Patent Application
20210139579
The present invention relates to a multispecific antibody product that binds to a first ROR1 epitope and to at least one other epitope of ROR1, and conjugates thereof, as well as to uses thereof.
Cancer is one of the leading causes of death. It is a class of diseases caused by malignant transformation of healthy cells, resulting from genetic alterations, like chromosomal translocations, mutations in tumor suppressor genes, transcription factors or growth-factor receptors, leading to the immortalization of the cells. If the immortalization is combined with excessive proliferation, the immortalized cells generate tumors, with or without metastasis (in case of solid tumors), or leukemias and lymphomas (cancers of the blood). Defective apoptosis, or programmed cell death, can further contribute to malignant transformation of cells leading to cancer.
A family of membrane associated receptor tyrosine kinases, consisting of the receptor tyrosine kinase orphan receptors-1 and -2 (ROR1 and ROR2) have been described as specifically associated with particular cancers (Rebagay et al. (2012) Front Oncol. 2(34):1-8; doi 10.3389/onc.2012.00034), while being largely absent in expression on healthy tissue with, a few exceptions e.g. in case of ROR1 (Balakrishnan et al. (2016) Clin Cancer Res. doi: 10.1158/1078-0432). Whether or not ROR expression is functionally associated with tumorigenesis remains unclear. However, due to the very tumor-selective expression of ROR family members, they represent relevant targets for targeted cancer therapies.
Receptor tyrosine kinase orphan receptors-1 and -2, ROR1 and ROR2, are the only two family members defining a new receptor tyrosine kinase family, based on the overall structural design and some functional similarities. Both ROR1 and ROR2 proteins are type I-single pass trans-membrane receptors with an extracellular domain (ECD) consisting of an immunoglobulin domain, a cysteine rich frizzled domain and a Kringle domain. These three extracellular domains are followed by a trans-membrane domain connecting the ECD to an intracellular portion of the protein comprising kinase domains (Rebagay et al. (2012) Frontiers Oncol. 2(34):1-8; doi 10.3389/onc.2012.00034).
The human ROR1 and ROR2 proteins are 58% homologous between each other, but each of the ROR proteins is highly conserved between species. This represents a challenge for the development of human ROR1 specific monoclonal antibodies and very few antibodies are known.
Further, it appears that anti-ROR1 antibodies, and antibody drug conjugates (ADCs) that encompass anti-ROR1 antibodies, show only limited efficacy, in particular on cell lines and tumors with low expression levels of ROR1.
It is hence one object of the present invention to provide antibody-based products that target ROR1 and demonstrate a better efficacy, in particular on cell lines and tumors with low expression levels of ROR1.
It is another object of the present invention to provide antibody drug conjugates (ADCs) that target ROR1 and demonstrate a better efficacy, in particular on cell lines with low expression levels.
These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments.
The present invention relates to a multi-specific product that binds to a first ROR1 epitope and to at least one other epitope on ROR1, and conjugates thereof, as well as the uses thereof. The invention and general advantages of its features will be discussed in detail below.
Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.
It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.
Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.
According to a first aspect of the invention, a multispecific antibody-based product comprising
As herein, the term “multispecific” means a product which has specificity for two or more different epitopes of ROR-1 antigens. In the context of the present invention, the terms “multispecific” and “multi-epitope reactive” are hence used simultaneously. In one embodiment, the multi-epitope reactive product is bi-epitope reactive.
The term “variable region”, as used herein, refers to the respective regions of the heavy and light chain of an antibody, abbreviated VH and VL, (sometimes also written VH and VL, or HCVD and LCVD), as opposed to the constant domains CH1, CH2 and CH3 of the heavy chain and CL of the light chain. The variable regions encompass the complementarity determining regions (CDRs). The term “variable domain” is used interchangeably with the term “variable region” herein.
While an antibody comprising an Fc region has specificity not only for antigen epitopes, via its VH/VL domains, but also binds, via its Fc region, to an Fc receptor. In case its VH/VL domains bind two different epitopes, such antibody will still be called “bispecific” or “bi-epitope reactive”, in case its VH/VL domains bind two different epitopes, despite the fact that its actually binds another target, namely an Fc receptor. In case its VH/VL domains bind only one epitope, such antibody will still be called “monospecific” or “mono-epitope reactive”, in case its VH/VL domains bind two different epitopes, despite the fact that its actually binds another target, namely an Fc receptor.
