (shown as item “A” in
In another embodiment, the protein binder does not bind to the juxtamembrane domain (JMD) located on the N-terminal side of Loop 1.
According to one embodiment of the invention, the protein binder binds within at least a region of human iRhom2 spanning from (and including) W526 to (and including) 1566, according to the numbering set forth in SEQ ID NO 181.
Preferably, the protein binder binds within a region which has at least 3 amino acids in length.
In one or more embodiments. the protein binder binds to ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, ≥12, 13, ≥14, ≥15, ≥16, ≥17, ≥18, ≥19, ≥20, ≥21, ≥22, ≥23, ≥24, or ≥25 amino acids within the above region. The respective amino acid residues can be present in a discrete, consecutive sequence, or in two or more clusters, each of which comprising one or more amino acid residues.
Preferably, the protein binder binds within a region of human iRhom2 spanning from (and including) P533 to (and including) K536, according to the numbering set forth in SEQ ID NO 181.
According to one embodiment of the invention, the protein binder binds a stretch of human iRhom2 comprising at least one residue selected from the group comprising W526; Q527; P532; P533; M534; D535; K536; S537; L539; K542; R543; T544; G546; R554; E557; S561; and/or 1566, according to the numbering set forth in SEQ ID NO 181.
In one or more embodiments, the protein binder binds to ≥2, ≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, or ≥12 amino acid residues from the above list. The respective amino acid residues can be present in a discrete, consecutive sequence, or in two or more clusters, each of which comprising one or more amino acid residues.
Preferably, the protein binder binds a stretch of iRhom2 comprising at least one residue selected from the group comprising P533; M534; D535; K536; and/or L539, according to the numbering set forth in SEQ ID NO 181.
In one or more embodiments. the protein binder binds to ≥2, ≥3 or ≥4 amino acid residues from the above list. The respective amino acid residues can be present in a discrete, consecutive sequence, or in two or more clusters, each of which comprising one or more amino acid residues.
According to one embodiment of the invention, the protein binder inhibits and/or reduces TACE/ADAM17 activity when bound to human iRhom2.
As used herein, the term “inhibits and/or reduces TACE/ADAM17 activity is meant to describe an effect caused by a protein binder that blocks or reduces the activity of TACE/ADAM17, as measured e.g. in a respective shedding assay (see., e.g.,
ADAM metallopeptidase domain 17 (ADAM17), also called TACE (tumor necrosis factor-α-converting enzyme), is an enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases. ADAM17 is understood to be involved in the processing of tumor necrosis factor alpha (TNF-α) at the surface of the cell, and from within the intracellular membranes of the trans-Golgi network. This process, which is also known as ‘shedding’, involves the cleavage and release of α soluble ectodomain from membrane-bound pro-proteins (such as pro-TNF-α), and is of known physiological importance. ADAM17 was the first ‘sheddase’ to be identified, and it is also understood to play a role in the release of a diverse variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands, and enzymes.
Cloning of the TNF-α gene revealed it to encode a 26 kDa type II transmembrane pro-polypeptide that becomes inserted into the cell membrane during its maturation. At the cell surface, pro-TNF-α is biologically active, and is able to induce immune responses via juxtacrine intercellular signaling. However, pro-TNF-α can undergo a proteolytic cleavage at its Ala76-Val77 amide bond, which releases α soluble 17 kDa extracellular domain (ectodomain) from the pro-TNF-α molecule. This soluble ectodomain is the cytokine commonly known as TNF-α, which is of pivotal importance in paracrine signaling. This proteolytic liberation of soluble TNF-α is catalyzed by ADAM17.
Recently, ADAM17 was discovered as a crucial mediator of resistance to radiotherapy. It was also shown that radiotherapy activates ADAM17 in non-small cell lung cancer, which results in shedding of multiple survival factors, growth factor pathway activation, and radiotherapy-induced treatment resistance.
ADAM17 also regulates the MAP kinase signaling pathway by regulating shedding of the EGFR ligand amphiregulin in the mammary gland. ADAM17 also has a role in the shedding of L-selectin, a cellular adhesion molecule.
According to one embodiment of the invention, the inhibition or reduction of TACE/ADAM17 activity is caused by interference of the protein binder with iRhom2-mediated TACE/ADAM17 activation or TACE/ADAM17 interaction with other proteins including substrate molecules.
According to one embodiment of the invention, the protein binder, when bound to human iRhom2, inhibits or reduces induced TNFα shedding.
According to one embodiment of the invention, the protein binder, when bound to human iRhom2, inhibits or reduces induced IL-6R shedding.
According to one embodiment of the invention, the protein binder, when bound to human iRhom2, inhibits or reduces induced HB-EGF shedding.
Tumor necrosis factor alpha (TNFα) shedding or release, as used herein, refers to a process in which membrane-anchored tumor necrosis factor alpha (mTNFα/pro-TNFα) upon cleavage is released into the environment to become soluble TNFα (sTNFα or simply TNFα). This process is, inter alia, triggered by TACE/ADAM17.
Release or shedding of Interleukin 6 receptor (IL-6R) refers to a process in which soluble IL-6R is produced by proteolytic cleavage of the membrane-bound IL-6R on the cell surface at a proteolytic site close to its transmembrane domain by TACE/ADAM17 Release or shedding of Heparin-binding EGF-like growth factor (HB-EGF) refers to a cleavage process in which the soluble form of HB-EGF is generated and set free from the cell surface. Heparin-binding EGF-like growth factor, an epidermal growth factor with an affinity for heparin, is synthesized as a membrane-anchored mitogenic and chemotactic glycoprotein. . First identified in the conditioned media of human macrophage-like cells, HB-EGF is an 87-amino acid glycoprotein that displays highly regulated gene expression.
Suitable Assays to determine the TNFα shedding effect are described, e.g., in
According to one embodiment of the invention, the human iRhom2 to which the protein binder binds comprises
In some embodiments, human iRhom2 comprises an amino acid sequence that has ≥81%, preferably 82%, more preferably 83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98 or most preferably ≥99% sequence identity with SEQ ID NO 181.
SEQ ID NO 181 represents the amino acid sequence of inactive rhomboid protein 2 (iRhom2) isoform 1 [Homo sapiens], accessible under NCBI reference NP_078875.4. Generally, different variants and isoforms of iRhom2 exist. Likewise, mutants comprising conservative or silent amino acid substitutions exist, or may exist, which maintain full or at least substantial iRhom2 activity. These isoforms, variants and mutants are encompassed by the identity range specified above, meaning however that dysfunctional, non-active variants and mutants are excluded.
According to one embodiment of the invention, the protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.
As used herein, the term “monoclonal antibody (mAb)” shall refer to an antibody composition having a homogenous antibody population, i.e., a homogeneous population consisting of a whole immunoglobulin, or a fragment or derivative thereof retaining target binding capacities.
Particularly preferred, such antibody is an IgG antibody, or a fragment or derivative thereof retaining target binding capacities. Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG has two antigen binding sites.
IgG antibodies are large molecules with a molecular weight of about 150 kDa made of four peptide chains. It contains two identical class 7 heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of the fork contains an identical antigen binding site. The Fc regions of IgGs bear a highly conserved N-glycosylation site. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans also bear bisecting GlcNAc and α-2,6-linked sialic acid residues.
There are four IgG subclasses (IgG1, 2, 3, and 4) in humans, named in order of their abundance in serum (IgG1 being the most abundant).
As used herein, the term “fragment” shall refer to fragments of such antibody retaining target binding capacities, e.g.
As used herein, the term “derivative” shall refer to protein constructs being structurally different from, but still having some structural relationship to, the common antibody concept, e.g., scFv, Fab and/or F(ab)2, as well as bi-, tri- or higher specific antibody constructs, and further retaining target binding capacities. All these items are explained below.
Other antibody derivatives known to the skilled person are Diabodies, Camelid Antibodies, Nanobodies, Domain Antibodies, bivalent homodimers with two chains consisting of scFvs, IgAs (two IgG structures joined by a J chain and a secretory component), shark antibodies, antibodies consisting of new world primate framework plus non-new world primate CDR, dimerized constructs comprising CH3+VL+VH, and antibody conjugates (e.g. antibody or fragments or derivatives linked to a toxin, a cytokine, a radioisotope or a label). These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, without adding further inventive activity.
Methods for the production of a hybridoma cell are disclosed in Köhler & Milstein (1975).
Methods for the production and/or selection of chimeric or humanised mAbs are known in the art. For example, U.S. Pat. No. 6,331,415 by Genentech describes the production of chimeric antibodies, while U.S. Pat. No. 6,548,640 by Medical Research Council describes CDR grafting techniques and U.S. Pat. No. 5,859,205 by Celltech describes the production of humanised antibodies. Methods for the production and/or selection of fully human mAbs are known in the art. These can involve the use of a transgenic animal which is immunized with the respective protein or peptide, or the use of a suitable display technique, like yeast display, phage display, B-cell display or ribosome display, where antibodies from a library are screened against human iRhom2 in a stationary phase.
In vitro antibody libraries are, among others, disclosed in U.S. Pat. No. 6,300,064 by MorphoSys and U.S. Pat. No. 6,248,516 by MRC/Scripps/Stratagene. Phage Display techniques are for example disclosed in U.S. Pat. No. 5,223,409 by Dyax. Transgenic mammal platforms are for example described in EP1480515A2 by TaconicArtemis.
IgG, IgM, scFv, Fab and/or F(ab)2 are antibody formats well known to the skilled person. Related enabling techniques are available from the respective textbooks.
As used herein, the term “Fab” relates to an IgG/IgM fragment comprising the antigen binding region, said fragment being composed of one constant and one variable domain from each heavy and light chain of the antibody
As used herein, the term “F(ab)2” relates to an IgG/IgM fragment consisting of two Fab fragments connected to one another by disulfide bonds.
As used herein, the term “scFv” relates to a single-chain variable fragment being a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide.
Modified antibody formats are for example bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates and the like. These types are well described in the literature and can be used by the skilled person on the basis of the present disclosure, with adding further inventive activity.
As used herein, the term “antibody mimetic” relates to an organic molecule, most often a protein that specifically binds to a target protein, similar to an antibody, but is not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. The definition encompasses, inter alia, Affibody molecules, Affilins, Affimers, Affitins, Alphabodies, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, Monobodies, and nanoCLAMPs.
In one or more embodiments, the protein binder is an isolated antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an isolated antibody mimetic
In one or more embodiments, the antibody is an engineered or recombinant antibody, or a target binding fragment or derivative thereof retaining target binding capacities, or an engineered or recombinant antibody mimetic.
According to one embodiment of the invention, the protein binder is an antibody in at least one of the formats selected from the group consisting of: IgG, scFv, Fab, or (Fab)2.
According to one embodiment of the invention, the protein binder is not cross-reactive with human iRhom1.
According to one embodiment of the invention, the protein binder is a murine, chimerized, humanized, or human antibody.
According to one embodiment of the invention, the protein binder is an antibody that
wherein the CDRs are embedded in a suitable protein framework so as to be capable to bind to human iRhom2 with sufficient binding affinity and to inhibit or reduce TACE/ADAM17 activity.
As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987) and MacCallum et al., (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. Note that this numbering may differ from the CDRs that are actually disclosed in the enclosed sequence listing, because CDR definitions vary from case to case.
As used herein, the term “framework” when used in reference to an antibody variable region is entered to mean all amino acid residues outside the CDR regions within the variable region of an antibody. Therefore, a variable region framework is between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs.
As used herein, the term “capable to bind to target X with sufficient binding affinity” has to be understood as meaning that respective binding domain binds the target with a KD of 10-4 or smaller. KD is the equilibrium dissociation constant, a ratio of koff/kon, between the protein binder and its antigen. KD and affinity are inversely related. The KD value relates to the concentration of protein binder (the amount of protein binder needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the binding domain. The following table shows typical KD ranges of monoclonal antibodies
Preferably, the protein binder has up to 2 amino acid substitutions, and more preferably up to 1 amino acid substitution.
Preferably, at least one of the CDRs of the protein binder has a sequence identity of ≥67%; ≥68%; ≥69%; ≥70%; ≥71%; ≥72%; ≥73%; ≥74%; ≥75%; ≥76%; ≥77%; ≥78%; ≥79%; ≥80%; ≥81%; ≥82%; ≥83%; ≥84%; ≥85%; ≥86%; ≥87%; ≥88%; ≥89%; ≥90%;≥91%;≥92%;≥93%;≥94%;≥95%;≥96%;≥97%;≥98%;≥99%, and most preferably 100% to the respective SEQ ID NO.
“Percentage of sequence identity” as used herein, is determined by comparing two optimally aligned biosequences (amino acid sequences or polynucleotide sequences) over a comparison window, wherein the portion of the corresponding sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 and multiplying the result by 100 to yield the percentage of sequence identity.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein.
Preferably, at least one of the CDRs has been subject to CDR sequence modification, including
Affinity maturation in the process by which the affinity of a given antibody is increased in vitro. Like the natural counterpart, in vitro affinity maturation is based on the principles of mutation and selection. It has successfully been used to optimize antibodies, antibody fragments or other peptide molecules like antibody mimetics. Random mutations inside the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, the genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods like phage display usually results in antibody fragments with affinities in the low nanomolar range. For principles see Eylenstein et al. (2016) or US20050169925A1, the content of which is incorporated herein by reference for enablement purposes.
Engineered antibodies contain murine-sequence derived CDR regions that have been engrafted, along with any necessary framework back-mutations, into sequence-derived V regions. Hence, the CDRs themselves can cause immunogenic reactions when the humanized antibody is administered to a patient. Methods of reducing immunogenicity caused by CDRs are disclosed in Harding et al. (2010), or US2014227251A1, the content of which is incorporated herein by reference for enablement purposes.
According to one embodiment of the invention, the protein binder is an antibody that
2and 7; 12and 17;22 and 27;32 and 37;42and 47;52 and 57; 62and 67;72 and 77; 82 and 87; 112 and 117; 152 and 157; 162 and 167; and/or 172 and 177;
said protein binder still being capable to bind to human iRhom2 with sufficient binding affinity and to inhibit or reduce TACE/ADAM17 activity.
A “variable domain” when used in reference to an antibody or a heavy or light chain thereof is intended to mean the portion of an antibody which confers antigen binding onto the molecule and which is not the constant region. The term is intended to include functional fragments thereof which maintain some of all of the binding function of the whole variable region.
Variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scfv) and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. Such terms are described in, for example, Huston et al., (1993) or Pluckthun and Skerra (1990).
Preferably, the HCVD and/or LCVD has a sequence identity of 81%; ≥82%; ≥83%; ≥84%; ≥85% 86%≥87%≥88%≥89%≥90%≥91%≥92%≥93%≥94%≥95%; ≥96%; ≥97%; ≥98%; ≥99%; or most preferably 100% to the respective SEQ ID NO.
According to one embodiment of the invention, at least one amino acid substitution is a conservative amino acid substitution.
A “conservative amino acid substitution”, as used herein, has a smaller effect on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups.
In some embodiments, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with
Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide. Conservative changes can further include substitution of chemically homologous non-natural amino acids (i.e. a synthetic non-natural hydrophobic amino acid in place of leucine, a synthetic non-natural aromatic amino acid in place of tryptophan).
According to one embodiment of the invention, the protein binder has at least one of
As used herein the term “binding affinity” is intended to mean the strength of a binding interaction and therefore includes both the actual binding affinity as well as the apparent binding affinity. The actual binding affinity is a ratio of the association rate over the disassociation rate. Therefore, conferring or optimizing binding affinity includes altering either or both of these components to achieve the desired level of binding affinity. The apparent affinity can include, for example, the avidity of the interaction. For example, a bivalent heteromeric variable region binding fragment can exhibit altered or optimized binding affinity due to its valency.
A suitable method for measuring the affinity of a binding agent is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal/liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or disassociation between a receptor-ligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allows for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (k on) and disassociation rates (koff).
Measurements of k on and koff values can be advantageous because they can identify altered variable regions or optimized variable regions that are therapeutically more efficacious. For example, an altered variable region, or heteromeric binding fragment thereof, can be more efficacious because it has, for example, a higher kon valued compared to variable regions and heteromeric binding fragments that exhibit similar binding affinity. Increased efficacy is conferred because molecules with higher kon values can specifically bind and inhibit their target at a faster rate. Similarly, a molecule of the invention can be more efficacious because it exhibits a lower koff value compared to molecules having similar binding affinity. Increased efficacy observed with molecules having lower koffrates can be observed because, once bound, the molecules are slower to dissociate from their target. Although described with reference to the altered variable regions and optimized variable regions of the invention including, heteromeric variable region binding fragments thereof, the methods described above for measuring associating and disassociation rates are applicable to essentially any protein binder or fragment thereof for identifying more effective binders for therapeutic or diagnostic purposes.
Another suitable method for measuring the affinity of a binding agent is through surface is by FACS/scatchard analysis. See inter alia example 10 for a respective description.
Methods for measuring the affinity, including association and disassociation rates using surface plasmon resonance are well known in the arts and can be found described in, for example, Jonsson and Malmquist, (1992) and Wu et al. (1998). Moreover, one apparatus well known in the art for measuring binding interactions is a BIAcore 2000 instrument which is commercially available through Pharmacia Biosensor, (Uppsala, Sweden).
Preferably said target binding affinity is ≥51%, ≥52%, ≥53%, ≥54%, ≥55%, ≥56%, ≥57%, 58%, ≥59%, ≥60%, ≥61%, ≥62%, ≥63%, ≥64%, ≥65%, ≥66%, ≥67%, ≥68%, ≥69%, 70%, ≥71%, ≥72%, ≥73%, ≥74%, ≥75%, ≥76%, ≥77%, ≥78%, ≥79%, ≥80%, ≥81%, 82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, and most preferably ≥99% compared to that of the reference binding agent.
As used herein, the quantification of the inhibiting or reducing effect on TACE/ADAM17 activity, compared to a benchmark binding agent, is determined with a suitable assay to determine the TNFα shedding effect, as, e.g., described, e.g., in
According to another aspect of the invention, a protein binder is provided that binds to human iRhom2, and competes for binding to human iRhom2 with
According to another aspect of the invention, a protein binder is provided that binds to essentially the same, or the same, region on human iRhom2 as
Clones #3, #5, #16, #22, #34, #42, #43, #44, #46, #47, #48, #49, #50, #51, #52, #54, #56, or #57 are identified in the sequence table herein.
As regards the format or structure of such protein binder, the same preferred embodiments as set forth above apply. In one embodiment, said protein binder is a monoclonal antibody, or a target-binding fragment or derivative thereof retaining target binding capacities, or an antibody mimetic.
As used herein, the term “competes for binding” is used in reference to one of the antibodies defined by the sequences as above, meaning that the actual protein binder as an activity which binds to the same target, or target epitope or domain or subdomain, as does said sequence defined protein binder, and is a variant of the latter. The efficiency (e.g., kinetics or thermodynamics) of binding may be the same as or greater than or less than the efficiency of the latter. For example, the equilibrium binding constant for binding to the substrate may be different for the two antibodies.
Such competition for binding can be suitably measured with a competitive binding assay. Such assays are disclosed in Finco et al. 2011, the content of which is incorporated herein by reference for enablement purposes, and their meaning for interpretation of a patent claim is disclosed in Deng et al 2018, the content of which is incorporated herein by reference for enablement purposes.
In order to test for this characteristic, suitable epitope mapping technologies are available, including, inter alia,
These methods are, inter alia, disclosed and discussed in Banik et al (2010), and DeLisser (1999), the content of which is herein incorporated by reference for enablement purposes.
According to another aspect of the invention, a nucleic acid is provided that encodes for at least one chain of the binding agent according to the above description.
In one embodiment, at least acids are provided which encode for the heavy chain and the light chain, respectively, of the binding agent, in case the later is a monoclonal antibody having a heteromeric stricture of at least one light chain and one heavy chain.