According to one embodiment of the respective aspect of the invention, the product is a multispecific antibody, alternative scaffold or antibody mimetic wherein
According to another embodiment of the respective aspect of the invention, the product comprises two or more antibodies, alternative scaffolds or antibody mimetics wherein
The two embodiments discussed above are shown, in principle, in
These two entities can either be on two different antibodies, or on a single multi-specific antibody molecule.
Additionally, the invention provides bi- or multispecific antibodies targeting ROR1 as well as at least one binding domain specific for another target, for instance, but not limited to targets that recruit and/or activate cells of the immune system, like T cells or NK cells. Such other binding domains may be specific for CD3, CD16, CD32, CD56, CD64 or other markers specific for T and NK cells.
SEQ ID NOs 2 and 3 belong to an anti-ROR1 antibody called ERR1-324. This antibody binds a specific epitope of ROR1, including human ROR1 (hROR1). In a preferred embodiment, the first entity comprises the CDRs of ERR1-324, as given in
In a preferred embodiment, the first entity comprises the variable domain sequences of ERR1-324, i.e., comprises the variable domain sequences of SEQ ID NO. 2 (HCVD) and SEQ ID NO. 3 (LCVD).
The invention shows that such multi-specific product has significant advantages over a product which only has a single epitope reactivity for the ROR1 target, like e.g. the epitope that an antibody having SEQ ID NO. 2 (HCVD) and SEQ ID NO. 3 (LCVD).
Without being bound to theory, it is conceivable that the bi-epitope reactivity may induce the formation of target-homodimers or even clusters of the target as schematically presented in
According to one embodiment, the entity comprising an antigen-binding domain is at least one selected from the group consisting of an antibody, an antibody-based binding protein, a modified antibody format retaining target binding capacity, an antibody derivative or a fragment retaining target binding capacity, an alternative scaffold and/or an antibody mimetic.
According to one other embodiment, the antibody is at least one selected from the group consisting of an an antibody, an antibody-based binding protein, a bi-epitope-reactive antibody, a modified antibody format retaining target binding capacity, an antibody derivative or a fragment retaining target binding capacity, an alternative scaffold and/or an antibody mimetic.
According to one other embodiment, the antibody is at least one selected from the group consisting of an antibody, an antibody-based binding protein, a bi-epitope-reactive antibody, a modified antibody format retaining target binding capacity, an antibody derivative or a fragment retaining target binding capacity.
The term “fully human antibody” refers to an antibody, antibody-based binding protein or antigen-binding fragment that contains sequences derived from human immunoglobulin genes, such that substantially all of the heavy and light chain CDR1 and CDR2 regions are of human origin, and substantially all of the heavy and light chain FR regions 1, 2, 3, and 4 correspond to those of a human immunoglobulin sequence either with or without a limited number of somatic mutations that may be introduced into individual heavy and light chain CDR1 and CDR2 and FR1, 2, 3, and 4 variable domain sequences.
The terms “antibody”, “antibody-based binding protein”, “modified antibody format retaining target binding capacity”, “antibody derivative or fragment retaining target binding capacity” refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Antibodies, antibody-based binding proteins and antigen-binding fragments used in the invention can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. Antibodies, antibody-based binding proteins and antigen-binding fragments of the invention include intact antibodies and antibody fragments or antigen-binding fragments that contain the antigen-binding portions of an intact antibody and retain the capacity to bind the cognate antigen. Unless otherwise specified herein, all peptide sequences, including all antibody and antigen-binding fragment sequences are referred to in N->C order.
An intact antibody typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region. In the case of IgG, the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system. Monoclonal antibodies (mAbs) consist of identical antibodies molecules.
The VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity-determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of CDR and FR regions and a numbering system have been defined, e.g., the IMGT system (Lefranc M P et al., 2015), or the Kabat numbering scheme.