Generally, due to the degeneracy of the genetic code, there is a large number of different nucleic acids that have the capacity to encode for such chain. The skilled person is perfectly able to determine if a given nucleic acid satisfies the above criterion. On the other hand, the skilled person is perfectly able to reverse engineer, from a given amino acid sequence, based on codon usage tables, a suitable nucleic acid encoding therefore. For this purpose, software tools such as “reverse translate” provided by the online tool “sequence manipulation suite”, (https://www.bioinfonnatics.org/sms2/rev_trans.html) can be used.
Such nucleic acid can be also be used for pharmaceutic purposes. In such case, it is an RNA-derived molecule that is administered to a patient, wherein the protein expression machinery of the patient expresses the respective binding agent. The mRNA can for example be delivered in suitable liposomes and comprises either specific sequences or modified uridine nucleosides to avoid immune responses and/or improve folding and translation efficiency, sometimes comprising cap modifications at the 5′—and/or 3′ terminus to target them to specific cell types. Such nucleic acid can be used for transfecting an expression host to then express the actual binding agent. In such case, the molecule can be a cDNA that is optionally integrated into a suitable vector.
According to another aspect of the invention, the use of the protein binder or nucleic acid according to the above description is provided (for the manufacture of a medicament) in the treatment of a human or animal subject
an inflammatory condition, or for the prevention of such condition.
In order to diagnose am inflammatory condition, the patient may have a physical exam and may also be asked about medical history. A practitioner may look for inflammation in the joints, joint stiffness and loss of function in the joint. In addition, the practitioner may order X-rays and/or Blood tests to detect inflammatory markers, like e.g. serum hs-CRP, IL-6, TNF-α, and IL-10, erythrocyte sedimentation rate, plasma viscosity, fibrinogen, and/or ferritin, as compared to healthy controls
According to another aspect of the invention, a pharmaceutical composition comprising the protein binder or nucleic acid according to the above description, and optionally one or more pharmaceutically acceptable excipients, is provided.
According to another aspect of the invention, a combination comprising (i) the protein binder or the nucleic acid or the pharmaceutical composition according to the above description and (ii) one or more therapeutically active compounds is provided.
According to another aspect of the invention, a method for treating or preventing an inflammatory condition is provided, which method comprises administration, to a human or animal subject, of (i) the protein binder according to the above description (ii) the nucleic acid according to the above description, (iii) the pharmaceutical composition according to the above description, or (iv) the combination according to the above description is provided in a therapeutically sufficient dose.
According to one embodiment of the invention, the inflammatory condition is Rheumatoid Arthritis (RA) According to another aspect of the invention, a therapeutic kit of parts comprising:
is provided.
Further embodiments of the present invention relate to antibodies 47, 48, 50, 51 and 52, which the inventors have shown to have specific effects, as shown in the following table (+ means inhibitory effect,—means no inhibitory effect)
The table above compares the properties of the antibodies 47, 48, 50, 51 and 52 of the invention on LPS-induced shedding of TNFα versus PMA-induced shedding of IL-6R and HB-EGF in THP-1 cells. In contrast to the LPS-induced release of TNFα in THP-1 cells, where the antibodies 47, 48, 50, 51 and 52 of the invention had no inhibitory effect, an inhibitory effect on the release of PMA-induced IL-6R and HB-EGF in THP-1 cells was observed for the antibodies 47, 48, 50, and 51 of the invention, but not for the antibody 52 of the invention.
While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.
All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′-≥3′.
In total 8 different expression vectors were generated for immunization, 3 of them coding for different human iRhom2 and the remaining five coding for different mouse iRhom2 variants.
Gene synthesis was performed at Thermo Fisher Scientific GeneArt GmbH, Regensburg, Germany. In brief, submitted DNA sequences were optimized using GeneOptimizer software for maximum protein production. After genes were synthesized using synthetic oligonucleotides, assembled by primer extension-based PCR, constructs were cloned into standard cloning vectors and subsequently verified by sequencing. The fragments were sub cloned into pcDNA 3.1(+) expression vector (Thermo Fisher Scientific, USA), plasmid DNA was purified from transformed bacteria and purity and concentration were determined by UV spectroscopy. The final constructs were verified by restriction mapping and sequencing.
Due to the high sequence homology of human versus mouse iRhom2 protein (referring to the NCBI reference sequence NP_078875.4 for human iRhom2 and the NCBI reference sequence NP_766160.2 for mouse iRhom2, the amino acid sequence identity for the extracellular loops 1, 2, 3 and the C-terminal tail of human versus mouse iRhom2 are calculated as 89.96%, 100.00%, 100.00% and 96.97%, respectively), iRhom2 knockout rather than wild type mice were bred for immunization.
In brief, the Rhbdf2tmlb(KOMP)Wtsi mouse strain (Rhbdf2 is an alternative name for iRhom2) was ordered for resuscitation from the KOMP Mouse Biology Program at University of California, Davis, and resulted in the availability of three heterozygous male mice. These three animals, which were in a C57BL/6N background (C57BL/6N-Rhbdf2tmlb(KOMP)Wtsi), were mated with wild type female mice of a 129Sv/J genetic background to produce heterozygous offspring. These heterozygous mice were mated with one another to generate male and female mice with homozygous knockout of the Rhbdf2 gene. The resulting homozygous Rhbdf2 knockout mouse colony was further expanded for immunization.
Ten cohorts of 8 to 12 weeks old male and female iRhom2 knockout mice (as described in Example 2) were genetically immunized with pBT2-8HAX3-vectors coding for hi2-FL-WT, hi2-FL-I186T, hi2-A1-242, mi2-FL-WT & mi2-FL-I156T, mi2-Δ1-212, mi2-A1-268, hi2-FL-WT & mi2-FL-WT, hi2-A1-242 & mi2-Δ1-212, hi2-A1-242 & mi2-A1-268, hi2-A1-242 & mi2-Δ1-212 & mi2-A1-268, respectively. Using the Helios™ Gene Gun System (Biorad, USA) DNA-coated (approximately five pg DNA) nano-goldparticles were administered by nonoverlapping shots on shaved skin of the animals.
Four to ten mice per cohort were injected every 7 days for four to nine times. Ten days after the last injection, blood (serum) was collected and tested for antibody titer.
Assessment of the immune response was conducted by serum antibody titer analysis applying the FACS method. In brief, sera, diluted 1:50 in PBS containing 3% FBS, were tested on murine L929 cells stably expressing human iRhom2 using goat F(ab′)2 anti-Mouse IgG (H+L)-R-phycoerythrin (RPE) conjugate (Dianova, Germany) as secondary antibody. As a negative control, parental L929 cells were used. Tests were performed on an Accuri C6 Plus (BD Biosciences, USA) flow cytometer. Pre-immune serum taken at day 0 of the immunization protocol served as negative control.
Four days after a final booster immunization, lymph nodes of selected animals were collected, lymphocytes were isolated and either directly used or cryopreserved for subsequent fusions.
Fresh or cryopreserved lymph node-derived lymphocytes from six to fifteen selected animals were fused with Ag8 mouse myeloma cells for the generation of hybridoma cells. Fusions and subsequent growth of cells were either carried out in liquid or semi-solid media. For fusion and growth on semi-solid media plates, IgM depletion and B-cell enrichment was performed before cells were plated. Advanced imaging with integrated robotics and data tracking for automated picking of the highest value clones using the CellCelector device (ALS, Germany) was applied for further propagation and testing. For fusion and growth using liquid media, IgM depletion and B-cell enrichment was not performed.
Fused cells were plated and grown on 96-well plates in the presence of hypoxanthine-aminopterin-thymidine (HAT) medium.
In this example, the approaches for screening of hybridoma supernatants are described. Both functional and binding screens were conducted. Thus, in the following sections, screens for assaying the effects of hybridoma supernatants on TNFα shedding in THP-1 cells, the generation of murine L929 cells expressing different forms of human iRhom2, and the binding screens on these engineered test systems will be described.
Functional Screen of Hybridoma Supernatants for Candidate Selection After 14 days of culture, supernatants of hybridoma cells were collected and subjected to an ELISA-based functional screen for iRhom2 activity-neutralizing antibodies. Since the crucial role of iRhom2 in TACE-mediated release of tumor necrosis factor alpha (TNFα) from macrophages is very well established (McIlwain et al., 2012, Adrain et al., 2012, Siggs et al., 2012), the human TNF-alpha DuoSet ELISA (R&D Systems, USA) was employed to compare the lipopolysaccharide (LPS)-induced release of endogenous TNFα from human THP-1 monocytic cells in the presence and absence of all DNA immunization-derived hybridoma supernatants.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked for 3 hours with 300 μl per well of TBS, 1% BSA at room temperature. 20,000 THP-1 (American Type Culture Collection, USA) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl of hybridoma supernatants at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium instead of hybridoma supernatants were added.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of LPS (Sigma-Aldrich, USA) at 300 ng/ml growth medium for a final concentration of 50 ng/ml at 37° C., 5% CO2 for 2 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl of TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl of cell-free supernatant per sample. Additionally, 100 μl of recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl per well of biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl of streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl of AttoPhos substrate solution (Promega, USA) was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 PRO (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In order to generate a cell system that is suited for comparable and reliable binding analyses of the antibodies, L929 (NCTC clone 929) mouse fibroblast cells (ATCC, USA) were genetically modified to knock-out the mouse iRhom2 gene. The resulting L929 mouse iRhom2 knock-out cell line was afterwards infected with different human iRhom2 constructs to obtain cell line derivatives, stably expressing different human iRhom2 proteins, that allow for binding analyses to different iRhom2 variants in the same genetic background.
In brief, mRhbdf2.3 IVT gRNA (AAGCATGCTATCCTGCTCGC) (SEQ ID NO 197) was synthesized at Thermo Fisher Scientific GeneArt GmbH, Regensburg, Germany. One day post seeding in 24 well plates, L929 parental cells were transfected according to GeneArt CRISPR Nuclease mRNA user guide (Thermo Fisher Scientific, USA) with the gRNA/GeneArt Platinium Cas9 Nucelase (Thermo Fisher Scientific, USA) mix using Lipofectamine CRISPRMAX Transfection Reagent (Thermo Fisher Scientific, USA). 3 days post transfection, cells were lysed and DNA was extracted for amplification of specific PCR products using the mRhbdf2.3 fwd (TCAATGAGCTCTTTATGGGGCA) (SEQ ID NO 195)/mRhbdf2.3 rev (AAGGTCTCCATCCCCTCAGGTC) (SEQ ID NO 196) 5primer pair (Thermo Fisher Scientific, USA). For selection of positive wells, GeneArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific, USA) was applied to those samples that had a prominent single band of the correct size in an Invitrogen 2% E-Gel Size Select agarose gel (Thermo Fisher Scientific, USA). Cleavage assay PCR products were also analyzed on Invitrogen 2% E-Gel Size Select agarose gels. Two rounds of subsequent sub cloning of the identified polyclonal L929 population using limited dilution technique were performed, using the Cleavage Detection Kit for identification of positive sub clones. Thereby, the most promising positive sub clone identified in the first round, named 1029, was further sub cloned in the second round to obtain the final clone, named 2041. The monoclonal cell population derived from this sub clone is named L929-2041 and was used for subsequent infections (according to the procedure described in Example 13) with the human iRhom2 constructs hiR2-Δ242-T7 and hiR2-FL-WT-T7 for the generation of the two cell lines L929-2041-hiR2-Δ242-T7 and L929-2041-hiR2-FL-WT-T7, respectively.
Upon selection, fluorescence activated cell sorting (FACS) analyses were conducted to verify the plasma membrane localization of the human iRhom2 variants ectopically expressed by the genetically engineered murine L929 cell populations.
In brief, murine L929-2041-EV control cells stably infected with pMSCV empty vector, L929-2041-hiR2-Δ242-T7 cells expressing a human iRhom2 variant deleted for amino acids 1-242 and C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and L929-2041-hiR2-FL-T7 cells expressing human iRhom2 full length wild type also C-terminally tagged with 3 consecutive copies of the T7 epitope were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Next, the validated L929 cell populations were applied to systematically screen hybridoma supernatants for iRhom2 binding antibodies.
In brief, L929-2041-EV control cells, L929-2041-hiR2-Δ242-T7 cells and L929-2041-hiR2-FL-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or hybridoma supernatants pre-diluted 1:50 in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Since most of the hybridoma cell populations appeared to be of oligoclonal origin, sub-cloning applying classical liquid dilution technique was performed to isolate monoclonal hybridoma cell pools.
In brief, cells of the oligoclonal hybridoma population were counted and the dilution factor to end up with an average of two cells per well of 96-well plates was calculated. Cells were diluted accordingly and wells with growth of a single cell population were identified by microscopy-based screening. After expansion of these monoclonal hybridoma populations for approximately 3 weeks, supernatants were collected and compared for inhibitory effects on LPS-induced release of TNFα from THP-1 cells as described in Example 5.1. Sub clones that turned out to significantly interfere with TNFα shedding were expanded and stocked.
Following the generation of monoclonal hybridoma populations, the antibodies were purified from their respective hybridoma supernatant applying affinity chromatography.
In brief, supernatants collected from the monoclonal hybridoma cells were loaded on equilibrated protein G sepharose prepacked gravity-flow columns (Protein G GraviTrap™, GE Healthcare, UK) for antibody capturing. Afterwards, columns were washed once with binding buffer and trapped antibodies were eluted with elution buffer (both buffers are provided as part of the Ab Buffer Kit; GE Healthcare, UK). Next, the eluate fractions were desalted using PD Miditrap G-25 columns (GE Healthcare, UK), and purified samples were concentrated via Amicon Ultra-4 Centrifugal Filter Units with a cutoff at 30 kDa (Sigma-Aldrich, USA).
Finally, the concentration of the purified proteins was determined applying a NanoDrop 2000/c spectrophotometer (Thermo Fisher Scientific, USA).
As a next step, a mouse IgG/IgM ELISA was performed to determine the isotype of the purified antibodies of the invention. In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of goat anti mouse IgG+ IgM (H+L) capture antibody (Sigma-Aldrich, USA) at 1 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl Pierce protein-free (TBS) blocking buffer (Thermo Fisher Scientific, USA) per well at room temperature for 2 hours. The blocking buffer was then removed and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). Afterwards, 100 μl TBS per well as blank and negative control, mouse IgG (Thermo Fisher Scientific, USA) and mouse IgM (Sigma-Aldrich, USA) antibody at defined concentrations (both 1:2 titrations starting at 1 μg/ml in TBS) as standard references, mouse IgG (Thermo Fisher Scientific, USA) and mouse IgM (Sigma-Aldrich, USA) antibody at 3 μg/ml in TBS each as positive and specificity controls, and the purified antibodies of the invention at 3 μg/ml in TBS were added to the wells and incubated at room temperature for 2 hours. Subsequently, the plates were washed 4 times with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland). For isotype detection, one half of the sample each were, protected from direct light, incubated with 100 μl per well of AP-conjugated goat anti mouse IgM (Sigma-Aldrich, USA) or AP-conjugated goat anti mouse IgG F(ab′)2 Fragment (Dianova, Germany) detection antibodies diluted 1:5,000 in TBS for 1.5 hours at room temperature. Following another round of 4 washing steps with 350 μl per well of TBS-T (Carl Roth, Germany) on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the last cycle, 100 μl of AttoPhos substrate solution (Promega, USA) were added for incubation in the dark and at room temperature for 10 minutes. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
All 18 antibodies of the invention were subjected to sequence determination. In brief, total RNA was isolated from the hybridoma cells following the technical manual of Ambion's TRIzol® Reagent (Thermo Fisher Scientific, USA). Total RNA was then reverse-transcribed into cDNA using either isotype-specific anti-sense primers or universal primers following the technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Japan). Antibody fragments of heavy chain and light chain were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript. Amplified antibody fragments were cloned into the pCE2 TA/Blunt-zero standard cloning vector (Vazyme Biotech Co.,Ltd, China) separately. Colony PCR was performed to screen for clones with inserts of correct sizes. In total five clones of each antibody were sequenced on an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific, USA).
In this study, affinity measurements of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention were performed by indirect FACS scatchard analysis on THP-1 cells, a human monocytic cell line endogenously expressing iRhom2.
In brief, human THP-1 cells (American Type Culture Collection, USA) were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. In order to pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or serial two-fold dilutions (in total 22 concentrations) of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention in FACS buffer starting at 40 μg/ml and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany). Applying Prism8 software (GraphPad Sowtware, USA), the respective KD value for each of the antibodies of the invention were calculated.
For various purposes, in particular binding studies, described in some of the following examples, cell systems expressing defined levels of particular iRhom variants of interest against a background lacking any endogenous iRhom1 or iRhom2 protein were required. For this purpose, mouse embryonic fibroblasts (MEFs) from double knockout (DKO) mice homozygously negative for both mouse iRhom1 and mouse iRhom2 (iRhom1/2−/−) were established. This example describes the mouse strains used for the establishment of iRhom1/2−/− DKO MEFs and the generation of an immortalized iRhom1/2−/− DKO MEF cell line.
Mouse Strains Used for the Establishment of iRhom1/2−/− DKO MEFs
In brief, the Rhbdf2tmlb(KOMP)Wtsi mouse strain on a C57BL/6N background (C57BL/6N-Rhbdf2tmlb(KOMP)Wtsi) was obtained from the Knockout Mouse Project (KOMP) Repository at the University of California, Davis, USA (Rhbdf2 is an alternative name for iRhom2). Heterozygous male Rhbdf2tmlb mice were mated with wild type female mice of a 129Sv/J genetic background to produce heterozygous offspring of mixed genetic background (129Sv/J-C57BL/6N). These heterozygous mice were mated with one another to generate male and female offspring that were homozygous for the deletion of the Rhbdf2 gene (Rhbdf2−/− mice, 129Sv/J-C57BL/6N). The resulting homozygous Rhbdf2 knockout mouse colony was further expanded by breeding of Rhbdf−/− male and female mice to generate sufficient numbers of mice. Homozygous Rhbdf2−/− mice are viable and fertile with no evident spontaneous pathological phenotypes. Rhbdf1 knockout mice were obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM) of the International Knockout Mouse Consortium (IKMC). The generation of these animals is described in Li et al., PNAS, 2015, doi: 10.1073/pnas.1505649112. Homozygous Rhbdf1-/− mice are viable and fertile with no evident spontaneous pathological phenotypes.
For the generation of DKO mice for Rhbdf1 and Rhbdf2 (Rhbdf1/2−/− mice), Rhbdf1-/− mice were mated with Rhbdf2−/− mice to generate Rhbdf1+/− Rhbdf2+/− doubly heterozygous mice. These were mated with Rhbdf2−/− mice to produce Rhbdf1+/− Rhbdf2−/− animals, which were mated with one another to generate E14.5 embryos lacking both Rhbdf genes (Rhbdf1/2−/− DKO embryos) at the expected Mendelian ratios (¼ of all embryos) for production of E13.5 Rhbdf1/2−/− DKO MEFs, as described below.
Generation of an Immortalized iRhom1/2−/− DKO MEF Cell Line
In brief, pregnant Rhbdf1+/− Rhbdf2−/− females were sacrificed at E13.5. The uterine horns were removed into a dish with ice-cold PBS. Using fine tip forceps, the embryos were released from maternal tissue and each embryo was removed from placenta. Each embryo was then decapitated with a sharp scalpel and all internal organs such as liver, heart, lung and intestines were removed. A 0.5 mm section of the tail was removed and transferred to a 1.5 ml Eppendorf tube for isolation of genomic DNA and subsequent PCR genotyping to confirm the correct genotype of the embryo. Afterwards, the remaining embryonic tissue was washed once with PBS and transferred into a tissue culture dish with 2 mL of 0.25% trypsin/EDTA. The tissue was extensively minced with two sterile scalpels, and the trypsin/cell mixture was incubated at 37° C. for 15 minutes. Trypsinization was stopped by the addition of FCS-containing growth medium. To generate a single cell suspension, the mixture was pipetted up and down, first five times with a 10 mL serum pipet, then five times with a 5 mL serum pipet and finally several times with a fire-polished Pasteur pipet to further dissociate any remaining cell clusters. Subsequently, cells obtained from one embryo were plated on two 10 cm tissue culture plates. The next day, the medium was replaced by fresh medium and the cells were allowed to grow until they reached 90% confluency. Finally, cells were expanded and stocked for future usage. For immortalization of primary Rhbdf1/2−/− DKO MEFs, cells were transduced with a retroviral system using the pMSCV expression system (Clontech, USA). Briefly, a pMSCV-Zeo-SV40 was generated as follows: the sequences coding for the puromycin resistance were removed from plasmid pMSCV-puro (Clontech, USA) and replaced with the sequences conferring the Zeocin resistance from pcDNA3.1(+) Zeo vector (Thermo Fisher Scientific, USA). The retroviral packaging cell line GP2-293 (Clontech, USA) was used in combination with the envelope vector pVSV-G (Clontech, USA) and the pMSCV-Zeo-SV40 plasmid to produce a retrovirus encoding the SV40 large T-antigen. The virus was filtered and added to primary Rhbdf1/2−/− DKO MEFs plated at 50% confluency for 24 hours. Afterwards, transduced Rhbdf1/2−/− DKO MEFs were allowed to grow in growth medium without selection pressure for 24 hours and were then shifted to growth medium containing 100 μg/ml of Zeocin. Cells were passaged when confluent and after ten passages were stocked for future usage.
Next, immortalized iRhom1/2−/− DKO MEFs were reconstituted with a tagged form of human iRhom2 in order to confirm target recognition by hybridoma supernatants (as described in example 5) for the respective purified antibodies of the invention and thereby to verify reconstituted iRhom1/2−/− DKO MEFs as suitable test systems. Additionally, iRhom1/2−/− DKO MEFs stably expressing a tagged form of mouse iRhom2 were generated in order to determine cross-reactivity of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention with the mouse orthologue of iRhom2.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing T7-Tagged Human or Mouse iRhom2 In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV (Clontech, USA) empty vector, pMSCV-hiR2-FL-WT-T7 encoding human iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG) or pMSCV-miR2-FL-WT-T7 encoding mouse iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV, pMSCV-hiR2-FL-WT-T7 or pMSCV-miR2-FL-WT-T7 ecotrophic virus were collected, filtered with 0.45 pm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, the virus containing supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-EV control cells stably infected with pMSCV empty vector, MEF-DKO-hiR2-FL-WT-T7 cells stably expressing human iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, and MEF-DKO-miR2-FL-WT-T7 cells stably expressing mouse iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-EV control cells, MEF-DKO-hiR2-FL-WT-T7 cells and MEF-DKO-miR2-FL-WT-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls), mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention also at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Due to the sequence homology of the human iRhom2 protein versus its closely related family member human iRhom1 (referring to the NCBI reference sequence NP_078875.4. for human iRhom2 and the NCBI reference sequence NP_071895.3 for human iRhom1, the amino acid sequence identity for the extracellular loops 1, 2, 3 and the C-terminal tail of human iRhom2 versus human iRhom1 are calculated as 67.4%, 100.00%, 80.00% and 63.64%, respectively), the binding specificity of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention for human iRhom2 versus human iRhom1 was assessed as a next step. For this purpose, iRhom1/2−/− DKO MEFs stably expressing a tagged form of human iRhom1 were generated.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing T7-Tagged Human iRhom1
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV-hiR1-FL-WT-T7 (SEQ ID NO 189) encoding human iRhom1 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV-hiR1-FL-WT-T7 ecotrophic virus were collected, filtered with 0.45 pm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-hiR1-FL-WT-T7 cells stably expressing human iRhom1 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, in addition to immortalized MEF-DKO-EV control cells and MEF-DKO-hiR2-FL-WT-T7 cells (as already described in example 12), MEF-DKO-miR2-FL-WT-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls), mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention also at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
In the following study, ELISA-based TNFα release assays were performed to verify the inhibitory effects of the purified antibodies of the invention on LPS-induced release of endogenous TNFα from human THP-1 monocytic cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 20,000 THP-1 (American Type Culture Collection, USA) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 50 μg/ml as isotype control (for a final concentration of 10 μg/ml in the resulting 100 μl sample volume) or purified antibodies of the invention at 50 μg/ml (for a final concentration of 10 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of LPS (Sigma-Aldrich, USA) at 300 ng/ml in growth medium for a final concentration of 50 ng/ml at 37° C., 5% CO2 for 2 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes.
Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Nowadays, several methods to map epitopes recognized by antibodies are available, including X-ray co-crystallography, array-based oligo-peptide scanning, hydrogen-deuterium exchange or cross-linking-coupled mass spectrometry. Genetic approaches such as site-directed mutagenesis or high-throughput shotgun mutagenesis allow epitope mapping at single amino acid resolution. However, amino acid substitutions at random positions of the protein or substitutions by non-related amino acids bear the risk of causing conformational changes and/or functional loss of the protein and, thus, may result in misinterpretations as to whether the substituted amino acid contributes to an antibody epitope. An elegant and generally accepted way to circumvent these risks is to replace individual amino acids of a given protein by the homologous amino acids of a structurally related protein, i.e. an orthologue or a closely related family member, provided these related proteins are not being recognized by the antibodies of interest. As described earlier, both is true for the purified anti-human iRhom2 antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention, since they were demonstrated to be neither cross-reactive with the mouse orthologue (example 12) nor to bind to the closely related family member human iRhom1 (example 13).
Thus, in a first approach to identify single amino acids that contribute to binding of the antibodies of the invention, plasmids for a set of 25 human iRhom2 variants with mouse iRhom2-related single amino acid substitutions were designed. These 25 substitutions reflect all amino acids in the extracellular parts, i.e. the juxtamembrane domain (JMD) and the large extracellular loop 1 as well as the C-terminus, that are non-identical in human versus mouse iRhom2. Instead of the amino acid of human iRhom2, the amino acid at the corresponding position of mouse iRhom2 was introduced, resulting in the variants hiR2-FL-R441K-T7, hiR2-FL-K443R-T7, hiR2-FL-V4591-T7, hiR2-FL-G481Q-T7, hiR2-FL-L488R-T7, hiR2-FL-L493I-T7, hiR2-FL-D496T-T7, hiR2-FL-H505R-T7, hiR2-FL-Q512L-T7, hiR2-FL- R513K-T7, hiR2-FL-D528N-T7, hiR2-FL-M534S-T7, hiR2-FL-G540S-T7, hiR2-FL-R543Q-T7, hiR2-FL-T544P-T7, hiR2-FL-G546A-T7, hiR2-FL-A547V-T7, hiR2-FL-R582Q-T7, hiR2-FL-F588L-T7, hiR2-FL-M591I-T7, hiR2-FL-E594K-T7, hiR2-FL-E626D-T7, hiR2-FL-L657I-T7, and hiR2-FL-H835Y-T7. In case no corresponding amino acid exists in mouse iRhom2, the respective amino acid of human iRhom2 was deleted, resulting in the variant hiR2-FL-P533-T7.
This example describes the generation of iRhom1/2−/− DKO MEF populations expressing the 25 mouse iRhom2-related single amino acid substitution variants as well as their characterization in terms of cell surface localization and functional activity as indicators of proper protein conformation. Subsequently, binding analyses of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention on the entire panel of 25 engineered MEF populations expressing human iRhom2 variants with mouse iRhom2-related single amino acid substitutions (including the variant deleted for P533) are described.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing 25 T7-Tagged Human iRhom2 Variants with Mouse iRhom2-Related Single Amino Acid Substitutions
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV-hiR2-FL-R441K-T7, pMSCV-hiR2-FL-K443R-T7, pMSCV-hiR2-FL-V459I-T7, pMSCV-hiR2-FL-G481Q-T7, pMSCV-hiR2-FL-L488R-T7, pMSCV-hiR2-FL-L493I-T7, pMSCV-hiR2-FL-D496T-T7, pMSCV-hiR2-FL-H505R-T7, pMSCV-hiR2-FL-Q512L-T7, pMSCV-hiR2-FL- R513K-T7, pMSCV-hiR2-FL-D528N-T7, pMSCV-hiR2-FL-P533-T7, pMSCV-hiR2-FL-M534S-T7, pMSCV-hiR2-FL-G540S-T7, pMSCV-hiR2-FL-R543Q-T7, pMSCV-hiR2-FL-T544P-T7, pMSCV-hiR2-FL-G546A-T7, pMSCV-hiR2-FL-A547V-T7, pMSCV-hiR2-FL-R582Q-T7, pMSCV-hiR2-FL-F588L-T7, pMSCV-hiR2-FL-M591I-T7, pMSCV-hiR2-FL-E594K-T7, pMSCV-hiR2-FL-E626D-T7, pMSCV-hiR2-FL-L657I-T7, and pMSCV-hiR2-FL-H835Y-T7 encoding human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV-hiR2-FL-R441K-T7, pMSCV-hiR2-FL-K443R-T7, pMSCV-hiR2-FL-V459I-T7, pMSCV-hiR2-FL-G481Q-T7, pMSCV-hiR2-FL-L488R-T7, pMSCV-hiR2-FL-L493I-T7, pMSCV-hiR2-FL-D496T-T7, pMSCV-hiR2-FL-H505R-T7, pMSCV-hiR2-FL-Q512L-T7, pMSCV-hiR2-FL- R513K-T7, pMSCV-hiR2-FL-D528N-T7, pMSCV-hiR2-FL-P533-T7, pMSCV-hiR2-FL-M534S-T7, pMSCV-hiR2-FL-G540S-T7, pMSCV-hiR2-FL-R543Q-T7, pMSCV-hiR2-FL-T544P-T7, pMSCV-hiR2-FL-G546A-T7, pMSCV-hiR2-FL-A547V-T7, pMSCV-hiR2-FL-R582Q-T7, pMSCV-hiR2-FL-F588L-T7, pMSCV-hiR2-FL-M591I-T7, pMSCV-hiR2-FL-E594K-T7, pMSCV-hiR2-FL-E626D-T7, pMSCV-hiR2-FL-L657I-T7, and pMSCV-hiR2-FL-H835Y-T7 ecotrophic virus were collected, filtered with 0.45 pm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA).
Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-hiR2-FL-R441K-T7, MEF-DKO-hiR2-FL-K443R-T7, MEF-DKO-hiR2-FL-V459I-T7, MEF-DKO-hiR2-FL-G481Q-T7, MEF-DKO-hiR2-FL-L488R-T7, MEF-DKO-hiR2-FL-L493I-T7, MEF-DKO-hiR2-FL-D496T-T7, MEF-DKO-hiR2-FL-H505R-T7, MEF-DKO-hiR2-FL-Q512L-T7, MEF-DKO-hiR2-FL- R513K-T7, MEF-DKO-hiR2-FL-D528N-T7, MEF-DKO-hiR2-FL-P533-T7, MEF-DKO-hiR2-FL-M534S-T7, MEF-DKO-hiR2-FL-G540S-T7, MEF-DKO-hiR2-FL-R543Q-T7, MEF-DKO-hiR2-FL-T544P-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547V-T7, MEF-DKO-hiR2-FL-R582Q-T7, MEF-DKO-hiR2-FL-F588L-T7, MEF-DKO-hiR2-FL-M591I-T7, MEF-DKO-hiR2-FL-E594K-T7, MEF-DKO-hiR2-FL-E626D-T7, MEF-DKO-hiR2-FL-L657I-T7, and MEF-DKO-hiR2-FL-H835Y-T7 cells stably expressing human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells, MEF-DKO-miR2-FL-WT-T7 cells and MEF-DKO-hiR2-FL-R441K-T7, MEF-DKO-hiR2-FL-K443R-T7, MEF-DKO-hiR2-FL-V459I-T7, MEF-DKO-hiR2-FL-G481Q-T7, MEF-DKO-hiR2-FL-L488R-T7, MEF-DKO-hiR2-FL-L493I-T7, MEF-DKO-hiR2-FL-D496T-T7, MEF-DKO-hiR2-FL-H505R-T7, MEF-DKO-hiR2-FL-Q512L-T7, MEF-DKO-hiR2-FL- R513K-T7, MEF-DKO-hiR2-FL-D528N-T7, MEF-DKO-hiR2-FL-P533-T7, MEF-DKO-hiR2-FL-M534S-T7, MEF-DKO-hiR2-FL-G540S-T7, MEF-DKO-hiR2-FL-R543Q-T7, MEF-DKO-hiR2-FL-T544P-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547V-T7, MEF-DKO-hiR2-FL-R582Q-T7, MEF-DKO-hiR2-FL-F588L-T7, MEF-DKO-hiR2-FL-M591I-T7, MEF-DKO-hiR2-FL-E594K-T7, MEF-DKO-hiR2-FL-E626D-T7, MEF-DKO-hiR2-FL-L657I-T7, and MEF-DKO-hiR2-FL-H835Y-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well.
To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer.
Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
To test all 25 human iRhom2 variants with mouse iRhom2-specific single amino acid substitutions, or single amino acid deletion as in the case of hiR2-FL-P533-, the respective MEF-DKO cell lines stably expressing these variants, generated as described in the example above, were subjected to TGFα shedding ELISA analysis. In order to demonstrate the functionality of all variants as an indicator that these variants are properly folded, PMA-induced release of nucleofected TGFα was assessed. As the cells used in this analysis are rescue variants of iRhom1/2−/− double knockout mouse embryonic fibroblasts (described in Example 11), that are rescued by the respective human iRhom2 variant with the mouse iRhom2-specific single amino acid substitution or deletion, the iRhom2 variant stably expressed is the only iRhom protein expressed in these cells at all and is therefore the only contributing iRhom to the shedding TGFα in these cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 400 ng/ml in TBS at 4° C. After MEF-DKO-hiR2-FL-R441K-T7, MEF-DKO-hiR2-FL-K443R-T7, MEF-DKO-hiR2-FL-V459I-T7, MEF-DKO-hiR2-FL-G481Q-T7, MEF-DKO-hiR2-FL-L488R-T7, MEF-DKO-hiR2-FL-L493I-T7, MEF-DKO-hiR2-FL-D496T-T7, MEF-DKO-hiR2-FL-H505R-T7, MEF-DKO-hiR2-FL-Q512L-T7, MEF-DKO-hiR2-FL- R513K-T7, MEF-DKO-hiR2-FL-D528N-T7, MEF-DKO-hiR2-FL-P533-T7, MEF-DKO-hiR2-FL-M534S-T7, MEF-DKO-hiR2-FL-G540S-T7, MEF-DKO-hiR2-FL-R543Q-T7, MEF-DKO-hiR2-FL-T544P-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547V-T7, MEF-DKO-hiR2-FL-R582Q-T7, MEF-DKO-hiR2-FL-F588L-T7, MEF-DKO-hiR2-FL-M591I-T7, MEF-DKO-hiR2-FL-E594K-T7, MEF-DKO-hiR2-FL-E626D-T7, MEF-DKO-hiR2-FL-L657I-T7, and MEF-DKO-hiR2-FL-H835Y-T7 cells were electroporated with the hTGFα-FL-WT construct in a pcDNA3.1 vector backbone, using an 4D-Nucleofector System (Lonza, Switzerland), approximately 35,000 MEF-DKO cells carrying the human iRhom2 variant with the mouse iRhom2-specific single amino acid substitution or deletion were seeded in 100 μl of normal growth medium in each well of F-bottom 96-well cell culture plates (Thermo Fisher Scientific, USA). On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for at least 1 hour. Meanwhile, the cells were washed once with PBS and afterwards 80 μl of OptiMEM medium (Thermo Fisher Scientific, USA) was added per well.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. 20 μl of OptiMEM medium was added to the unstimulated control cells. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells, MEF-DKO-miR2-FL-WT-T7 cells and MEF-DKO-hiR2-FL-R441K-T7, MEF-DKO-hiR2-FL-K443R-T7, MEF-DKO-hiR2-FL-V459I-T7, MEF-DKO-hiR2-FL-G481Q-T7, MEF-DKO-hiR2-FL-L488R-T7, MEF-DKO-hiR2-FL-L493I-T7, MEF-DKO-hiR2-FL-D496T-T7, MEF-DKO-hiR2-FL-H505R-T7, MEF-DKO-hiR2-FL-Q512L-T7, MEF-DKO-hiR2-FL- R513K-T7, MEF-DKO-hiR2-FL-D528N-T7, MEF-DKO-hiR2-FL-P533-T7, MEF-DKO-hiR2-FL-M534S-T7, MEF-DKO-hiR2-FL-G540S-T7, MEF-DKO-hiR2-FL-R543Q-T7, MEF-DKO-hiR2-FL-T544P-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547V-T7, MEF-DKO-hiR2-FL-R582Q-T7, MEF-DKO-hiR2-FL-F588L-T7, MEF-DKO-hiR2-FL-M591I-T7, MEF-DKO-hiR2-FL-E594K-T7, MEF-DKO-hiR2-FL-E626D-T7, MEF-DKO-hiR2-FL-L657I-T7, and MEF-DKO-hiR2-FL-H835Y-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well.
To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Binding of each antibody to human iRhom2 wild type is considered 100 percent. A respective drop of antibody binding to any variant by 30-59% is indicated by cells held in light gray (and marked with “1”), an impaired binding by 60-95% is illustrated by cells colored in gray (and marked with “2”), and a loss of binding by ≥95% is highlighted by dark gray cells (marked with “3”). These data reveal related (except for antibody 16 of the invention) patterns of amino acid positions relevant for binding of the antibodies 3, 5, 16, 22, 34, 42, 43, and 44 of the invention with inhibitory effects on TNFα release to human iRhom2, which are different from patterns of amino acid positions contributing to binding of the antibodies 48 and 50 without inhibitory effects on TNFα release.
Complementary to Example 15, plasmids for a set of 30 human iRhom2 variants with human iRhom1-related single amino acid substitutions to identify single amino acids that contribute to binding of the antibodies of the invention, were designed in a second approach. These 30 substitutions reflect amino acids in the central region of the large extracellular loop 1 that are non-identical in human iRhom2 versus human iRhom1. Instead of the amino acid of human iRhom2, the amino acid at the corresponding position of human iRhom1 was introduced, resulting in the variants hiR2-FL-G498A-T7, hiR2-FL-Q502R-T7, hiR2-FL-1509V-T7, hiR2-FL-Q512S-T7, hiR2-FL-R513E-T7, hiR2-FL-K514E-T7, hiR2-FL-D515E-T7, hiR2-FL-E518S-T7, hiR2-FL-T522V-T7, hiR2-FL-F523W-T7, hiR2-FL-Q527P-T7, hiR2-FL-D528I-T7, hiR2-FL-D529H-T7, hiR2-FL-T530P-T7, hiR2-FL-G531S-T7, hiR2-FL-P532A-T7, hiR2-FL-S537E-T7, hiR2-FL-D538L-T7, hiR2-FL-L539A-T7, hiR2-FL-Q541H-T7, hiR2-FL-T544Q-T7, hiR2-FL-S545F-T7, hiR2-FL-A547S-T7, hiR2-FL-T555V-T7, hiR2-FL-E557D-T7, hiR2-FL-A560S-T7 and hiR2-FL-S562E-T7. In case no corresponding amino acid exists in human iRhom1, the respective amino acid of human iRhom2 was deleted, resulting in the variants hiR2-FL-M534-T7, hiR2-FL-D535-T7 and hiR2-FL-K536-T7.