Antibodies, antibody-based binding proteins and antigen-binding fragments of the invention also encompass single chain antibodies. The term “single chain antibody” refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the VL and VH domains of the Fv fragment that are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
Examples of antibody-based binding proteins are polypeptides in which the binding domains of the antibodies are combined with other polypeptides or polypeptide domains, e.g. alternative molecular scaffolds, Fc-regions, other functional or binding domains of other polypeptides or antibodies resulting in molecules with addition binding properties, e.g. bi- or multispecific proteins or antibodies. Such polypeptides can create an arrangement of binding or functional domains normally not found in naturally occurring antibodies or antibody fragments.
Examples of antigen-binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a VH or VL domain (see, e.g., Ward et al., Nature 341:544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide.
Antigen-binding fragments of the present invention also encompass single domain antigen-binding units that have a camelid scaffold. Animals in the camelid family include camels, llamas, and alpacas. Camelids produce functional antibodies devoid of light chains. The heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies.
The terms “alternative scaffold” and “antibody mimetic” refer to proteins not belonging to the immunoglobulin family, and even non-proteins such as aptamers, or synthetic polymers. Some types have an antibody-like beta-sheet structure. Potential advantages of “antibody mimetics” or “alternative scaffolds” over antibodies are better solubility, higher tissue penetration, higher stability towards heat and enzymes, and comparatively low production costs.
Some antibody mimetics can be provided in large libraries, which offer specific binding candidates against every conceivable target. Just like with antibodies, target specific antibody mimetics can be developed by use of High Throughput Screening (HTS) technologies as well as with established display technologies, just like phage display, bacterial display, yeast or mammalian display. Currently developed antibody mimetics encompass, for example, ankyrin repeat proteins (called DARPins), C-type lectins, A-domain proteins of S. aureus, transferrins, lipocalins, 10th type III domains of fibronectin, Kunitz domain protease inhibitors, ubiquitin derived binders (called affilins), gamma crystallin derived binders, cysteine knots or knottins, thioredoxin A scaffold based binders, nucleic acid aptamers, artificial antibodies produced by molecular imprinting of polymers, peptide libraries from bacterial genomes, SH-3 domains, stradobodies, “A domains” of membrane receptors stabilised by disulfide bonds and Ca2+, CTLA4-based compounds, Fyn SH3, and aptamers (oligonucleic acid or peptide molecules that bind to a specific target molecules)
The anti-ROR1 antibodies, antibody-based binding proteins and antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies. Methods for generating these antibodies, antibody-based binding proteins and antigen-binding molecules are all well known in the art. In particular, scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Fv antibody fragments can be generated as described in Skerra and Plückthun, Science 240:1038-41, 1988. Disulfide-stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67:113-23, 1996. Similarly, single domain antibodies (dAbs) can be produced by a variety of methods described in, e.g., Ward et al., Nature 341:544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nat. Struct. Biol. 11:500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J. Mol. Biol. 332:643-55, 2003. Other types of antigen-binding fragments (e.g., Fab, F(ab′)2 or Fd fragments) can also be readily produced with routinely practiced immunology methods. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998.
The anti-ROR1 antibodies, antibody-based binding proteins or antigen-binding fragments of the invention can be produced by any suitable technique, for example, using any suitable eukaryotic or non-eukaryotic expression system. In certain embodiments, the antibody, antibody-based binding protein or antigen-binding fragment is produced using a mammalian expression system. Some specific techniques for generating the antibodies, antibody-based binding proteins or antigen-binding fragments or antigen-binding fragments of the invention are exemplified herein. In some embodiments, the antibodies, antibody-based binding proteins or antigen-binding fragments of the invention can be produced using a suitable non-eukaryotic expression system such as a bacterial expression system. Bacterial expression systems can be used to produce fragments such as a F(ab)2, Fv, scFv, IgGACH2, F(ab′)2, scFv2CH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv)2, and diabodies. Techniques for altering DNA coding sequences to produce such fragments are known in the art.
According to one preferred embodiment, the first and/or the second antibody, alternative scaffold or antibody mimetic is monospecific or mono epitope-reactive.
According to one embodiment of the respective aspect of the invention, the second antibody, alternative scaffold or antibody mimetic, or the second antigen-binding domain binds to ROR1, yet
the antibody comprising a heavy chain variable region sequence shown in SEQ ID NO. 2 and a light chain variable region sequence shown in SEQ ID NO. 3.