This example describes the generation of iRhom1/2−/− DKO MEF populations expressing the 30 human iRhom1-related single amino acid substitution variants as well as their characterization in terms of cell surface localization and functional activity as indicators of proper protein conformation. Subsequently, binding analyses of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention on the entire panel of 30 engineered MEF populations expressing human iRhom2 variants with human iRhom1-related single amino acid substitutions (including the variants deleted for M534, D535 and K536) are described.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing 30 T7-Tagged Human iRhom2 Variants with Human iRhom1-Related Single Amino Acid Substitutions
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV-hiR2-FL-G498A-T7, pMSCV-hiR2-FL-Q502R-T7, pMSCV-hiR2-FL-1509V-T7, pMSCV-hiR2-FL-Q512S-T7, pMSCV-hiR2-FL-R513E-T7, pMSCV-hiR2-FL-K514E-T7, pMSCV-hiR2-FL-D515E-T7, pMSCV-hiR2-FL-E518S-T7, pMSCV-hiR2-FL-T522V-T7, pMSCV-hiR2-FL-F523W-T7, pMSCV-hiR2-FL-Q527P-T7, pMSCV-hiR2-FL-D528I-T7, pMSCV-hiR2-FL-D529H-T7, pMSCV-hiR2-FL-T530P-T7, pMSCV-hiR2-FL-G531S-T7, pMSCV-hiR2-FL-P532A-T7, pMSCV-hiR2-FL-M534-T7, pMSCV-hiR2-FL-D535-T7, pMSCV-hiR2-FL-K536-T7, pMSCV-hiR2-FL-S537E-T7, pMSCV-hiR2-FL-D538L-T7, pMSCV-hiR2-FL-L539A-T7, pMSCV-hiR2-FL-Q541H-T7, pMSCV-hiR2-FL-T544Q-T7, pMSCV-hiR2-FL-S545F-T7, pMSCV-hiR2-FL-A547S-T7, pMSCV-hiR2-FL-T555V-T7, pMSCV-hiR2-FL-E557D-T7, pMSCV-hiR2-FL-A560S-T7 and pMSCV-hiR2-FL-S562E-T7 encoding human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV-hiR2-FL-G498A-T7, pMSCV-hiR2-FL-Q502R-T7, pMSCV-hiR2-FL-1509V-T7, pMSCV-hiR2-FL-Q512S-T7, pMSCV-hiR2-FL-R513E-T7, pMSCV-hiR2-FL-K514E-T7, pMSCV-hiR2-FL-D515E-T7, pMSCV-hiR2-FL-E518S-T7, pMSCV-hiR2-FL-T522V-T7, pMSCV-hiR2-FL-F523W-T7, pMSCV-hiR2-FL-Q527P-T7, pMSCV-hiR2-FL-D528I-T7, pMSCV-hiR2-FL-D529H-T7, pMSCV-hiR2-FL-T530P-T7, pMSCV-hiR2-FL-G531S-T7, pMSCV-hiR2-FL-P532A-T7, pMSCV-hiR2-FL-M534-T7, pMSCV-hiR2-FL-D535-T7, pMSCV-hiR2-FL-K536-T7, pMSCV-hiR2-FL-S537E-T7, pMSCV-hiR2-FL-D538L-T7, pMSCV-hiR2-FL-L539A-T7, pMSCV-hiR2-FL-Q541H-T7, pMSCV-hiR2-FL-T544Q-T7, pMSCV-hiR2-FL-S545F-T7, pMSCV-hiR2-FL-A547S-T7, pMSCV-hiR2-FL-T555V-T7, pMSCV-hiR2-FL-E557D-T7, pMSCV-hiR2-FL-A560S-T7 and pMSCV-hiR2-FL-S562E-T7 ecotrophic virus were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-hiR2-FL-G498A-T7, MEF-DKO-hiR2-FL-Q502R-T7, MEF-DKO-hiR2-FL-1509V-T7, MEF-DKO-hiR2-FL-Q512S-T7, MEF-DKO-hiR2-FL-R513E-T7, MEF-DKO-hiR2-FL-K514E-T7, MEF-DKO-hiR2-FL-D515E-T7, MEF-DKO-hiR2-FL-E518S-T7, MEF-DKO-hiR2-FL-T522V-T7, MEF-DKO-hiR2-FL-F523W-T7, MEF-DKO-hiR2-FL-Q527P-T7, MEF-DKO-hiR2-FL-D528I-T7, MEF-DKO-hiR2-FL-D529H-T7, MEF-DKO-hiR2-FL-T530P-T7, MEF-DKO-hiR2-FL-G531S-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-M534-T7, MEF-DKO-hiR2-FL-D535-T7, MEF-DKO-hiR2-FL-K536-T7, MEF-DKO-hiR2-FL-S537E-T7, MEF-DKO-hiR2-FL-D538L-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-Q541H-T7, MEF-DKO-hiR2-FL-T544Q-T7, MEF-DKO-hiR2-FL-S545F-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-T555V-T7, MEF-DKO-hiR2-FL-E557D-T7, MEF-DKO-hiR2-FL-A560S-T7 and MEF-DKO-hiR2-FL-S562E-T7 cells stably expressing human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells, MEF-DKO-hiR1-FL-WT-T7 cells and MEF-DKO-hiR2-FL-G498A-T7, MEF-DKO-hiR2-FL-Q502R-T7, MEF-DKO-hiR2-FL-I509V-T7, MEF-DKO-hiR2-FL-Q512S-T7, MEF-DKO-hiR2-FL-R513E-T7, MEF-DKO-hiR2-FL-K514E-T7, MEF-DKO-hiR2-FL-D515E-T7, MEF-DKO-hiR2-FL-E518S-T7, MEF-DKO-hiR2-FL-T522V-T7, MEF-DKO-hiR2-FL-F523W-T7, MEF-DKO-hiR2-FL-Q527P-T7, MEF-DKO-hiR2-FL-D528I-T7, MEF-DKO-hiR2-FL-D529H-T7, MEF-DKO-hiR2-FL-T530P-T7, MEF-DKO-hiR2-FL-G531S-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-M534-T7, MEF-DKO-hiR2-FL-D535-T7, MEF-DKO-hiR2-FL-K536-T7, MEF-DKO-hiR2-FL-S537E-T7, MEF-DKO-hiR2-FL-D538L-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-Q541H-T7, MEF-DKO-hiR2-FL-T544Q-T7, MEF-DKO-hiR2-FL-S545F-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-T555V-T7, MEF-DKO-hiR2-FL-E557D-T7, MEF-DKO-hiR2-FL-A560S-T7 and MEF-DKO-hiR2-FL-S562E-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
TGFα ELISA for Test System Validation To test all 30 human iRhom2 variants with human iRhom1-specific single amino acid substitutions, or single amino acid deletion as in the case of hiR2-FL-M534-, hiR2-FL-D535—and hiR2-FL-K536-, the respective MEF-DKO cell lines stably expressing these variants, generated as described in the example above, were subjected to TGFα shedding ELISA analysis. In order to demonstrate the functionality of all variants as an indicator that these variants are properly folded, PMA-induced release of nucleofected TGFα was assessed. As the cells used in this analysis are rescue variants of iRhom1/2−/− double knockout mouse embryonic fibroblasts (described in Example 11), that are rescued by the respective human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion, the iRhom2 variant stably expressed is the only iRhom protein expressed in these cells at all and is therefore the only contributing iRhom to the shedding TGFα in these cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 400 ng/ml in TBS at 4° C. After MEF-DKO-hiR2-FL-G498A-T7, MEF-DKO-hiR2-FL-Q502R-T7, MEF-DKO-hiR2-FL-I509V-T7, MEF-DKO-hiR2-FL-Q512S-T7, MEF-DKO-hiR2-FL-R513E-T7, MEF-DKO-hiR2-FL-K514E-T7, MEF-DKO-hiR2-FL-D515E-T7, MEF-DKO-hiR2-FL-E518S-T7, MEF-DKO-hiR2-FL-T522V-T7, MEF-DKO-hiR2-FL-F523W-T7, MEF-DKO-hiR2-FL-Q527P-T7, MEF-DKO-hiR2-FL-D528I-T7, MEF-DKO-hiR2-FL-D529H-T7, MEF-DKO-hiR2-FL-T530P-T7, MEF-DKO-hiR2-FL-G531S-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-M534-T7, MEF-DKO-hiR2-FL-D535-T7, MEF-DKO-hiR2-FL-K536-T7, MEF-DKO-hiR2-FL-S537E-T7, MEF-DKO-hiR2-FL-D538L-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-Q541H-T7, MEF-DKO-hiR2-FL-T544Q-T7, MEF-DKO-hiR2-FL-S545F-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-T555V-T7, MEF-DKO-hiR2-FL-E557D-T7, MEF-DKO-hiR2-FL-A560S-T7 and MEF-DKO-hiR2-FL-S562E-T7 cells were electroporated with the hTGFα-FL-WT construct in a pcDNA3.1 vector backbone, using an 4D-Nucleofector System (Lonza, Switzerland), approximately 35,000 MEF-DKO cells carrying the human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion were seeded in 100 μl of normal growth medium in each well of F-bottom 96-well cell culture plates (Thermo Fisher Scientific, USA). On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for at least 1 hour. Meanwhile, the cells were washed once with PBS and afterwards 80 μl of OptiMEM medium (Thermo Fisher Scientific, USA) was added per well.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. 20 μl of OptiMEM medium was added to the unstimulated control cells. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In brief, immortalized MEF-DKO-hiR2-FL-G498A-T7, MEF-DKO-hiR2-FL-Q502R-T7, MEF-DKO-hiR2-FL-I509V-T7, MEF-DKO-hiR2-FL-Q512S-T7, MEF-DKO-hiR2-FL-R513E-T7, MEF-DKO-hiR2-FL-K514E-T7, MEF-DKO-hiR2-FL-D515E-T7, MEF-DKO-hiR2-FL-E518S-T7, MEF-DKO-hiR2-FL-T522V-T7, MEF-DKO-hiR2-FL-F523W-T7, MEF-DKO-hiR2-FL-Q527P-T7, MEF-DKO-hiR2-FL-D528I-T7, MEF-DKO-hiR2-FL-D529H-T7, MEF-DKO-hiR2-FL-T530P-T7, MEF-DKO-hiR2-FL-G531S-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-M534-T7, MEF-DKO-hiR2-FL-D535-T7, MEF-DKO-hiR2-FL-K536-T7, MEF-DKO-hiR2-FL-S537E-T7, MEF-DKO-hiR2-FL-D538L-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-Q541H-T7, MEF-DKO-hiR2-FL-T544Q-T7, MEF-DKO-hiR2-FL-S545F-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-T555V-T7, MEF-DKO-hiR2-FL-E557D-T7, MEF-DKO-hiR2-FL-A560S-T7 and MEF-DKO-hiR2-FL-S562E-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention at 3 μg/ml in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
In contrast to Example 14, where the hybridoma supernatant-derived purified antibodies of the invention were tested in ELISA-based TNFα release assays, this analysis was conducted with recombinant produced antibodies of the invention to verify their inhibitory effects on LPS-induced release of endogenous TNFα from human THP-1 monocytic cells.
To produce the recombinant antibody material, target DNA sequence was designed, optimized and synthesized. The complete sequence was sub-cloned into pcDNA3.4 vector (Thermo Fisher Scientific, USA) and the transfection grade plasmid was maxi-prepared for Expi293F (Thermo Fisher Scientific, USA) cell expression. Expi293F cells were grown in serum-free Expi293FTM expression medium (Thermo Fisher Scientific, USA) in Erlenmeyer flasks (Coming Inc., USA) at 37° C. with 8% CO2 on an orbital shaker (VWR Scientific, Germany). One day before transfection, the cells were seeded at an appropriate density in new Erlenmeyer flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and then added into the flask with cells ready for transfection. The recombinant plasmids encoding target protein were transiently transfected into suspension Expi293F cell cultures. The cell culture supernatant collected on day 6 post-transfection was used for purification. Cell culture broth was centrifuged and filtrated. Filtered cell culture supernatant was loaded onto either HiTrap MabSelect SuRe (GE Healthcare, UK), MabSelect SuRe™ LX (GE Healthcare, UK) or RoboColumn Eshmuno A (Merck Millipore, USA) affinity purification columns at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to final formulation buffer. The purified protein was analyzed by SDS-PAGE analysis for molecular weight and purity measurements. Finally, the concentration was determined applying a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
The ELISA-based TNFα release assay that was used in this example is identical to the one described in Example 14.
Next, iRhom1/2−/− DKO MEFs stably expressing a tagged form of rhesus monkey, cynomolgus monkey, dog or rabbit iRhom2 were generated in order to determine cross-reactivity of the antibodies of the invention with the respective orthologue of iRhom2. iRhom1/2−/− DKO MEFs stably expressing a tagged form of rhesus monkey, cynomolgus monkey, dog or rabbit iRhom1 were generated to confirm specificity for iRhom2 versus iRhom1 of these species.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing T7-Tagged Rhesus Monkey, Cynomolgus Monkey, Dog or Rabbit iRhom2
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV (Clontech, USA) empty vector, pMSCV-rhesus-iR2-FL-WT-T7 encoding rhesus monkey iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, pMSCV-cyno-iR2-FL-WT-T7 encoding cynomolgus monkey iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, pMSCV-dog-iR2-FL-WT-T7 encoding dog iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope or pMSCV-rabbit-iR2-FL-WT-T7 encoding rabbit iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, respectively, and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV, pMSCV-rhesus-iR2-FL-WT-T7, pMSCV-cyno-iR2-FL-WT-T7, pMSCV-dog-iR2-FL-WT-T7 or pMSCV-rabbit-iR2-FL-WT-T7 ecotrophic virus, respectively were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, the virus containing supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-EV control cells stably infected with pMSCV empty vector, pMSCV-rhesus-iR2-FL-WT-T7 cells stably expressing rhesus monkey iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, pMSCV-cyno-iR2-FL-WT-T7 cells stably expressing cynomolgus monkey iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, pMSCV-dog-iR2-FL-WT-T7 cells stably expressing dog iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope or pMSCV-rabbit-iR2-FL-WT-T7 cells stably expressing rabbit iRhom2 full length wild type C-terminally tagged with 3 consecutive copies of the T7 epitope, respectively. Upon propagation, cells were stocked for future usage. In parallel, iRhom1/2−/− DKO MEFs stably expressing a tagged form of rhesus monkey, cynomolgus monkey, dog or rabbit iRhom1 were generated in an analogous manner.
In brief, immortalized MEF-DKO-EV control cells, MEF-DKO-rhesus-iR2-FL-WT-T7 cells, MEF-DKO-cyno-iR2-FL-WT-T7 cells, MEF-DKO-dog-iR2-FL-WT-T7 cells and MEF-DKO-rabbit-iR2-FL-WT-T7 cells, as well as their respective iRhom1 counterparts, were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls), mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer or the antibodies of the invention also at 3 μg/ml FACS buffer and incubated on ice for 1 hour.
Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
In addition to Example 12, where T7-tagged iRhom variants were described, immortalized iRhom1/2−/− DKO MEFs were reconstituted with a FLAG-tagged form of human iRhom2 in order to confirm target recognition for the antibodies of the invention. Additionally, iRhom1/2-/− DKO MEFs stably expressing a FLAG-tagged form of mouse iRhom2 were generated in order to determine cross-reactivity of the antibodies of the invention with the mouse orthologue of iRhom2.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing FLAG-Tagged Human or Mouse iRhom2
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV (Clontech, USA) empty vector, pMSCV-hiR2-FL-WT-FLAG encoding human iRhom2 full length wild type C-terminally tagged with the triple-FLAG epitope (DYKDHDGDYKDHDIDYKDDDDK) or pMSCV-miR2-FL-WT-FLAG encoding mouse iRhom2 full length wild type C-terminally tagged with the triple-FLAG epitope, and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV, pMSCV-hiR2-FL-WT-FLAG or pMSCV-miR2-FL-WT-FLAG ecotrophic virus were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, the virus containing supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-EV control cells stably infected with pMSCV empty vector, MEF-DKO-hiR2-FL-WT-FLAG cells stably expressing human iRhom2 full length wild type C-terminally tagged with the triple-FLAG epitope, and MEF-DKO-miR2-FL-WT-FLAG cells stably expressing mouse iRhom2 full length wild type C-terminally tagged with the triple-FLAG epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-EV control cells, MEF-DKO-hiR2-FL-WT-FLAG cells and MEF-DKO-miR2-FL-WT-FLAG cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 3×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls), mouse monoclonal anti-FLAG IgG (Sigma-Aldrich, USA) at 3 μg/ml FACS buffer or the antibodies of the invention also at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
In this study, binding specificity analyses of the hybridoma supernatant leading to the antibody 16 of the invention as a representative example of antibodies 3, 16, 22 and 42 of the invention as well as of the antibody 16 of the invention as a representative example of antibodies 16, 22 and 42 of the invention in cell lines endogenously expressing iRhom2 were performed. The studies were conducted on RPMI-8226 cells, a human B lymphocytic cell line endogenously expressing iRhom2 but being endogenously negative for iRhom1, on THP-1 cells, a human monocytic cell line endogenously expressing both iRhom2 and iRhom1 and on RH-30 cells, a human fibroblastic cell line endogenously negative for iRhom2 but endogenously expressing iRhom1.
In brief, human RPMI-8226 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany), THP-1 cells (American Type Culture Collection, USA) and RH-30 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany) were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 2×105 cells per well. In order to pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls), a 1:60 dilution of the hybridoma supernatant, the primary material leading to the antibodies 3, 16, 22 and 42 of the invention in FACS buffer or 3 μg/ml of the antibodies 16, 22 and 42 of the invention in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 or goat anti-human IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Complementary to Examples 15 and 16, plasmids for a set of 23 human iRhom2 variants with human iRhom1-related single amino acid substitutions to identify single amino acids that contribute to binding of the antibodies of the invention, were designed in a third approach.
These 23 substitutions reflect amino acids N-terminal of the central region of the large extracellular loop 1 that are non-identical in human iRhom2 versus human iRhom1. Instead of the amino acid of human iRhom2, the amino acid at the corresponding position of human iRhom1 was introduced, resulting in the variants hiR2-FL-A431S-T7, hiR2-FL-V434E-T7, hiR2-FL-T436V-T7, hiR2-FL-Q437D-T7, hiR2-FL-L438S-T7, hiR2-FL-S448N-T7, hiR2-FL-I452V-T7, hiR2-FL-1464E-T7, hiR2-FL-D465A-T7, hiR2-FL-1477M-T7, hiR2-FL-K479Q-T7, hiR2-FL-G481P-T7, hiR2-FL-1483V-T7, hiR2-FL-E484H-T7, hiR2-FL-Q485S-T7, hiR2-FL-L486F-T7, hiR2-FL-V4871-T7, hiR2-FL-R489S-T7, hiR2-FL-E490A-T7, hiR2-FL-D492E-T7, hiR2-FL-L493R-T7, hiR2-FL-R495K-T7 and hiR2-FL-D496H-T7.
This example describes the generation of iRhom1/2−/− DKO MEF populations expressing the 23 human iRhom1-related single amino acid substitution variants as well as their characterization in terms of cell surface localization and functional activity as indicators of proper protein conformation. Subsequently, binding analyses of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention on the entire panel of 23 engineered MEF populations expressing human iRhom2 variants with human iRhom1-related single amino acid substitutions are described.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing 23 T7-Tagged Human iRhom2 Variants with Human iRhom1-Related Single Amino Acid Substitutions
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV-hiR2-FL-A431S-T7, pMSCV-hiR2-FL-V434E-T7, pMSCV-hiR2-FL-T436V-T7, pMSCV-hiR2-FL-Q437D-T7, pMSCV-hiR2-FL-L438S-T7, pMSCV-hiR2-FL-S448N-T7, pMSCV-hiR2-FL-I452V-T7, pMSCV-hiR2-FL-1464E-T7, pMSCV-hiR2-FL-D465A-T7, pMSCV-hiR2-FL-I477M-T7, pMSCV-hiR2-FL-K479Q-T7, pMSCV-hiR2-FL-G481P-T7, pMSCV-hiR2-FL-I483V-T7, pMSCV-hiR2-FL-E484H-T7, pMSCV-hiR2-FL-Q485S-T7, pMSCV-hiR2-FL-L486F-T7, pMSCV-hiR2-FL-V487I-T7, pMSCV-hiR2-FL-R489S-T7, pMSCV-hiR2-FL-E490A-T7, pMSCV-hiR2-FL-D492E-T7, pMSCV-hiR2-FL-L493R-T7, pMSCV-hiR2-FL-R495K-T7 and pMSCV-hiR2-FL-D496H-T7 encoding human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV-hiR2-FL-A431S-T7, pMSCV-hiR2-FL-V434E-T7, pMSCV-hiR2-FL-T436V-T7, pMSCV-hiR2-FL-Q437D-T7, pMSCV-hiR2-FL-L438S-T7, pMSCV-hiR2-FL-S448N-T7, pMSCV-hiR2-FL-1452V-T7, pMSCV-hiR2-FL-1464E-T7, pMSCV-hiR2-FL-D465A-T7, pMSCV-hiR2-FL-1477M-T7, pMSCV-hiR2-FL-K479Q-T7, pMSCV-hiR2-FL-G481P-T7, pMSCV-hiR2-FL-1483V-T7, pMSCV-hiR2-FL-E484H-T7, pMSCV-hiR2-FL-Q485S-T7, pMSCV-hiR2-FL-L486F-T7, pMSCV-hiR2-FL-V487I-T7, pMSCV-hiR2-FL-R489S-T7, pMSCV-hiR2-FL-E490A-T7, pMSCV-hiR2-FL-D492E-T7, pMSCV-hiR2-FL-L493R-T7, pMSCV-hiR2-FL-R495K-T7 and pMSCV-hiR2-FL-D496H-T7 ecotrophic virus were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-hiR2-FL-A431S-T7, MEF-DKO-hiR2-FL-V434E-T7, MEF-DKO-hiR2-FL-T436V-T7, MEF-DKO-hiR2-FL-Q437D-T7, MEF-DKO-hiR2-FL-L438S-T7, MEF-DKO-hiR2-FL-S448N-T7, MEF-DKO-hiR2-FL-I452V-T7, MEF-DKO-hiR2-FL-I464E-T7, MEF-DKO-hiR2-FL-D465A-T7, MEF-DKO-hiR2-FL-I477M-T7, MEF-DKO-hiR2-FL-K479Q-T7, MEF-DKO-hiR2-FL-G481P-T7, MEF-DKO-hiR2-FL-I483V-T7, MEF-DKO-hiR2-FL-E484H-T7, MEF-DKO-hiR2-FL-Q485S-T7, MEF-DKO-hiR2-FL-L486F-T7, MEF-DKO-hiR2-FL-V487I-T7, MEF-DKO-hiR2-FL-R489S-T7, MEF-DKO-hiR2-FL-E490A-T7, MEF-DKO-hiR2-FL-D492E-T7, MEF-DKO-hiR2-FL-L493R-T7, MEF-DKO-hiR2-FL-R495K-T7 and MEF-DKO-hiR2-FL-D496H-T7 cells stably expressing human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells and MEF-DKO-hiR2-FL-A431S-T7, MEF-DKO-hiR2-FL-V434E-T7, MEF-DKO-hiR2-FL-T436V-T7, MEF-DKO-hiR2-FL-Q437D-T7, MEF-DKO-hiR2-FL-L438S-T7, MEF-DKO-hiR2-FL-S448N-T7, MEF-DKO-hiR2-FL-I452V-T7, MEF-DKO-hiR2-FL-I464E-T7, MEF-DKO-hiR2-FL-D465A-T7, MEF-DKO-hiR2-FL-I477M-T7, MEF-DKO-hiR2-FL-K479Q-T7, MEF-DKO-hiR2-FL-G481P-T7, MEF-DKO-hiR2-FL-I483V-T7, MEF-DKO-hiR2-FL-E484H-T7, MEF-DKO-hiR2-FL-Q485S-T7, MEF-DKO-hiR2-FL-L486F-T7, MEF-DKO-hiR2-FL-V487I-T7, MEF-DKO-hiR2-FL-R489S-T7, MEF-DKO-hiR2-FL-E490A-T7, MEF-DKO-hiR2-FL-D492E-T7, MEF-DKO-hiR2-FL-L493R-T7, MEF-DKO-hiR2-FL-R495K-T7 and MEF-DKO-hiR2-FL-D496H-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
To test all 23 human iRhom2 variants with human iRhom1-specific single amino acid substitutions, the respective MEF-DKO cell lines stably expressing these variants, generated as described in the example above, were subjected to TGFα shedding ELISA analysis. In order to demonstrate the functionality of all variants as an indicator that these variants are properly folded, PMA-induced release of nucleofected TGFα was assessed. As the cells used in this analysis are rescue variants of iRhom1/2−/− double knockout mouse embryonic fibroblasts (described in Example 11), that are rescued by the respective human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion, the iRhom2 variant stably expressed is the only iRhom protein expressed in these cells at all and is therefore the only contributing iRhom to the shedding TGFα in these cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 400 ng/ml in TBS at 4° C. After MEF-DKO-hiR2-FL-A431S-T7, MEF-DKO-hiR2-FL-V434E-T7, MEF-DKO-hiR2-FL-T436V-T7, MEF-DKO-hiR2-FL-Q437D-T7, MEF-DKO-hiR2-FL-L438S-T7, MEF-DKO-hiR2-FL-S448N-T7, MEF-DKO-hiR2-FL-I452V-T7, MEF-DKO-hiR2-FL-I464E-T7, MEF-DKO-hiR2-FL-D465A-T7, MEF-DKO-hiR2-FL-I477M-T7, MEF-DKO-hiR2-FL-K479Q-T7, MEF-DKO-hiR2-FL-G481P-T7, MEF-DKO-hiR2-FL-I483V-T7, MEF-DKO-hiR2-FL-E484H-T7, MEF-DKO-hiR2-FL-Q485S-T7, MEF-DKO-hiR2-FL-L486F-T7, MEF-DKO-hiR2-FL-V487I-T7, MEF-DKO-hiR2-FL-R489S-T7, MEF-DKO-hiR2-FL-E490A-T7, MEF-DKO-hiR2-FL-D492E-T7, MEF-DKO-hiR2-FL-L493R-T7, MEF-DKO-hiR2-FL-R495K-T7 and MEF-DKO-hiR2-FL-D496H-T7 cells were electroporated with the hTGFα-FL-WT construct in a pcDNA3.1 vector backbone, using an 4D-Nucleofector System (Lonza, Switzerland), approximately 35,000 MEF-DKO cells carrying the human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion were seeded in 100 μl of normal growth medium in each well of F-bottom 96-well cell culture plates (Thermo Fisher Scientific, USA). On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for at least 1 hour. Meanwhile, the cells were washed once with PBS and afterwards 80 μl of OptiMEM medium (Thermo Fisher Scientific, USA) was added per well.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. 20 μl of OptiMEM medium was added to the unstimulated control cells. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In brief, immortalized MEF-DKO-hiR2-FL-A431S-T7, MEF-DKO-hiR2-FL-V434E-T7, MEF-DKO-hiR2-FL-T436V-T7, MEF-DKO-hiR2-FL-Q437D-T7, MEF-DKO-hiR2-FL-L438S-T7, MEF-DKO-hiR2-FL-S448N-T7, MEF-DKO-hiR2-FL-I452V-T7, MEF-DKO-hiR2-FL-I464E-T7, MEF-DKO-hiR2-FL-D465A-T7, MEF-DKO-hiR2-FL-I477M-T7, MEF-DKO-hiR2-FL-K479Q-T7, MEF-DKO-hiR2-FL-G481P-T7, MEF-DKO-hiR2-FL-I483V-T7, MEF-DKO-hiR2-FL-E484H-T7, MEF-DKO-hiR2-FL-Q485S-T7, MEF-DKO-hiR2-FL-L486F-T7, MEF-DKO-hiR2-FL-V487I-T7, MEF-DKO-hiR2-FL-R489S-T7, MEF-DKO-hiR2-FL-E490A-T7, MEF-DKO-hiR2-FL-D492E-T7, MEF-DKO-hiR2-FL-L493R-T7, MEF-DKO-hiR2-FL-R495K-T7 and MEF-DKO-hiR2-FL-D496H-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention at 3 μg/ml in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Complementary to Examples 15 and 16 and also to Example 21 described above, plasmids for a set of 33 human iRhom2 variants with human iRhom1-related single amino acid substitutions to identify single amino acids that contribute to binding of the antibodies of the invention, were designed in a fourth approach. These 33 substitutions reflect amino acids C-terminal of the central region of the large extracellular loop 1, in loop 5 and in the C-terminus that are non-identical in human iRhom2 versus human iRhom1. Instead of the amino acid of human iRhom2, the amino acid at the corresponding position of human iRhom1 was introduced, resulting in the variants hiR2-FL-G563D-T7, hiR2-FL-A564P-T7, hiR2-FL-1566E-T7, hiR2-FL-D569E-T7, hiR2-FL-E579K-T7, hiR2-FL-Q580N-T7, hiR2-FL-A581S-T7, hiR2-FL-R582A-T7, hiR2-FL-S583G-T7, hiR2-FL-G587N-T7, hiR2-FL-F588H-T7, hiR2-FL-L589P-T7, hiR2-FL-E594V-T7, hiR2-FL-K596T-T7, hiR2-FL-S607R-T7, hiR2-FL-T612S-T7, hiR2-FL-E617D-T7, hiR2-FL-H620R-T7, hiR2-FL-L636M-T7, hiR2-FL-K638D-T7, hiR2-FL-I771F-T7, hiR2-FL-1825V-T7, hiR2-FL-1828V-T7, hiR2-FL-N829R-T7, hiR2-FL-W830C-T7, hiR2-FL-P831E-T7, hiR2-FL-1833C-T7, hiR2-FL-H835F-T7, hiR2-FL-F8391-T7, hiR2-FL-S843D-T7, hiR2-FL-Q853A-T7, hiR2-FL-V854Q-T7 and hiR2-FL-R844K-T7.
This example describes the generation of iRhom1/2−/− DKO MEF populations expressing the 33 human iRhom1-related single amino acid substitution variants as well as their characterization in terms of cell surface localization and functional activity as indicators of proper protein conformation. Subsequently, binding analyses of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention on the entire panel of 33 engineered MEF populations expressing human iRhom2 variants with human iRhom1-related single amino acid substitutions are described.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing 33 T7-Tagged Human iRhom2 Variants with Human iRhom1-Related Single Amino Acid Substitutions
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV hiR2-FL-G563D-T7, pMSCV hiR2-FL-A564P-T7, pMSCV hiR2-FL-1566E-T7, pMSCV hiR2-FL-D569E-T7, pMSCV hiR2-FL-E579K-T7, pMSCV hiR2-FL-Q580N-T7, pMSCV hiR2-FL-A581S-T7, pMSCV hiR2-FL-R582A-T7, pMSCV hiR2-FL-S583G-T7, pMSCV hiR2-FL-G587N-T7, pMSCV hiR2-FL-F588H-T7, pMSCV hiR2-FL-L589P-T7, pMSCV hiR2-FL-E594V-T7, pMSCV hiR2-FL-K596T-T7, pMSCV hiR2-FL-S607R-T7, pMSCV hiR2-FL-T612S-T7, pMSCV hiR2-FL-E617D-T7, pMSCV hiR2-FL-H620R-T7, pMSCV hiR2-FL-L636M-T7, pMSCV hiR2-FL-K638D-T7, pMSCV hiR2-FL-1771F-T7, pMSCV hiR2-FL-1825V-T7, pMSCV hiR2-FL-1828V-T7, pMSCV hiR2-FL-N829R-T7, pMSCV hiR2-FL-W830C-T7, pMSCV hiR2-FL-P831E-T7, pMSCV hiR2-FL-1833C-T7, pMSCV hiR2-FL-H835F-T7, pMSCV hiR2-FL-F8391-T7, pMSCV hiR2-FL-S843D-T7, pMSCV hiR2-FL-Q853A-T7, pMSCV hiR2-FL-V854Q-T7 and pMSCV hiR2-FL-R844K-T7 encoding human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV hiR2-FL-G563D-T7, pMSCV hiR2-FL-A564P-T7, pMSCV hiR2-FL-1566E-T7, pMSCV hiR2-FL-D569E-T7, pMSCV hiR2-FL-E579K-T7, pMSCV hiR2-FL-Q580N-T7, pMSCV hiR2-FL-A581S-T7, pMSCV hiR2-FL-R582A-T7, pMSCV hiR2-FL-S583G-T7, pMSCV hiR2-FL-G587N-T7, pMSCV hiR2-FL-F588H-T7, pMSCV hiR2-FL-L589P-T7, pMSCV hiR2-FL-E594V-T7, pMSCV hiR2-FL-K596T-T7, pMSCV hiR2-FL-S607R-T7, pMSCV hiR2-FL-T612S-T7, pMSCV hiR2-FL-E617D-T7, pMSCV hiR2-FL-H620R-T7, pMSCV hiR2-FL-L636M-T7, pMSCV hiR2-FL-K638D-T7, pMSCV hiR2-FL-1771F-T7, pMSCV hiR2-FL-1825V-T7, pMSCV hiR2-FL-1828V-T7, pMSCV hiR2-FL-N829R-T7, pMSCV hiR2-FL-W830C-T7, pMSCV hiR2-FL-P831E-T7, pMSCV hiR2-FL-1833C-T7, pMSCV hiR2-FL-H835F-T7, pMSCV hiR2-FL-F8391-T7, pMSCV hiR2-FL-S843D-T7, pMSCV hiR2-FL-Q853A-T7, pMSCV hiR2-FL-V854Q-T7 and pMSCV hiR2-FL-R844K-T7 ecotrophic virus were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO hiR2-FL-G563D-T7, MEF-DKO hiR2-FL-A564P-T7, MEF-DKO hiR2-FL-1566E-T7, MEF-DKO hiR2-FL-D569E-T7, MEF-DKO hiR2-FL-E579K-T7, MEF-DKO hiR2-FL-Q580N-T7, MEF-DKO hiR2-FL-A581S-T7, MEF-DKO hiR2-FL-R582A-T7, MEF-DKO hiR2-FL-S583G-T7, MEF-DKO hiR2-FL-G587N-T7, MEF-DKO hiR2-FL-F588H-T7, MEF-DKO hiR2-FL-L589P-T7, MEF-DKO hiR2-FL-E594V-T7, MEF-DKO hiR2-FL-K596T-T7, MEF-DKO hiR2-FL-S607R-T7, MEF-DKO hiR2-FL-T612S-T7, MEF-DKO hiR2-FL-E617D-T7, MEF-DKO hiR2-FL-H620R-T7, MEF-DKO hiR2-FL-L636M-T7, MEF-DKO hiR2-FL-K638D-T7, MEF-DKO hiR2-FL-1771F-T7, MEF-DKO hiR2-FL-1825V-T7, MEF-DKO hiR2-FL-I828V-T7, MEF-DKO hiR2-FL-N829R-T7, MEF-DKO hiR2-FL-W830C-T7, MEF-DKO hiR2-FL-P831E-T7, MEF-DKO hiR2-FL-1833C-T7, MEF-DKO hiR2-FL-H835F-T7, MEF-DKO hiR2-FL-F8391-T7, MEF-DKO hiR2-FL-S843D-T7, MEF-DKO hiR2-FL-Q853A-T7, MEF-DKO hiR2-FL-V854Q-T7 and MEF-DKO hiR2-FL-R844K-T7 cells stably expressing human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells and MEF-DKO hiR2-FL-G563D-T7, MEF-DKO hiR2-FL-A564P-T7, MEF-DKO hiR2-FL-1566E-T7, MEF-DKO hiR2-FL-D569E-T7, MEF-DKO hiR2-FL-E579K-T7, MEF-DKO hiR2-FL-Q580N-T7, MEF-DKO hiR2-FL-A581S-T7, MEF-DKO hiR2-FL-R582A-T7, MEF-DKO hiR2-FL-S583G-T7, MEF-DKO hiR2-FL-G587N-T7, MEF-DKO hiR2-FL-F588H-T7, MEF-DKO hiR2-FL-L589P-T7, MEF-DKO hiR2-FL-E594V-T7, MEF-DKO hiR2-FL-K596T-T7, MEF-DKO hiR2-FL-S607R-T7, MEF-DKO hiR2-FL-T612S-T7, MEF-DKO hiR2-FL-E617D-T7, MEF-DKO hiR2-FL-H620R-T7, MEF-DKO hiR2-FL-L636M-T7, MEF-DKO hiR2-FL-K638D-T7, MEF-DKO hiR2-FL-1771F-T7, MEF-DKO hiR2-FL-1825V-T7, MEF-DKO hiR2-FL-I828V-T7, MEF-DKO hiR2-FL-N829R-T7, MEF-DKO hiR2-FL-W830C-T7, MEF-DKO hiR2-FL-P831E-T7, MEF-DKO hiR2-FL-1833C-T7, MEF-DKO hiR2-FL-H835F-T7, MEF-DKO hiR2-FL-F8391-T7, MEF-DKO hiR2-FL-S843D-T7, MEF-DKO hiR2-FL-Q853A-T7, MEF-DKO hiR2-FL-V854Q-T7 and MEF-DKO hiR2-FL-R844K-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
To test all 33 human iRhom2 variants with human iRhom1-specific single amino acid substitutions, the respective MEF-DKO cell lines stably expressing these variants, generated as described in the example above, were subjected to TGFα shedding ELISA analysis. In order to demonstrate the functionality of all variants as an indicator that these variants are properly folded, PMA-induced release of nucleofected TGFα was assessed. As the cells used in this analysis are rescue variants of iRhom1/2−/− double knockout mouse embryonic fibroblasts (described in Example 11), that are rescued by the respective human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion, the iRhom2 variant stably expressed is the only iRhom protein expressed in these cells and is therefore the only iRhom contributing to the shedding TGFα in these cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 400 ng/ml in TBS at 4° C. After MEF-DKO hiR2-FL-G563D-T7, MEF-DKO hiR2-FL-A564P-T7, MEF-DKO hiR2-FL-I566E-T7, MEF-DKO hiR2-FL-D569E-T7, MEF-DKO hiR2-FL-E579K-T7, MEF-DKO hiR2-FL-Q580N-T7, MEF-DKO hiR2-FL-A581S-T7, MEF-DKO hiR2-FL-R582A-T7, MEF-DKO hiR2-FL-S583G-T7, MEF-DKO hiR2-FL-G587N-T7, MEF-DKO hiR2-FL-F588H-T7, MEF-DKO hiR2-FL-L589P-T7, MEF-DKO hiR2-FL-E594V-T7, MEF-DKO hiR2-FL-K596T-T7, MEF-DKO hiR2-FL-S607R-T7, MEF-DKO hiR2-FL-T612S-T7, MEF-DKO hiR2-FL-E617D-T7, MEF-DKO hiR2-FL-H620R-T7, MEF-DKO hiR2-FL-L636M-T7, MEF-DKO hiR2-FL-K638D-T7, MEF-DKO hiR2-FL-I771F-T7, MEF-DKO hiR2-FL-I825V-T7, MEF-DKO hiR2-FL-I828V-T7, MEF-DKO hiR2-FL-N829R-T7, MEF-DKO hiR2-FL-W830C-T7, MEF-DKO hiR2-FL-P831E-T7, MEF-DKO hiR2-FL-I833C-T7, MEF-DKO hiR2-FL-H835F-T7, MEF-DKO hiR2-FL-F839I-T7, MEF-DKO hiR2-FL-S843D-T7, MEF-DKO hiR2-FL-Q853A-T7, MEF-DKO hiR2-FL-V854Q-T7 and MEF-DKO hiR2-FL-R844K-T7 cells were electroporated with the hTGFα-FL-WT construct in a pcDNA3.1 vector backbone, using an 4D-Nucleofector System (Lonza, Switzerland), approximately 35,000 MEF-DKO cells carrying the human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion were seeded in 100 μl of normal growth medium in each well of F-bottom 96-well cell culture plates (Thermo Fisher Scientific, USA). On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for at least 1 hour. Meanwhile, the cells were washed once with PBS and afterwards 80 μl of OptiMEM medium (Thermo Fisher Scientific, USA) was added per well. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. 20 μl of OptiMEM medium was added to the unstimulated control cells. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In brief, immortalized MEF-DKO hiR2-FL-G563D-T7, MEF-DKO hiR2-FL-A564P-T7, MEF-DKO hiR2-FL-1566E-T7, MEF-DKO hiR2-FL-D569E-T7, MEF-DKO hiR2-FL-E579K-T7, MEF-DKO hiR2-FL-Q580N-T7, MEF-DKO hiR2-FL-A581S-T7, MEF-DKO hiR2-FL-R582A-T7, MEF-DKO hiR2-FL-S583G-T7, MEF-DKO hiR2-FL-G587N-T7, MEF-DKO hiR2-FL-F588H-T7, MEF-DKO hiR2-FL-L589P-T7, MEF-DKO hiR2-FL-E594V-T7, MEF-DKO hiR2-FL-K596T-T7, MEF-DKO hiR2-FL-S607R-T7, MEF-DKO hiR2-FL-T612S-T7, MEF-DKO hiR2-FL-E617D-T7, MEF-DKO hiR2-FL-H620R-T7, MEF-DKO hiR2-FL-L636M-T7, MEF-DKO hiR2-FL-K638D-T7, MEF-DKO hiR2-FL-1771F-T7, MEF-DKO hiR2-FL-1825V-T7, MEF-DKO hiR2-FL-1828V-T7, MEF-DKO hiR2-FL-N829R-T7, MEF-DKO hiR2-FL-W830C-T7, MEF-DKO hiR2-FL-P831E-T7, MEF-DKO hiR2-FL-I833C-T7, MEF-DKO hiR2-FL-H835F-T7, MEF-DKO hiR2-FL-F8391-T7, MEF-DKO hiR2-FL-S843D-T7, MEF-DKO hiR2-FL-Q853A-T7, MEF-DKO hiR2-FL-V854Q-T7 and MEF-DKO hiR2-FL-R844K-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention at 3 μg/ml in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
Complementary to Examples 15 and 16 and also to Examples 21 & 22 described above, plasmids for a set of 91 human iRhom2 variants with single amino acid substitutions to alanine to identify single amino acids that contribute to binding of the antibodies of the invention, were designed in a fifth approach. Instead of the amino acid of human iRhom2, the amino acid alanine was introduced at the corresponding position, resulting in the variants hiR2-FL-N503A-T7, hiR2-FL-D504A-T7, hiR2-FL-H505A-T7, hiR2-FL-S506A-T7, hiR2-FL-G507A-T7, hiR2-FL-C508A-T7, hiR2-FL-1509A-T7, hiR2-FL-Q510A-T7, hiR2-FL-T511A-T7, hiR2-FL-Q512A-T7, hiR2-FL-R513A-T7, hiR2-FL-K514A-T7, hiR2-FL-D515A-T7, hiR2-FL-C516A-T7, hiR2-FL-S517A-T7, hiR2-FL-E518A-T7, hiR2-FL-T519A-T7, hiR2-FL-L520A-T7, hiR2-FL-A521S-T7, hiR2-FL-T522A-T7, hiR2-FL-F523A-T7, hiR2-FL-V524A-T7, hiR2-FL-K525A-T7, hiR2-FL-W526A-T7, hiR2-FL-Q527A-T7, hiR2-FL-D528A-T7, hiR2-FL-D529A-T7, hiR2-FL-T530A-T7, hiR2-FL-G531A-T7, hiR2-FL-P532A-T7, hiR2-FL-P533A-T7, hiR2-FL-M534A-T7, hiR2-FL-D535A-T7, hiR2-FL-K536A-T7, hiR2-FL-S537A-T7, hiR2-FL-D538A-T7, hiR2-FL-L539A-T7, hiR2-FL-G540A-T7, hiR2-FL-Q541A-T7, hiR2-FL-K542A-T7, hiR2-FL-R543A-T7, hiR2-FL-T544A-T7, hiR2-FL-S545A-T7, hiR2-FL-G546A-T7, hiR2-FL-A547S-T7, hiR2-FL-V548A-T7, hiR2-FL-C549A-T7, hiR2-FL-H550A-T7, hiR2-FL-Q551A-T7, hiR2-FL-D552A-T7, hiR2-FL-P553A-T7, hiR2-FL-R554A-T7, hiR2-FL-T555A-T7, hiR2-FL-C556A-T7, hiR2-FL-E557A-T7, hiR2-FL-E558A-T7, hiR2-FL-P559A-T7, hiR2-FL-A560S-T7, hiR2-FL-S561A-T7, hiR2-FL-S562A-T7, hiR2-FL-G563A-T7, hiR2-FL-A564S-T7, hiR2-FL-H565A-T7, hiR2-FL-1566A-T7, hiR2-FL-W567A-T7, hiR2-FL-P568A-T7, hiR2-FL-D569A-T7, hiR2-FL-D570A-T7, hiR2-FL-I571A-T7, hiR2-FL-T572A-T7, hiR2-FL-K573A-T7, hiR2-FL-W574A-T7, hiR2-FL-P575A-T7, hiR2-FL-1576A-T7, hiR2-FL-C577A-T7, hiR2-FL-T578A-T7, hiR2-FL-E579A-T7, hiR2-FL-Q580A-T7, hiR2-FL-A581S-T7, hiR2-FL-R582A-T7, hiR2-FL-S583A-T7, hiR2-FL-N584A-T7, hiR2-FL-H585A-T7, hiR2-FL-T586A-T7, hiR2-FL-G587A-T7, hiR2-FL-F588A-T7, hiR2-FL-L589A-T7, hiR2-FL-H590A-T7, hiR2-FL-M591A-T7, hiR2-FL-D592A-T7 and hiR2-FL-C593A-T7.