According to one embodiment of the respective aspect of the invention, the second antibody, alternative scaffold or antibody mimetic, or antigen-binding domain
at least one antibody selected from the group consisting of
According to one embodiment of the invention, ROR1 as mentioned herein is human ROR1 (hROR1).
According to one embodiment of the respective aspect of the invention, the multispecific product is in a format selected from the group consisting of
The bispecific scFv-Fc format consists of two scFv fragments of different specificity genetically fused to an Fc fragment. The bispecific scFv-IgG format consists of an IgG shaped antibody with a given specificity with two scFv fragments of different specificity fused to the N-terminus of the VH domain of the IgG. The DVD-Ig format consists of an Ig shaped antibody with a given specificity, wherein each VL/VH pair carries, N-terminally, another VH/VL pair of different specificity.
Examples for the three formats are shown in the following table, and in
According to another embodiment, the first entity comprises the following CDRs:
These are the CDRs of antibody ERR1-324. The CDRs are comprised in a suitable protein framework so as to be capable to bind to ROR1.
According to another embodiment, the second entity comprises one of the following CDR sets:
GYTFSDYEMH
AIDPETGGTAY
NQKFKG
YYDYDSFTY
KASQNVDAAVA
SASNRYT
QQYDIYPYT
Again, the CDRs are comprised in a suitable protein framework so as to be capable to bind to ROR1.
According to another embodiment, the first entity comprises the heavy chain variable region sequence of antibody ERR1-324 shown in SEQ ID NO. 2 and the light chain variable region sequence of antibody ERR1-324 shown in SEQ ID NO. 3.
According to another embodiment, the second entity comprises at least one of the following sequence pairs:
According to another aspect of the invention, an antibody drug conjugate (ADC) having the general formula A-(L)n-(T)m is provided, in which
and in which n and m are integers between >1 and <10.
According to another aspect of the invention, an antibody effector conjugate (AEC) having the general formula A-(L)n-(E)m is provided, in which
and in which n and m are integers between >1 and <10.
Such label can be a detectable label, can be at least one selected from the group consisting of: a fluorescent label (including a fluorescent dye or a fluorescent protein), a chromophore label, a radioisotope label containing iodine (e.g., 125I), gallium (67Ga), indium (111I), technetium (99mTc), phosphorus (32P), carbon (14C), tritium (3H), other radioisotope (e.g., a radioactive ion), and/or a protein label such as avidin or streptavidin.
According to one embodiment of the respective aspect of the invention, the antibody is a multi-specific, preferably a bi-epitope reactive antibody according to the above description.
According to another aspect of the invention, a composition comprising at least two antibody effector conjugates (AEC) or antibody drug conjugates (ADC) according to the above description, wherein each of the two conjugates comprises one of the monospecific antibodies, alternative scaffolds or antibody mimetics according to the above description.
According to yet another embodiment of the invention, the ADC is a bi-epitope reactive ADC (abbreviated BETR-ADC™) binding to two different epitopes of the ROR1 target.
According to one embodiment of the respective aspect of the invention, the linker is at least one selected from the group consisting of
According to one embodiment, the linker has at least one of the following amino acid sequences: -LPXTGn-, -LPXAGn-, -LPXSGn-, -LAXTGn-, -LPXTGn-, -LPXTAn- or -NPQTGn-, with Gn being an oligo- or polyglycine with n being an integer between ≥1 and ≤21, An being an oligo- or polyalanine with n being an integer between ≥1 and ≤21, and X being any conceivable amino acid sequence.
Gn (also called Gly(n)) is the oligoglycin discussed elsewhere herein In a preferred embodiment its length n can be between ≥1 and ≤21, preferably between ≥1 and ≤5.
It is important to understand that, in one specific embodiment (where Streptococcus pyogenes sortase A is used, see below), the oligo-glycine (Gly)n can optionally be replaced by an oligo-alanine (Ala)n.
According to one embodiment of the respective aspect of the invention, the linker is conjugated to the C-terminus of at least one subdomain of the antibody.