This example describes the generation of iRhom1/2−/− DKO MEF populations expressing the 91 single alanine amino acid substitution variants as well as their characterization in terms of cell surface localization and functional activity as indicators of proper protein conformation. Subsequently, binding analyses of the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention on the entire panel of 91 engineered MEF populations expressing human iRhom2 variants with single amino acid substitutions to alanine are described.
Generation of iRhom1/2−/− DKO MEFs Stably Expressing 91 T7-Tagged Human iRhom2 Variants with Single Amino Acid Substitutions to Alanine
In brief, on day 1, Phoenix-ECO cells (American Type Culture Collection, USA) were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 8×105 cells per well and kept overnight at 37° C., 5% CO2. On day 2, the medium was replaced by fresh medium supplemented with chloroquine (Sigma-Aldrich, USA) at a final concentration of 25 μM. Applying the calcium phosphate method, cells were transfected with 2 μg/ml of pMSCV-hiR2-FL-N503A-T7, pMSCV-hiR2-FL-D504A-T7, pMSCV-hiR2-FL-H505A-T7, pMSCV-hiR2-FL-S506A-T7, pMSCV-hiR2-FL-G507A-T7, pMSCV-hiR2-FL-C508A-T7, pMSCV-hiR2-FL-I509A-T7, pMSCV-hiR2-FL-Q510A-T7, pMSCV-hiR2-FL-T511A-T7, pMSCV-hiR2-FL-Q512A-T7, pMSCV-hiR2-FL-R513A-T7, pMSCV-hiR2-FL-K514A-T7, pMSCV-hiR2-FL-D515A-T7, pMSCV-hiR2-FL-C516A-T7, pMSCV-hiR2-FL-S517A-T7, pMSCV-hiR2-FL-E518A-T7, pMSCV-hiR2-FL-T519A-T7, pMSCV-hiR2-FL-L520A-T7, pMSCV-hiR2-FL-A521S-T7, pMSCV-hiR2-FL-T522A-T7, pMSCV-hiR2-FL-F523A-T7, pMSCV-hiR2-FL-V524A-T7, pMSCV-hiR2-FL-K525A-T7, pMSCV-hiR2-FL-W526A-T7, pMSCV-hiR2-FL-Q527A-T7, pMSCV-hiR2-FL-D528A-T7, pMSCV-hiR2-FL-D529A-T7, pMSCV-hiR2-FL-T530A-T7, pMSCV-hiR2-FL-G531A-T7, pMSCV-hiR2-FL-P532A-T7, pMSCV-hiR2-FL-P533A-T7, pMSCV-hiR2-FL-M534A-T7, pMSCV-hiR2-FL-D535A-T7, pMSCV-hiR2-FL-K536A-T7, pMSCV-hiR2-FL-S537A-T7, pMSCV-hiR2-FL-D538A-T7, pMSCV-hiR2-FL-L539A-T7, pMSCV-hiR2-FL-G540A-T7, pMSCV-hiR2-FL-Q541A-T7, pMSCV-hiR2-FL-K542A-T7, pMSCV-hiR2-FL-R543A-T7, pMSCV-hiR2-FL-T544A-T7, pMSCV-hiR2-FL-S545A-T7, pMSCV-hiR2-FL-G546A-T7, pMSCV-hiR2-FL-A547S-T7, pMSCV-hiR2-FL-V548A-T7, pMSCV-hiR2-FL-C549A-T7, pMSCV-hiR2-FL-H550A-T7, pMSCV-hiR2-FL-Q551A-T7, pMSCV-hiR2-FL-D552A-T7, pMSCV-hiR2-FL-P553A-T7, pMSCV-hiR2-FL-R554A-T7, pMSCV-hiR2-FL-T555A-T7, pMSCV-hiR2-FL-C556A-T7, pMSCV-hiR2-FL-E557A-T7, pMSCV-hiR2-FL-E558A-T7, pMSCV-hiR2-FL-P559A-T7, pMSCV-hiR2-FL-A560S-T7, pMSCV-hiR2-FL-S561A-T7, pMSCV-hiR2-FL-S562A-T7, pMSCV-hiR2-FL-G563A-T7, pMSCV-hiR2-FL-A564S-T7, pMSCV-hiR2-FL-H565A-T7, pMSCV-hiR2-FL-I566A-T7, pMSCV-hiR2-FL-W567A-T7, pMSCV-hiR2-FL-P568A-T7, pMSCV-hiR2-FL-D569A-T7, pMSCV-hiR2-FL-D570A-T7, pMSCV-hiR2-FL-1571A-T7, pMSCV-hiR2-FL-T572A-T7, pMSCV-hiR2-FL-K573A-T7, pMSCV-hiR2-FL-W574A-T7, pMSCV-hiR2-FL-P575A-T7, pMSCV-hiR2-FL-1576A-T7, pMSCV-hiR2-FL-C577A-T7, pMSCV-hiR2-FL-T578A-T7, pMSCV-hiR2-FL-E579A-T7, pMSCV-hiR2-FL-Q580A-T7, pMSCV-hiR2-FL-A581S-T7, pMSCV-hiR2-FL-R582A-T7, pMSCV-hiR2-FL-S583A-T7, pMSCV-hiR2-FL-N584A-T7, pMSCV-hiR2-FL-H585A-T7, pMSCV-hiR2-FL-T586A-T7, pMSCV-hiR2-FL-G587A-T7, pMSCV-hiR2-FL-F588A-T7, pMSCV-hiR2-FL-L589A-T7, pMSCV-hiR2-FL-H590A-T7, pMSCV-hiR2-FL-M591A-T7, pMSCV-hiR2-FL-D592A-T7 and pMSCV-hiR2-FL-C593A-T7 encoding human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope (MASMTGGQQMG), and were kept at 37° C., 5% CO2. After 7 hours, the transfections were stopped by replacing cell supernatants with standard growth medium lacking chloroquine, and cells were incubated at 37° C., 5% CO2 to allow virus production overnight. In parallel, immortalized iRhom1/2−/− DKO MEFs as target cells for retroviral infection were seeded on 6-well tissue culture plates (Greiner, Germany) in standard growth medium at 1×105 cells per well and were also kept overnight at 37° C., 5% CO2. On day 3, the supernatants of Phoenix-ECO cells releasing pMSCV-hiR2-FL-N503A-T7, pMSCV-hiR2-FL-D504A-T7, pMSCV-hiR2-FL-H505A-T7, pMSCV-hiR2-FL-S506A-T7, pMSCV-hiR2-FL-G507A-T7, pMSCV-hiR2-FL-C508A-T7, pMSCV-hiR2-FL-1509A-T7, pMSCV-hiR2-FL-Q510A-T7, pMSCV-hiR2-FL-T511A-T7, pMSCV-hiR2-FL-Q512A-T7, pMSCV-hiR2-FL-R513A-T7, pMSCV-hiR2-FL-K514A-T7, pMSCV-hiR2-FL-D515A-T7, pMSCV-hiR2-FL-C516A-T7, pMSCV-hiR2-FL-S517A-T7, pMSCV-hiR2-FL-E518A-T7, pMSCV-hiR2-FL-T519A-T7, pMSCV-hiR2-FL-L520A-T7, pMSCV-hiR2-FL-A521S-T7, pMSCV-hiR2-FL-T522A-T7, pMSCV-hiR2-FL-F523A-T7, pMSCV-hiR2-FL-V524A-T7, pMSCV-hiR2-FL-K525A-T7, pMSCV-hiR2-FL-W526A-T7, pMSCV-hiR2-FL-Q527A-T7, pMSCV-hiR2-FL-D528A-T7, pMSCV-hiR2-FL-D529A-T7, pMSCV-hiR2-FL-T530A-T7, pMSCV-hiR2-FL-G531A-T7, pMSCV-hiR2-FL-P532A-T7, pMSCV-hiR2-FL-P533A-T7, pMSCV-hiR2-FL-M534A-T7, pMSCV-hiR2-FL-D535A-T7, pMSCV-hiR2-FL-K536A-T7, pMSCV-hiR2-FL-S537A-T7, pMSCV-hiR2-FL-D538A-T7, pMSCV-hiR2-FL-L539A-T7, pMSCV-hiR2-FL-G540A-T7, pMSCV-hiR2-FL-Q541A-T7, pMSCV-hiR2-FL-K542A-T7, pMSCV-hiR2-FL-R543A-T7, pMSCV-hiR2-FL-T544A-T7, pMSCV-hiR2-FL-S545A-T7, pMSCV-hiR2-FL-G546A-T7, pMSCV-hiR2-FL-A547S-T7, pMSCV-hiR2-FL-V548A-T7, pMSCV-hiR2-FL-C549A-T7, pMSCV-hiR2-FL-H550A-T7, pMSCV-hiR2-FL-Q551A-T7, pMSCV-hiR2-FL-D552A-T7, pMSCV-hiR2-FL-P553A-T7, pMSCV-hiR2-FL-R554A-T7, pMSCV-hiR2-FL-T555A-T7, pMSCV-hiR2-FL-C556A-T7, pMSCV-hiR2-FL-E557A-T7, pMSCV-hiR2-FL-E558A-T7, pMSCV-hiR2-FL-P559A-T7, pMSCV-hiR2-FL-A560S-T7, pMSCV-hiR2-FL-S561A-T7, pMSCV-hiR2-FL-S562A-T7, pMSCV-hiR2-FL-G563A-T7, pMSCV-hiR2-FL-A564S-T7, pMSCV-hiR2-FL-H565A-T7, pMSCV-hiR2-FL-1566A-T7, pMSCV-hiR2-FL-W567A-T7, pMSCV-hiR2-FL-P568A-T7, pMSCV-hiR2-FL-D569A-T7, pMSCV-hiR2-FL-D570A-T7, pMSCV-hiR2-FL-I571A-T7, pMSCV-hiR2-FL-T572A-T7, pMSCV-hiR2-FL-K573A-T7, pMSCV-hiR2-FL-W574A-T7, pMSCV-hiR2-FL-P575A-T7, pMSCV-hiR2-FL-1576A-T7, pMSCV-hiR2-FL-C577A-T7, pMSCV-hiR2-FL-T578A-T7, pMSCV-hiR2-FL-E579A-T7, pMSCV-hiR2-FL-Q580A-T7, pMSCV-hiR2-FL-A581S-T7, pMSCV-hiR2-FL-R582A-T7, pMSCV-hiR2-FL-S583A-T7, pMSCV-hiR2-FL-N584A-T7, pMSCV-hiR2-FL-H585A-T7, pMSCV-hiR2-FL-T586A-T7, pMSCV-hiR2-FL-G587A-T7, pMSCV-hiR2-FL-F588A-T7, pMSCV-hiR2-FL-L589A-T7, pMSCV-hiR2-FL-H590A-T7, pMSCV-hiR2-FL-M591A-T7, pMSCV-hiR2-FL-D592A-T7 and pMSCV-hiR2-FL-C593A-T7 ecotrophic virus were collected, filtered with 0.45 μm CA filters, and supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, USA). Upon removal of medium from the immortalized iRhom1/2−/− DKO MEFs, these supernatants were added to the target cells for 4 hours at 37° C., 5% CO2 for first infection. Simultaneously, Phoenix-ECO cells were re-incubated with fresh medium, which, after another 4 hours, was filtered and used for the second infection of the respective target cell populations, again in the presence of 4 μg/ml of polybrene. Likewise, a third, but overnight infection cycle was performed. On day 4, virus containing cell supernatants were replaced by fresh standard growth medium. From day 5 onwards, cells were grown in the presence of 2 mg/ml of geneticin (G418, Thermo Fisher Scientific, USA) for the selection of immortalized MEF-DKO-hiR2-FL-N503A-T7, MEF-DKO-hiR2-FL-D504A-T7, MEF-DKO-hiR2-FL-H505A-T7, MEF-DKO-hiR2-FL-S506A-T7, MEF-DKO-hiR2-FL-G507A-T7, MEF-DKO-hiR2-FL-C508A-T7, MEF-DKO-hiR2-FL-I509A-T7, MEF-DKO-hiR2-FL-Q510A-T7, MEF-DKO-hiR2-FL-T511A-T7, MEF-DKO-hiR2-FL-Q512A-T7, MEF-DKO-hiR2-FL-R513A-T7, MEF-DKO-hiR2-FL-K514A-T7, MEF-DKO-hiR2-FL-D515A-T7, MEF-DKO-hiR2-FL-C516A-T7, MEF-DKO-hiR2-FL-S517A-T7, MEF-DKO-hiR2-FL-E518A-T7, MEF-DKO-hiR2-FL-T519A-T7, MEF-DKO-hiR2-FL-L520A-T7, MEF-DKO-hiR2-FL-A521S-T7, MEF-DKO-hiR2-FL-T522A-T7, MEF-DKO-hiR2-FL-F523A-T7, MEF-DKO-hiR2-FL-V524A-T7, MEF-DKO-hiR2-FL-K525A-T7, MEF-DKO-hiR2-FL-W526A-T7, MEF-DKO-hiR2-FL-Q527A-T7, MEF-DKO-hiR2-FL-D528A-T7, MEF-DKO-hiR2-FL-D529A-T7, MEF-DKO-hiR2-FL-T530A-T7, MEF-DKO-hiR2-FL-G531A-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-P533A-T7, MEF-DKO-hiR2-FL-M534A-T7, MEF-DKO-hiR2-FL-D535A-T7, MEF-DKO-hiR2-FL-K536A-T7, MEF-DKO-hiR2-FL-S537A-T7, MEF-DKO-hiR2-FL-D538A-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-G540A-T7, MEF-DKO-hiR2-FL-Q541A-T7, MEF-DKO-hiR2-FL-K542A-T7, MEF-DKO-hiR2-FL-R543A-T7, MEF-DKO-hiR2-FL-T544A-T7, MEF-DKO-hiR2-FL-S545A-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-V548A-T7, MEF-DKO-hiR2-FL-C549A-T7, MEF-DKO-hiR2-FL-H550A-T7, MEF-DKO-hiR2-FL-Q551A-T7, MEF-DKO-hiR2-FL-D552A-T7, MEF-DKO-hiR2-FL-P553A-T7, MEF-DKO-hiR2-FL-R554A-T7, MEF-DKO-hiR2-FL-T555A-T7, MEF-DKO-hiR2-FL-C556A-T7, MEF-DKO-hiR2-FL-E557A-T7, MEF-DKO-hiR2-FL-E558A-T7, MEF-DKO-hiR2-FL-P559A-T7, MEF-DKO-hiR2-FL-A560S-T7, MEF-DKO-hiR2-FL-S561A-T7, MEF-DKO-hiR2-FL-S562A-T7, MEF-DKO-hiR2-FL-G563A-T7, MEF-DKO-hiR2-FL-A564S-T7, MEF-DKO-hiR2-FL-H565A-T7, MEF-DKO-hiR2-FL-1566A-T7, MEF-DKO-hiR2-FL-W567A-T7, MEF-DKO-hiR2-FL-P568A-T7, MEF-DKO-hiR2-FL-D569A-T7, MEF-DKO-hiR2-FL-D570A-T7, MEF-DKO-hiR2-FL-1571A-T7, MEF-DKO-hiR2-FL-T572A-T7, MEF-DKO-hiR2-FL-K573A-T7, MEF-DKO-hiR2-FL-W574A-T7, MEF-DKO-hiR2-FL-P575A-T7, MEF-DKO-hiR2-FL-1576A-T7, MEF-DKO-hiR2-FL-C577A-T7, MEF-DKO-hiR2-FL-T578A-T7, MEF-DKO-hiR2-FL-E579A-T7, MEF-DKO-hiR2-FL-Q580A-T7, MEF-DKO-hiR2-FL-A581S-T7, MEF-DKO-hiR2-FL-R582A-T7, MEF-DKO-hiR2-FL-S583A-T7, MEF-DKO-hiR2-FL-N584A-T7, MEF-DKO-hiR2-FL-H585A-T7, MEF-DKO-hiR2-FL-T586A-T7, MEF-DKO-hiR2-FL-G587A-T7, MEF-DKO-hiR2-FL-F588A-T7, MEF-DKO-hiR2-FL-L589A-T7, MEF-DKO-hiR2-FL-H590A-T7, MEF-DKO-hiR2-FL-M591A-T7, MEF-DKO-hiR2-FL-D592A-T7 and MEF-DKO-hiR2-FL-C593A-T7 cells stably expressing human iRhom2 full length single amino acid substitutions C-terminally tagged with 3 consecutive copies of the T7 epitope. Upon propagation, cells were stocked for future usage.
In brief, immortalized MEF-DKO-hiR2-FL-WT-T7 cells and MEF-DKO-hiR2-FL-N503A-T7, MEF-DKO-hiR2-FL-D504A-T7, MEF-DKO-hiR2-FL-H505A-T7, MEF-DKO-hiR2-FL-S506A-T7, MEF-DKO-hiR2-FL-G507A-T7, MEF-DKO-hiR2-FL-C508A-T7, MEF-DKO-hiR2-FL-I509A-T7, MEF-DKO-hiR2-FL-Q510A-T7, MEF-DKO-hiR2-FL-T511A-T7, MEF-DKO-hiR2-FL-Q512A-T7, MEF-DKO-hiR2-FL-R513A-T7, MEF-DKO-hiR2-FL-K514A-T7, MEF-DKO-hiR2-FL-D515A-T7, MEF-DKO-hiR2-FL-C516A-T7, MEF-DKO-hiR2-FL-S517A-T7, MEF-DKO-hiR2-FL-E518A-T7, MEF-DKO-hiR2-FL-T519A-T7, MEF-DKO-hiR2-FL-L520A-T7, MEF-DKO-hiR2-FL-A521S-T7, MEF-DKO-hiR2-FL-T522A-T7, MEF-DKO-hiR2-FL-F523A-T7, MEF-DKO-hiR2-FL-V524A-T7, MEF-DKO-hiR2-FL-K525A-T7, MEF-DKO-hiR2-FL-W526A-T7, MEF-DKO-hiR2-FL-Q527A-T7, MEF-DKO-hiR2-FL-D528A-T7, MEF-DKO-hiR2-FL-D529A-T7, MEF-DKO-hiR2-FL-T530A-T7, MEF-DKO-hiR2-FL-G531A-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-P533A-T7, MEF-DKO-hiR2-FL-M534A-T7, MEF-DKO-hiR2-FL-D535A-T7, MEF-DKO-hiR2-FL-K536A-T7, MEF-DKO-hiR2-FL-S537A-T7, MEF-DKO-hiR2-FL-D538A-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-G540A-T7, MEF-DKO-hiR2-FL-Q541A-T7, MEF-DKO-hiR2-FL-K542A-T7, MEF-DKO-hiR2-FL-R543A-T7, MEF-DKO-hiR2-FL-T544A-T7, MEF-DKO-hiR2-FL-S545A-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-V548A-T7, MEF-DKO-hiR2-FL-C549A-T7, MEF-DKO-hiR2-FL-H550A-T7, MEF-DKO-hiR2-FL-Q551A-T7, MEF-DKO-hiR2-FL-D552A-T7, MEF-DKO-hiR2-FL-P553A-T7, MEF-DKO-hiR2-FL-R554A-T7, MEF-DKO-hiR2-FL-T555A-T7, MEF-DKO-hiR2-FL-C556A-T7, MEF-DKO-hiR2-FL-E557A-T7, MEF-DKO-hiR2-FL-E558A-T7, MEF-DKO-hiR2-FL-P559A-T7, MEF-DKO-hiR2-FL-A560S-T7, MEF-DKO-hiR2-FL-S561A-T7, MEF-DKO-hiR2-FL-S562A-T7, MEF-DKO-hiR2-FL-G563A-T7, MEF-DKO-hiR2-FL-A564S-T7, MEF-DKO-hiR2-FL-H565A-T7, MEF-DKO-hiR2-FL-1566A-T7, MEF-DKO-hiR2-FL-W567A-T7, MEF-DKO-hiR2-FL-P568A-T7, MEF-DKO-hiR2-FL-D569A-T7, MEF-DKO-hiR2-FL-D570A-T7, MEF-DKO-hiR2-FL-I571A-T7, MEF-DKO-hiR2-FL-T572A-T7, MEF-DKO-hiR2-FL-K573A-T7, MEF-DKO-hiR2-FL-W574A-T7, MEF-DKO-hiR2-FL-P575A-T7, MEF-DKO-hiR2-FL-1576A-T7, MEF-DKO-hiR2-FL-C577A-T7, MEF-DKO-hiR2-FL-T578A-T7, MEF-DKO-hiR2-FL-E579A-T7, MEF-DKO-hiR2-FL-Q580A-T7, MEF-DKO-hiR2-FL-A581S-T7, MEF-DKO-hiR2-FL-R582A-T7, MEF-DKO-hiR2-FL-S583A-T7, MEF-DKO-hiR2-FL-N584A-T7, MEF-DKO-hiR2-FL-H585A-T7, MEF-DKO-hiR2-FL-T586A-T7, MEF-DKO-hiR2-FL-G587A-T7, MEF-DKO-hiR2-FL-F588A-T7, MEF-DKO-hiR2-FL-L589A-T7, MEF-DKO-hiR2-FL-H590A-T7, MEF-DKO-hiR2-FL-M591A-T7, MEF-DKO-hiR2-FL-D592A-T7 and MEF-DKO-hiR2-FL-C593A-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or mouse monoclonal anti-T7 IgG (Merck Millipore, USA) at 3 μg/ml FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
To test all 91 human iRhom2 variants with human iRhom1-specific single amino acid substitutions, the respective MEF-DKO cell lines stably expressing these variants, generated as described in the example above, were subjected to TGFα shedding ELISA analysis. In order to demonstrate the functionality of all variants as an indicator that these variants are properly folded, PMA-induced release of nucleofected TGFα was assessed. As the cells used in this analysis are rescue variants of iRhom1/2−/− double knockout mouse embryonic fibroblasts (described in Example 11), that are rescued by the respective human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion, the iRhom2 variant stably expressed is the only iRhom protein expressed in these cells at all and is therefore the only contributing iRhom to the shedding TGFα in these cells.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 400 ng/ml in TBS at 4° C. After MEF-DKO-hiR2-FL-N503A-T7, MEF-DKO-hiR2-FL-D504A-T7, MEF-DKO-hiR2-FL-H505A-T7, MEF-DKO-hiR2-FL-S506A-T7, MEF-DKO-hiR2-FL-G507A-T7, MEF-DKO-hiR2-FL-C508A-T7, MEF-DKO-hiR2-FL-1509A-T7, MEF-DKO-hiR2-FL-Q510A-T7, MEF-DKO-hiR2-FL-T511A-T7, MEF-DKO-hiR2-FL-Q512A-T7, MEF-DKO-hiR2-FL-R513A-T7, MEF-DKO-hiR2-FL-K514A-T7, MEF-DKO-hiR2-FL-D515A-T7, MEF-DKO-hiR2-FL-C516A-T7, MEF-DKO-hiR2-FL-S517A-T7, MEF-DKO-hiR2-FL-E518A-T7, MEF-DKO-hiR2-FL-T519A-T7, MEF-DKO-hiR2-FL-L520A-T7, MEF-DKO-hiR2-FL-A521S-T7, MEF-DKO-hiR2-FL-T522A-T7, MEF-DKO-hiR2-FL-F523A-T7, MEF-DKO-hiR2-FL-V524A-T7, MEF-DKO-hiR2-FL-K525A-T7, MEF-DKO-hiR2-FL-W526A-T7, MEF-DKO-hiR2-FL-Q527A-T7, MEF-DKO-hiR2-FL-D528A-T7, MEF-DKO-hiR2-FL-D529A-T7, MEF-DKO-hiR2-FL-T530A-T7, MEF-DKO-hiR2-FL-G531A-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-P533A-T7, MEF-DKO-hiR2-FL-M534A-T7, MEF-DKO-hiR2-FL-D535A-T7, MEF-DKO-hiR2-FL-K536A-T7, MEF-DKO-hiR2-FL-S537A-T7, MEF-DKO-hiR2-FL-D538A-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-G540A-T7, MEF-DKO-hiR2-FL-Q541A-T7, MEF-DKO-hiR2-FL-K542A-T7, MEF-DKO-hiR2-FL-R543A-T7, MEF-DKO-hiR2-FL-T544A-T7, MEF-DKO-hiR2-FL-S545A-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-V548A-T7, MEF-DKO-hiR2-FL-C549A-T7, MEF-DKO-hiR2-FL-H550A-T7, MEF-DKO-hiR2-FL-Q551A-T7, MEF-DKO-hiR2-FL-D552A-T7, MEF-DKO-hiR2-FL-P553A-T7, MEF-DKO-hiR2-FL-R554A-T7, MEF-DKO-hiR2-FL-T555A-T7, MEF-DKO-hiR2-FL-C556A-T7, MEF-DKO-hiR2-FL-E557A-T7, MEF-DKO-hiR2-FL-E558A-T7, MEF-DKO-hiR2-FL-P559A-T7, MEF-DKO-hiR2-FL-A560S-T7, MEF-DKO-hiR2-FL-S561A-T7, MEF-DKO-hiR2-FL-S562A-T7, MEF-DKO-hiR2-FL-G563A-T7, MEF-DKO-hiR2-FL-A564S-T7, MEF-DKO-hiR2-FL-H565A-T7, MEF-DKO-hiR2-FL-1566A-T7, MEF-DKO-hiR2-FL-W567A-T7, MEF-DKO-hiR2-FL-P568A-T7, MEF-DKO-hiR2-FL-D569A-T7, MEF-DKO-hiR2-FL-D570A-T7, MEF-DKO-hiR2-FL-1571A-T7, MEF-DKO-hiR2-FL-T572A-T7, MEF-DKO-hiR2-FL-K573A-T7, MEF-DKO-hiR2-FL-W574A-T7, MEF-DKO-hiR2-FL-P575A-T7, MEF-DKO-hiR2-FL-1576A-T7, MEF-DKO-hiR2-FL-C577A-T7, MEF-DKO-hiR2-FL-T578A-T7, MEF-DKO-hiR2-FL-E579A-T7, MEF-DKO-hiR2-FL-Q580A-T7, MEF-DKO-hiR2-FL-A581S-T7, MEF-DKO-hiR2-FL-R582A-T7, MEF-DKO-hiR2-FL-S583A-T7, MEF-DKO-hiR2-FL-N584A-T7, MEF-DKO-hiR2-FL-H585A-T7, MEF-DKO-hiR2-FL-T586A-T7, MEF-DKO-hiR2-FL-G587A-T7, MEF-DKO-hiR2-FL-F588A-T7, MEF-DKO-hiR2-FL-L589A-T7, MEF-DKO-hiR2-FL-H590A-T7, MEF-DKO-hiR2-FL-M591A-T7, MEF-DKO-hiR2-FL-D592A-T7 and MEF-DKO-hiR2-FL-C593A-T7 cells were electroporated with the hTGFα-FL-WT construct in a pcDNA3.1 vector backbone, using an 4D-Nucleofector System (Lonza, Switzerland), approximately 35,000 MEF-DKO cells carrying the human iRhom2 variant with the human iRhom1-specific single amino acid substitution or deletion were seeded in 100 μl of normal growth medium in each well of F-bottom 96-well cell culture plates (Thermo Fisher Scientific, USA). On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for at least 1 hour. Meanwhile, the cells were washed once with PBS and afterwards 80 μl of OptiMEM medium (Thermo Fisher Scientific, USA) was added per well.
Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. 20 μl of OptiMEM medium was added to the unstimulated control cells. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In brief, immortalized MEF-DKO-hiR2-FL-N503A-T7, MEF-DKO-hiR2-FL-D504A-T7, MEF-DKO-hiR2-FL-H505A-T7, MEF-DKO-hiR2-FL-S506A-T7, MEF-DKO-hiR2-FL-G507A-T7, MEF-DKO-hiR2-FL-C508A-T7, MEF-DKO-hiR2-FL-1509A-T7, MEF-DKO-hiR2-FL-Q510A-T7, MEF-DKO-hiR2-FL-T511A-T7, MEF-DKO-hiR2-FL-Q512A-T7, MEF-DKO-hiR2-FL-R513A-T7, MEF-DKO-hiR2-FL-K514A-T7, MEF-DKO-hiR2-FL-D515A-T7, MEF-DKO-hiR2-FL-S517A-T7, MEF-DKO-hiR2-FL-E518A-T7, MEF-DKO-hiR2-FL-T519A-T7, MEF-DKO-hiR2-FL-L520A-T7, MEF-DKO-hiR2-FL-A521S-T7, MEF-DKO-hiR2-FL-T522A-T7, MEF-DKO-hiR2-FL-V524A-T7, MEF-DKO-hiR2-FL-K525A-T7, MEF-DKO-hiR2-FL-W526A-T7, MEF-DKO-hiR2-FL-Q527A-T7, MEF-DKO-hiR2-FL-D528A-T7, MEF-DKO-hiR2-FL-D529A-T7, MEF-DKO-hiR2-FL-T530A-T7, MEF-DKO-hiR2-FL-G531A-T7, MEF-DKO-hiR2-FL-P532A-T7, MEF-DKO-hiR2-FL-P533A-T7, MEF-DKO-hiR2-FL-M534A-T7, MEF-DKO-hiR2-FL-D535A-T7, MEF-DKO-hiR2-FL-K536A-T7, MEF-DKO-hiR2-FL-S537A-T7, MEF-DKO-hiR2-FL-D538A-T7, MEF-DKO-hiR2-FL-L539A-T7, MEF-DKO-hiR2-FL-G540A-T7, MEF-DKO-hiR2-FL-Q541A-T7, MEF-DKO-hiR2-FL-K542A-T7, MEF-DKO-hiR2-FL-R543A-T7, MEF-DKO-hiR2-FL-T544A-T7, MEF-DKO-hiR2-FL-S545A-T7, MEF-DKO-hiR2-FL-G546A-T7, MEF-DKO-hiR2-FL-A547S-T7, MEF-DKO-hiR2-FL-V548A-T7, MEF-DKO-hiR2-FL-H550A-T7, MEF-DKO-hiR2-FL-Q551A-T7, MEF-DKO-hiR2-FL-P553A-T7, MEF-DKO-hiR2-FL-R554A-T7, MEF-DKO-hiR2-FL-T555A-T7, MEF-DKO-hiR2-FL-E557A-T7, MEF-DKO-hiR2-FL-E558A-T7, MEF-DKO-hiR2-FL-P559A-T7, MEF-DKO-hiR2-FL-A560S-T7, MEF-DKO-hiR2-FL-S561A-T7, MEF-DKO-hiR2-FL-S562A-T7, MEF-DKO-hiR2-FL-G563A-T7, MEF-DKO-hiR2-FL-A564S-T7, MEF-DKO-hiR2-FL-H565A-T7, MEF-DKO-hiR2-FL-1566A-T7, MEF-DKO-hiR2-FL-P568A-T7, MEF-DKO-hiR2-FL-D569A-T7, MEF-DKO-hiR2-FL-D570A-T7, MEF-DKO-hiR2-FL-1571A-T7, MEF-DKO-hiR2-FL-T572A-T7, MEF-DKO-hiR2-FL-K573A-T7, MEF-DKO-hiR2-FL-P575A-T7, MEF-DKO-hiR2-FL-I576A-T7, MEF-DKO-hiR2-FL-T578A-T7, MEF-DKO-hiR2-FL-E579A-T7, MEF-DKO-hiR2-FL-Q580A-T7, MEF-DKO-hiR2-FL-A581S-T7, MEF-DKO-hiR2-FL-R582A-T7, MEF-DKO-hiR2-FL-S583A-T7, MEF-DKO-hiR2-FL-N584A-T7, MEF-DKO-hiR2-FL-H585A-T7, MEF-DKO-hiR2-FL-T586A-T7, MEF-DKO-hiR2-FL-G587A-T7, MEF-DKO-hiR2-FL-F588A-T7, MEF-DKO-hiR2-FL-L589A-T7, MEF-DKO-hiR2-FL-H590A-T7, MEF-DKO-hiR2-FL-M591A-T7, MEF-DKO-hiR2-FL-D592A-T7 and MEF-DKO-hiR2-FL-C593A-T7 cells were harvested with 10 mM EDTA in PBS, washed and resuspended in FACS buffer (PBS, 3% FBS, 0.05% sodium azide), and seeded in Nunc U-bottom 96-well plates (Thermo Fisher Scientific, USA) at approximately 1×105 cells per well. To pellet cells and remove supernatants, the plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes. For primary staining, cells were resuspended in 100 μl per well of either FACS buffer alone (controls) or the purified antibodies 3, 5, 16, 22, 34, 42, 43, 44, 48, and 50 of the invention at 3 μg/ml in FACS buffer and incubated on ice for 1 hour. Afterwards, plates were centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed twice with 200 μl per well of FACS buffer. For secondary staining, cells were spun down and resuspended in 100 μl per well of PE-conjugated goat anti-mouse IgG F(ab′)2 detection fragment (Dianova, Germany) diluted 1:100 in FACS buffer. Protected from light, the cell suspensions were incubated on ice for 1 hour. Plates were then centrifuged at 1,500 rpm and 4° C. for 3 minutes and washed three times with 200 μl per well of FACS buffer. Finally, cells were resuspended in 150 μl per well of FACS buffer and analyzed using a BD Accuri™ C6 Plus flow cytometer (Becton Dickinson, Germany).
In contrast to Example 14 and 17, where the inhibitory effects of the antibodies of the invention on LPS-induced release of endogenous TNFα from human THP-1 cells were tested, this analysis was conducted with recombinantly produced murine antibodies of the invention to verify their inhibitory effects on PMA-induced release of endogenous TNFα from human monocytic U-937 cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based TNFα release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1-2 hours. Meanwhile, 80,000 U-937 (European Collection of Authenticated Cell Cultures, UK) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Complementary to Example 24 described above, ELISA-based TNFα release assays were performed to verify the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous TNFα from human U-937 cells. However, this analysis was conducted with both recombinantly produced murine and recombinantly produced chimeric antibodies of the invention.