According to one embodiment of the respective aspect of the invention, prior to conjugation
According to another embodiment, said sortase enzyme recognition motif comprises at least one of the following amino acid sequences: LPXTG, LPXAG, LPXSG, LAXTG, LPXTA or NPQTN, with X being any conceivable amino acid sequence.
The following table shows the recognition tags and the peptides derived therefrom to be part of the linker:
Staphylococcus aureus sortase A recognition sequence,
Staphylococcus aureus sortase A recognition sequence,
aureus, with X being any amino acid
Staphylococcus aureus, with X being any amino acid
Streptococcus pyogenes sortase A recognition sequence,
Staphylococcus aureus sortase recognition sequence
Staphylococcus aureus sortase A or engineered sortase
Engineered sortases, including but not limited to sortase A mutant 2A-9 and sortase A mutant 4S-9 from Staphylococcus aureus, are described in Dorr et al. (2014) and mutants described in Chen et al. (2011).
As background and to exemplify the general concept of sortase transpeptidation, Sortase A uses an oligo-glycine-stretch as a nucleophile to catalyze a transpeptidation by which the terminal amino group of the oligo-glycine effects a nucleophilic attack on the peptide bond joining the last two C-terminal residues of the sortase tag. This results in breakage of that peptide bond and formation of a new peptide bond between the C-terminally second-to-last residue of the sortase tag and the N-terminal glycine of the oligo-glycine peptide, i.e. resulting in a transpeptidation.
It is important to understand that, in one specific embodiment (where Streptococcus pyogenes sortase A is used, see above), the oligo-glycine (Gly)n can optionally be replaced by an oligo-alanine (Ala)n.
Prior to sortase conjugation, the sortase recognition motif may, at its C-terminus, furthermore carry other tags, like His-tags, Myc-tags or Strep-tags (see
The sortase tag may, for example, be fused to a C-terminus of a binding protein, or to a domain or subunit thereof, by genetic fusion and co-expressed therewith. In another preferred embodiment, the sortase tag may directly be appended to the last naturally occurring C-terminal amino acid of the immunoglobulin light chains or heavy chains, which in case of the human immunoglobulin kappa light chain is the C-terminal cysteine residue, and which in the case of the human immunoglobulin IgG1 heavy chain may be the C-terminal lysine residue encoded by human Fcγ1 cDNA. However, another preferred embodiment is also to directly append the sortase tag to the second last C-terminal glycine residue encoded by human Fcγ1 cDNA, because usually terminal lysine residues of antibody heavy chains are clipped off by posttranslational modification in mammalian cells. Therefore, in more than 90% of the cases naturally occurring human IgG1 lacks the C-terminal lysine residues of the IgG1 heavy chains.
Therefore, one preferred embodiment of the invention is to omit the C-terminal lysine amino acid residues of human IgG1 heavy chain constant regions in expression constructs for sortase recognition-motif tagged Igγ1 heavy chains. Another preferred embodiment is to include the C-terminal lysine amino acid residues of human IgG1 heavy chain constant regions in expression constructs for sortase recognition-motif tagged Igγ1 heavy chains.
In another preferred embodiment the sortase or oligoglycine tag may be appended to the C-terminus of a human immunoglobulin IgG1 heavy chain where the C-terminal lysine residue encoded by human Fcγ1 cDNA is replaced by an amino acid residue other than lysine to prevent unproductive reactions of sortase with the ε-amino group of said C-terminal lysine residue leading to inter-heavy chain crosslinking.
We have described previously that in some cases (e.g. at the C-terminus of the Ig kappa light chains, see: Beerli et al. (2015) PloS One 10, e131177) it is beneficial to add additional amino acids between the C-terminus of the binding protein and the sortase tag. This has been shown to improve sortase enzyme conjugation efficiencies of payloads to the binding protein. In the case of Ig kappa light chains, it was observed that by adding 5 amino acids between the last C-terminal cysteine amino acid of the Ig kappa light chain and the sortase pentapeptide motif improved the kinetic of conjugation, so that the C-termini of Ig kappa light chains and Ig heavy chains could be conjugated with similar kinetics (see: Beerli et al. (2015) PloS One 10, e131177). Therefore, it is another preferred embodiment that optionally ≥1 and ≤11 amino acids are added in between the last C-terminal amino acid of a binding protein or antibody subunit and the sortase tag. In a preferred embodiment, a GnS peptide (wherein n is from 1 to 10, preferably 1 to 5) is added between the last C-terminal amino acid of a binding protein or antibody subunit and the sortase tag. Finally, in another preferred embodiment, additional amino acids between the C-terminus of the binding protein and the sortase or oligoglycine tag may beneficially be included that comprise a sequence and/or linker that is cleavable by hydrolysis, by a pH change or by a change in redox potential, or that is cleavable by a non-sortase enzyme, e.g., by proteases.