To produce the recombinant antibody material, target DNA sequence was designed, optimized and synthesized. The complete sequence was sub-cloned into pcDNA3.4 vector (Thermo Fisher Scientific, USA), for the murine material, or into pTT5 vector (Thermo Fisher Scientific, USA), for the chimeric material and the transfection grade plasmid was maxi-prepared for Expi293F (Thermo Fisher Scientific, USA), for the murine material or for CHO-3E7 or HD CHO-S(Thermo Fisher Scientific, USA) for the chimeric material, cell expression. Expi293F cells were grown in serum-free Expi293FTM expression medium (Thermo Fisher Scientific, USA), CHO cells were grown in serum-free FreeStyle™ CHO Expression Medium (Thermo Fisher Scientific, USA) in Erlenmeyer flasks (Corning Inc., USA) at 37° C. with 5-8% CO2 on an orbital shaker (VWR Scientific, Germany). One day before transfection, the cells were seeded at an appropriate density in new Erlenmeyer flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and then added into the flask with cells ready for transfection. The recombinant plasmids encoding target protein were transiently transfected into suspension Expi293F cell cultures, for the murine material or into suspension CHO cell cultures, for the chimeric material. The cell culture supernatant collected on day 6 post-transfection was used for purification. Cell culture broth was centrifuged and filtrated. Filtered cell culture supernatant was loaded onto either HiTrap MabSelect SuRe (GE Healthcare, UK), MabSelect SuRe™ LX (GE Healthcare, UK) or RoboColumn Eshmuno A (Merck Millipore, USA) affinity purification columns at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to final formulation buffer. The purified protein was analyzed by SDS-PAGE analysis for molecular weight and purity measurements. Finally, the concentration was determined applying a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1-2 hours. Meanwhile, 80,000 U-937 (European Collection of Authenticated Cell Cultures, UK) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse or human IgG antibody (Thermo Fisher Scientific, USA) at 15 μg/ml as isotype control (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 15 μg/ml (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In the following study, ELISA-based IL-6R release assays were performed to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from human THP-1 monocytic cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based IL-6R release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated for 7 hours with 100 μl per well of mouse anti-human IL-6R capture antibody (provided as part of the DuoSet ELISA kit) at 2 μg/ml TBS at room temperature. Capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1.5 hours. Meanwhile, 40,000 THP-1 (American Type Culture Collection, USA) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human IL-6R protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human IL-6R detection antibody (provided as part of the DuoSet ELISA kit) at 100 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Complementary to Example 26 described above, ELISA-based IL-6R release assays were performed to verify the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from human U-937 cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based IL-6R release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human IL-6R capture antibody (provided as part of the DuoSet ELISA kit) at 2 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1-2 hours. Meanwhile, 80,000 U-937 (European Collection of Authenticated Cell Cultures, UK) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 375 ng/ml in growth medium for a final concentration of 62.5 ng/ml at 37° C., 5% CO2 for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human IL-6R protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human IL-6R detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Complementary to Example 27 described above, ELISA-based IL-6R release assays were performed to verify the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from human U-937 cells. However, this analysis was conducted with both recombinant produced murine and recombinant produced chimeric antibodies of the invention.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 25. The ELISA-based IL-6R release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated for 7 hours with 100 μl per well of mouse anti-human IL-6R capture antibody (provided as part of the DuoSet ELISA kit) at 2 μg/ml TBS at room temperature. Capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1 hour. Meanwhile, 80,000 U-937 (European Collection of Authenticated Cell Cultures, UK) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse or human IgG antibody (Thermo Fisher Scientific, USA) at 15 μg/ml as isotype control (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 15 μg/ml (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 375 ng/ml in growth medium for a final concentration of 62.5 ng/ml at 37° C., 5% CO2 for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human IL-6R protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human IL-6R detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In the following study, ELISA-based HB-EGF release assays were performed to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous HB-EGF from human THP-1 monocytic cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 25. The ELISA-based HB-EGF release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated for 7 hours with 100 μl per well of rat anti-human HB-EGF capture antibody (provided as part of the DuoSet ELISA kit) at 2 μg/ml TBS at room temperature. Capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 80,000 THP-1 (American Type Culture Collection, USA) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse or human IgG antibody (Thermo Fisher Scientific, USA) at 15 μg/ml as isotype control (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 15 μg/ml (for a final concentration of 3 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 6 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human HB-EGF protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human HB-EGF detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes.
Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Complementary to Example 29 described above, ELISA-based HB-EGF release assays were performed to verify the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous HB-EGF from human U-937 cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 25. The ELISA-based HB-EGF release assay that was used in this example is identical with the one described in Example 30, with the only difference, that U-937 (European Collection of Authenticated Cell Cultures, UK) cells were used instead of THP-1 (American Type Culture Collection, USA) cells.
Highly comparable to the results obtained with the murine antibodies of the invention, an equal concentration of the chimeric antibodies chl6, ch22, ch34, ch42 and ch44 of the invention inhibits PMA-induced release of HB-EGF from U-937 cells by 100.8%, 103.2%, 98.1%, 103.0% and 99.2%, respectively.
In the following study, ELISA-based TGFα release assays were performed to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous TGFα from human PC3 prostate cancer cells.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based TGFα release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of goat anti-human TGFα capture antibody (provided as part of the DuoSet ELISA kit) at 0.4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 100,000 PC3 (European Collection of Authenticated Cell Cultures, UK) cells in 80 μl of normal growth medium were seeded in each well of F-bottom 96-well cell culture plates (Coming, USA) and pre-incubated with 20 μl per well of OptiMEM medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of OptiMEM medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in OptiMEM for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 2 hours. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TGFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TGFα detection antibody (provided as part of the DuoSet ELISA kit) at 37.5 ng/ml in TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In the following study, ELISA-based TNFα release assays were performed to analyze the inhibitory effects of the antibodies of the invention on LPS-induced release of endogenous TNFα from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs).
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based TNFα release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 20,000 PBMC from healthy donors (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of LPS (Sigma-Aldrich, USA) at 300 ng/ml in growth medium for a final concentration of 50 ng/ml at 37° C., 5% CO2 for 2 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
Complementary to Example 32 described above, ELISA-based TNFα release assays were performed to test the inhibitory effects of the antibodies on LPS-induced release of endogenous TNFα from human macrophages from healthy donors.
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The isolation of macrophages and the ELISA-based TNFα release assay that was used in this example are described below.
To obtain human macrophages, peripheral blood mononuclear cells (PBMCs) from 4 healthy donors were used. Cells from 5 ml human blood were resuspended in 20 ml of DMEM medium (Corning, USA) supplemented with 10% FCS (Atlanta Biological, USA) and Penicillin/Streptomycin (Corning, USA) (DMEM/FCS) and were then incubated on two 10 cm petri dishes (Thermo Fisher, USA) at 37° C., 5% CO2 for 3h. Non-adherent cells were subsequently removed and 6 ml of DMEM/FCS supplemented with 10 ng/ml human MCSF (Peprotech, USA) (DMEM/FCS/MCSF) was added per plate. Both plates were incubated at 37° C., 5% CO2 for 2 days, then 2 ml of DMEM/FCS/MCSF was added to each plate on day 2 and 4. On day 5, all medium was removed and attached cells were washed once with 5 ml PBS (Corning, USA). 2 ml of Accutase (Promocell, USA) solution was added to each plate and incubated at 37° C. for 15 min. 10 ml of DMEM/FCS was added to each plate and detached cells were collected in a 15 ml Falcon tube. Cells were subsequently pelleted in a tabletop centrifuge (Eppendorf, USA) at 1,500 rpm for 5 min. The medium was removed and the cells were resuspended in 2 ml of DMEM/FCS. 10 μl from this cell suspension was manually counted in a hemocytometer (Thermo Fisher, USA). The cell number was adjusted to 2.5×105 cells/ml and 200 μl of the suspension was plated in one well of a 48-well tissue culture plate (Corning, USA) resulting in a cell density of 0.5×105 cells per 48-well. Cells were then incubated for 16 h at 37° C. For measuring TNFα released from PBMC cells, a OptEIA human TNFα ELISA (BD, USA) was used. Briefly, on day 1, Costar® 96-well plates (Corning, USA) were coated overnight with 100 μl per well of anti-human TNFα capture antibody (provided as part of the OptEIA® ELISA kit) at 1:250 in PBS at 4° C. On day 2, the capture antibody solution was removed, Costar® plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and plates were then blocked with 300 μl per well of PBS, 10% FCS at room temperature for 2 hours.
Meanwhile, on day 2, human macrophages were pre-incubated with 200 μl per well of DMEM/FCS supplemented with Batimastat (BB94, Abeam, MA, USA) at 20 μM as positive control (for a final concentration of 10 μM in the resulting 400 μl sample volume), or purified antibodies of the invention at 20 μg/ml (for a final concentration of 10 μg/ml in the resulting 400 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of unstimulated controls, 200 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 200 μl per well of LPS (Sigma-Aldrich, USA) at 10 ng/ml in growth medium for a final concentration of 5 ng/ml at 37° C., 5% CO2 for 2 hours. After 2 h, supernatants were removed and debris was spun out at 13,000 rpm at 4° C. in a tabletop centrifuge (Eppendorf, USA) and clarified supernatants were diluted 1:10 with PBS-T (Boston Bio, USA). Diluted supernatants were kept on ice until added to ELISA plates. In parallel, blocking buffer was removed from the Costar® plates and plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA). Immediately after, 100 μl clear, diluted supernatant was added to wells. Additionally, 100 μl recombinant human TNFα protein (provided as part of the OptEIA ELISA kit) diluted in PBS-BSA at defined concentrations were added to the plate as standard references. Samples and standards were incubated for 2h at room temperature. Thereafter, plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA). Then, 100 μl biotinylated anti-human TNFα detection antibody (provided as part of OptEIA ELISA kit) at a dilution of 1:250 in PBS-FCS was added per well and plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-horseradish peroxidase conjugate (provided as part of OptEIA ELISA kit) diluted 1:40 in PBS-FCS were added to each well and plates were incubated at room temperature for 20 minutes. Following another round of 4 washes with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and careful removal of all buffer traces after the fourth cycle, 100 μl TMB substrate solution (BD, USA) per well was added for incubation for 20 minutes. The color reaction was stopped by the addition of 50 μl 2N sulfuric acid (Boston Bio, USA) and the ELISA plate was read at the wavelength of 450 nm using a Multiskan Titertek Plate reader (VWR, USA).
In the following study, ELISA-based IL-6R release assays were performed to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs).
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based IL-6R release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human IL-6R capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 40,000 PBMC from healthy donors (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 6 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human IL-6R protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human IL-6R detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In the following study, ELISA-based HB-EGF release assays were performed to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous HB-EGF from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs).
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 17. The ELISA-based HB-EGF release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human HB-EGF capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 80,000 PBMC from healthy donors (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 6 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human HB-EGF protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human HB-EGF detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In the following study, ELISA-based TNFα release assays were performed to verify the inhibitory effects of the antibodies of the invention on LPS-induced release of endogenous TNFα in a mouse model for septic shock. Humanized huNOG-EXL mice (human CD34+) were obtained from Taconic, USA. Upon arrival, mice were housed for a minimum of 12 h to acclimatize before any treatments were initiated. All mouse experiments were approved by and were in compliance with the institutional animal care and use committee (IACUC) regulations of HSS/Weill Cornell Medicine.
On day 1, one group of mice was injected with the antibodies of the invention at a concentration of 500 μg/200 μl PBS per mouse (25 mg/kg). A second group was injected with the same volume of PBS only (200 μl PBS per mouse). 12h later all mice were subjected to an injection of LPS (Sigma, USA) at a concentration of 500 ng/200 μl per mouse. All mice were closely monitored and euthanized after 2h by CO2 inhalation. Blood was removed from the chest cavity and was centrifuged at 2000 g for 10 min at room temperature to remove cells and debris. Clear serum was transferred to a new tube and subsequently diluted 1:100 in PBS for ELISA measurements.
For measuring TNFα release, an OptEIA human TNFα ELISA (BD, USA) was used. Briefly, on day 1, Costar® 96-well plates (Coming, USA) were coated overnight with 100 μl per well of anti-human TNFα capture antibody (provided as part of the OptEIA® ELISA kit) at 1:250 in PBS at 4° C. On day 2, the capture antibody solution was removed, Costar® plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and plates were blocked with 300 μl per well of PBS, 10% FCS at room temperature for 2 hours. Then, blocking buffer was removed from the Costar® plates and plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA). Immediately after, 100 μl clear, diluted serum was added to wells. Additionally, 100 μl recombinant human TNFα protein (provided as part of the OptEIA ELISA kit) diluted in PBS-BSA at defined concentrations were added to the plate as standard references. Samples and standards were incubated for 2h at room temperature.
Thereafter, plates were washed 4 times with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA). Then, 100 μl biotinylated anti-human TNFα detection antibody (provided as part of OptEIA ELISA kit) at a dilution of 1:250 in PBS-FCS were added per well and plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-horseradish peroxidase conjugate (provided as part of OptEIA ELISA kit) diluted 1:40 in PBS-FCS were added to each well and plates were incubated at room temperature for 20 minutes. Following another round of 4 washes with 350 μl PBS-T (Boston Bio, USA) per well with a Nunc Immunowash plate washer (VWR, USA) and careful removal of all buffer traces after the fourth cycle, 100 μl TMB substrate solution (BD, USA) per well was added for incubation for 20 minutes. The color reaction was stopped by the addition of 50 μl 2N sulfuric acid (Boston Bio, USA) and the ELISA plate was read at the wavelength of 450 nm using a Multiskan Titertek Plate reader (VWR, USA).
In contrast to Example 32, where the inhibitory effects of the antibodies of the invention on LPS-induced release of endogenous TNFα from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs) were tested, this analysis was conducted to analyze the inhibitory effects of the antibodies of the invention on LPS-induced release of endogenous TNFα from primary human material obtained from patients suffering from rheumatoid arthritis (RA-patients).
To produce the recombinant chimeric antibody material, target DNA sequence was designed, optimized and synthesized. The complete sequence was sub-cloned into into pTT5 vector (Thermo Fisher Scientific, USA) and the transfection grade plasmid was maxi-prepared for CHO-3E7 or HD CHO-S(Thermo Fisher Scientific, USA) cell expression. CHO cells were grown in serum-free FreeStyle™ CHO Expression Medium (Thermo Fisher Scientific, USA) in Erlenmeyer flasks (Coming Inc., USA) at 37° C. with 5-8% CO2 on an orbital shaker (VWR Scientific, Germany). One day before transfection, the cells were seeded at an appropriate density in new Erlenmeyer flasks. On the day of transfection, DNA and transfection reagent were mixed at an optimal ratio and then added into the flask with cells ready for transfection. The recombinant plasmids encoding target protein were transiently transfected into suspension CHO cell cultures. The cell culture supernatant collected on day 6 post-transfection was used for purification. Cell culture broth was centrifuged and filtrated. Filtered cell culture supernatant was loaded onto MabSelect SuRe™ LX (GE Healthcare, UK) affinity purification columns at an appropriate flowrate. After washing and elution with appropriate buffers, the eluted fractions were pooled and buffer exchanged to final formulation buffer. The purified protein was analyzed by SDS-PAGE analysis for molecular weight and purity measurements. Finally, the concentration was determined applying a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA).
The ELISA-based TNFα release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human TNFα capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 20,000 PBMC from patients suffering from rheumatoid arthritis (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), human IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of LPS (Sigma-Aldrich, USA) at 6 ng/ml in growth medium for a final concentration of 1 ng/ml at 37° C., 5% CO2 for 1.5 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human TNFα protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human TNFα detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In contrast to Example 34, where the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs) were tested, this analysis was conducted to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous IL-6R from primary human material obtained from patients suffering from rheumatoid arthritis (RA-patients).
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 37. The ELISA-based IL-6R release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of mouse anti-human IL-6R capture antibody (provided as part of the DuoSet ELISA kit) at 4 μg/ml TBS at 4° C. Meanwhile, 40,000 PBMC from patients suffering from rheumatoid arthritis (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), mouse IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 24 hours. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 1 hour. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human IL-6R protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human IL-6R detection antibody (provided as part of the DuoSet ELISA kit) at 100 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
In contrast to Example 35, where the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous HB-EGF from primary human material obtained from healthy donors using peripheral blood mononuclear cells (PBMCs) were tested, this analysis was conducted to analyze the inhibitory effects of the antibodies of the invention on PMA-induced release of endogenous HB-EGF from primary human material obtained from patients suffering from rheumatoid arthritis (RA-patients).
The production of the recombinant antibody material that was used in this example is identical to the one described in Example 37. The ELISA-based HB-EGF release assay that was used in this example is described below.
In brief, on day 1, Nunc black MaxiSorp® 96-well plates (Thermo Fisher Scientific, USA) were coated overnight with 100 μl per well of rat anti-human HB-EGF capture antibody (provided as part of the DuoSet ELISA kit) at 2 μg/ml TBS at 4° C. On day 2, the capture antibody solution was removed and MaxiSorp® plates were blocked with 300 μl per well of TBS, 1% BSA at room temperature for 3 hours. Meanwhile, 80,000 PBMC from patients suffering from rheumatoid arthritis (STEMCELL Technologies, Canada) cells in 80 μl of normal growth medium were seeded in each well of Greiner CELLSTAR V-bottom 96-well plates (Thermo Fisher Scientific, USA) and pre-incubated with 20 μl per well of standard growth medium supplemented with Batimastat (BB94, Abcam, UK) at 50 μM as positive control (for a final concentration of 10 μM in the resulting 100 μl sample volume), human IgG antibody (Thermo Fisher Scientific, USA) at 5 μg/ml as isotype control (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) or antibodies of the invention at 5 μg/ml (for a final concentration of 1 μg/ml in the resulting 100 μl sample volume) at 37° C., 5% CO2 for 30 minutes. In case of stimulation controls, 20 μl of standard growth medium without test articles were added. Subsequently, cells (except those for unstimulated controls) were stimulated with 20 μl per well of PMA (Sigma-Aldrich, USA) at 150 ng/ml in growth medium for a final concentration of 25 ng/ml at 37° C., 5% CO2 for 6 hours. Afterwards, the 96-well plates were centrifuged to pellet cells. In parallel, blocking buffer was removed from the MaxiSorp® plates and plates were washed 4 times with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland). To avoid drying-up, 30 μl TBS were added to each well of the MaxiSorp® plates immediately, followed by the transfer of 70 μl cell-free supernatant per sample. Additionally, 100 μl recombinant human HB-EGF protein (provided as part of the DuoSet ELISA kit) diluted in TBS at defined concentrations were added to the plate as standard references. Thereafter, 100 μl biotinylated goat anti-human HB-EGF detection antibody (provided as part of the DuoSet ELISA kit) at 50 ng/ml TBS were added per well and, protected from direct light, plates were incubated at room temperature for 2 hours. After 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl streptavidin-AP (R&D Systems, USA) diluted 1:10,000 in TBS were added to each well and, again protected from direct light, plates were incubated at room temperature for 30 minutes. Following another round of 4 times washing with 350 μl TBS-T (Carl Roth, Germany) per well on a 96-head plate washer (Tecan Group, Switzerland) and careful removal of all buffer traces after the fourth cycle, 100 μl AttoPhos substrate solution (Promega, USA) per well was added for incubation in the dark at room temperature for 1 hour. Using an infinite M1000 (Tecan Group, Switzerland) microplate reader, the fluorescence of each well was collected at an excitation wavelength of 435 nm and an emission wavelength of 555 nm.
The following sequences form part of the disclosure of the present application. A WIPO ST 25 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19200589.0 | Sep 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/077346 | 9/30/2020 | WO |