According to one embodiment of the respective aspect of the invention, the toxin or derivative thereof is at least one selected from the group consisting of:
In a preferred embodiment of the ADC, the toxin is selected from PNU-159682 as described in Quintieri et al. (2005) and derivatives thereof, maytansine, monomethyl auristatin MMAE, and monomethyl auristatin MMAF. In a preferred embodiment of the ADC, the toxin, joined to the linker at its wavy line, is of formula (i), as described in WO 2016/102679:
In the embodiment where the toxin is of formula (i), the linker may optionally comprise an alkyldiamino group of the form NH2—(CH2)m—NH2, where m≥1 and ≤11, preferably m=2, such that one amino group is directly linked at the wavy line of formula (i) to form an amide bond. It is moreover preferred that the second amino group is linked to an oligopeptide linker, which is more preferably an oligoglycine.
According to one embodiment of the respective aspect of the invention, the conjugate is created by sortase-mediated conjugation of (i) an antibody carrying one or more sortase recognition tags and (ii) one or more toxins or labels carrying an oligoglycine tag.
According to another aspect of the invention, a method of producing a conjugate according to the above description is provided, which method comprises the following steps:
a) providing an antibody, alternative scaffold or antibody mimetic according to the above description, which antibody carries a sortase recognition tag,
b) providing one or more toxins or labels carrying an oligoglycine tag, and
c) conjugating the antibody, alternative scaffold or antibody mimetic and the toxin or label by means of sortase-mediated
conjugation.
According to another embodiment of the invention, the use of the multispecific product according to the above description or the antibody drug conjugate according to the above description, for the treatment of a patient that is
a neoplastic disease is provided.
As an alternative, a method of treating a patient suffering from, at risk of developing, and/or being diagnosed for a neoplastic disease is provided, which method comprises the administration of one or more therapeutically active doses of the multispecific product according to the above description or the antibody drug conjugate according to the above description.
According to another aspect of the invention, the neoplastic disease is a neoplastic disease characterized by expression of ROR1. In one embodiment, ROR1 is overexpressed in said neoplastic disease.
As used herein, the term “overexpression of ROR1” refers to the expression level of ROR1 mRNA and/or protein expressed in cells of a given tissue being elevated in comparison to the levels of ROR1 as measured in normal cells (free from disease) of the same type of tissue, under analogous conditions. Said ROR1 mRNA and/or protein expression level may be determined by a number of techniques known in the art including, but not limited to, quantitative RT-PCR, western blotting, immunohistochemistry, and suitable derivatives of the above.
According to another aspect of the invention, the neoplastic disease is at least one selected from the group consisting of sarcoma, renal cell carcinoma, breast cancer, incl. triple-negative breast cancer, lung cancer, colon carcinoma, testicular cancer, ovarian cancer, pancreatic cancer, kidney cancer, gastric cancer, prostate cancer, head and neck cancer, melanoma, squamous cell carcinoma, multiple myeloma and other cancers, mesothelioma, chronic lymphoblastic leukemia, mantle cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, preB acute lymphocytic leukemia, acute myeloid leukemia, multiple myeloma, and other types of leukemias and lymphomas as well as solid tumors.
According to another aspect of the invention, a pharmaceutical composition comprising the multi-specific product according to the above description, the antibody drug conjugate according to the above description together with one or more pharmaceutically acceptable ingredients, is provided.
According to another aspect of the invention, a method of killing or inhibiting the growth of a cell expressing or overexpressing ROR1 in vitro or in a patient is provided, which method comprises administering to the cell a pharmaceutically effective amount or dose of the multi-specific or bi-epitope reactive product according to the above description, the antibody drug conjugate according to the above description, or of the pharmaceutical composition according to the above description.
According to one embodiment, the cell expressing ROR1 is a cancer cell.
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′.
The mouse Abelson murine pre-B cell line 63-12 (Shinkai et al. (1992) Cell 68:855-67) was cultured in culture media (17.7 g/L Gibco® IMDM (Life Technologies, 42200-030), 3.024 g/L NaHCO3(Sigma-Aldrich, p.a., >99.7%), 10 mL/L 100× non-essential amino acids (Life Technologies, 11140035), 5 mg/L insulin (Sigma-Aldrich, 1-5500), 3 mL/L of 10% primatone RL/UF in H2O (Sheffield Bioscience), and 1 mL/L of 50 mM 2-mercaptoethanol (Sigma-Aldrich, M-3148) in H2O), supplemented with 2% (v/v) FCS, 100 IU/mL Pen/Strep/Fungizone (Amimed, 4-02F00-H), 200 mM L-glutamine (Amimed, 5-10K00-H) and 50 μM 2-mercaptoethanol (Amresco, 0482) at 37° C. and 7.5% CO2.
Cells were engineered to overexpress hROR1 and hROR2 by transposition as follows: cells were centrifuged (6 min, 1200 rpm, 4° C.) and resuspended in RPMI-1640 media (5×106 cells/mL). 400 μL of cell suspension was then added to 400 μL of RPMI containing 10 μg of transposable vector pPB-PGK-Puro-ROR1 (directing co-expression of full-length ROR1 (NP 005003.2) and the puromycin-resistance gene), or 10 μg of transposable vector pPB-PGK-Puro-ROR2 (directing co-expression of full-length ROR2 (NP_004551.2) and the puromycin-resistance gene), along with 10 μg of transposase-containing vector pCDNA3.1_hy_mPB. DNA/63-12 cell mixtures were transferred to electroporation cuvettes (0.4 cm-gap, 165-2088, BioRad, Cressier, Switzerland) and electroporated using the Biorad Gene Pulser II with capacitance extender at 300V and 950 μf. Then, cells were incubated for 5-10 min at room temperature. Following the incubation, cells were centrifuged at 1200 rpm for 6 min (4° C.), washed once and subsequently resuspended in aqueous culture media (17.7 g/L Gibco® IMDM (Life Technologies, 42200-030), 3.024 g/L NaHCO3(Sigma-Aldrich, p.a., >99.7%), 10 mL/L 100× non-essential amino acids (Life Technologies, 11140035), 5 mg/L insulin (Sigma-Aldrich, 1-5500), 3 mL/L of 10% primatone RL/UF in H2O (Sheffield Bioscience), and 1 mL/L of 50 mM 2-mercaptoethanol (Sigma-Aldrich, M-3148) in H2O), supplemented with 2% (v/v) FCS, 100 IU/mL Pen/Strep/Fungizone (Amimed, 4-02F00-H), 200 mM L-glutamine (Amimed, 5-10K00-H) and 50 μM 2-mercaptoethanol (Amresco, 0482). After two days incubation at 37° C. in a humidified incubator at 5% CO2 atmosphere, cell pools stably expressing hROR1 or hROR2 were selected by adding 2 μg/mL puromycin (Sigma-Aldrich, P8833).
After 4 to 5 days, hROR1 or hROR2 expression on engineered cells were confirmed by flow cytometry. Briefly, following trypzinization, 106 cells were centrifuged in FACS tubes; obtained pellets were resuspended in buffer (PBS with 2% (v/v) FCS). In the case of hROR1-engineered cells, cells were then incubated with 2A2 (mAb066 antibody targeting ROR1, final concentration 2 μg/mL) for 30 min at 4° C., followed by centrifugation and washing. Cells were resuspended as previously and incubated with anti-human IgG antibody (Fc gamma-specific) PE (eBioscience, Vienna, Austria, 12-4998-82), at a 1:100 dilution, in the dark (30 min, 4° C.), washed once in buffer and kept on ice until FACS sorting. For hROR2-engineered 63-12 cells, the same protocol was followed but using EPR3779 (Abcam antibody targeting ROR2; 1:100 dilution) as primary antibody and allophycocyanin-conjugated AffiniPure F(ab′)2 goat anti-rabbit IgG (H+L) (Jackson Immunoresearch, 111-136-144) as secondary antibody.
In the case of hROR1-engineered 63-12 cells, cells were single cell sorted into 96-well flat-bottom plates containing 200 μL of supplemented culture media per well using a FACS Aria II. Plates were incubated at 37° C. and clones were expanded to 6-well plates before analysis. Target expression was confirmed by flow cytometry using a FACSCalibur instrument (BD Biosciences) and FlowJo analytical software (Tree Star, Ashland, Oreg.).
63-12, 63-12/hROR1 and 63-12/hROR2 transfectants were cultured in DMEM (Invitrogen; Carlsbad, Calif.) supplemented with 10% (v/v) heat inactivated FBS (Thermo Scientific; Logan, Utah), 100 IU/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). HEK 293F cells were purchased from Invitrogen and maintained in FreeStyle Medium supplemented with 1% (v/v) heat inactivated FBS (Thermo Scientific) to support adherent culture or without FBS for suspension culture, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen).
Construction, expression, and purification of recombinant human ROR1 proteins: Construction, expression, purification and biotinylation of hFc fusion proteins containing different domains of human ROR1 or mouse ROR1 were described (Yang et al., PloS One 6:e21018, 2011). For hROR1-AVI-6×HIS fusion protein, the extracellular domain of human ROR1 (24-403) was PCR amplified with primers pCEP4-hROR1-F and pCEP4-hROR1-Avi tag-R (note that the AVI tag was introduced to the C terminus of ROR1 by primer pCEP4-hROR1-Avi tag-R), followed by extension PCR with primers pCEP4-signal-F-KpnI and pCEP4-6HIS-R-XhoI to add a signal peptide and 6×HIS tag to the N and C terminus separately before cloning into pCEP4 via KpnI/XhoI. This construct was then transiently transfected into HEK 293F cells (Invitrogen) using 293fectin (Invitrogen), and the protein was purified by Immobilized Metal Ion Affinity Chromatography using a 1-mL HisTrap column (GE Healthcare) as described in Kwong and Rader, Curr Protoc Protein Sci Chapter 6:Unit 6 10, 2009. The quality and quantity of purified hROR1-AVI-6×HIS was analyzed by SDS-PAGE and A280 absorbance, respectively. Subsequently, the fusion protein was biotinylated by BirA enzyme kit from Avidity (Aurora, Colo.) following the protocol. Briefly, 2 mg ROR1-AVI-6×HIS at 40 μM in 10 mM Tris-HCl (pH 8) was biotinylated in the presence of biotin using 10 μg BirA after incubation for 30 min at 37° C., followed by purification again using a 1-mL HisTrap column (GE Healthcare) as described above.
Construction, expression, and purification of recombinant human ROR1 (hROR1-His) and human ROR2 (hROR2-His) proteins: hROR1-His was PCR-amplified with primers
using pCEP4-hFc-hROR1 (Yang et al., PloS One 6:e21018, 2011) as template, while hROR2-His was PCR-amplified with primers
using pCEP4-hFc-hROR2 as template. Then they are cloned into pCEP4 (Invitrogen) separately via KpnI/XhoI. These constructs were then separately and transiently transfected into HEK 293F cells (Invitrogen) using 293fectin (Invitrogen), and the corresponding proteins were purified by Immobilized Metal Ion Affinity Chromatography using a 1-mL HisTrap column (GE Healthcare) as described in Kwong and Rader, Curr Protoc Protein Sci Chapter 6:Unit 6 10, 2009. The quality and quantity of purified hROR1-His and hROR2-His were analyzed by SDS-PAGE and A280 absorbance, respectively.
Generation and selection of naïve chimeric rabbit/human Fab libraries: All rabbit handling was carried out by veterinary personnel at Pocono Rabbit Farm & Laboratory (Canadensis, Pa.) or R & R Research (Stanwood, Wash.). A total of nine rabbits (ages 3-4 months) were used. Five of these rabbits were of the New Zealand White (NZW) strain, with three obtained from Pocono Rabbit Farm & Laboratory (Canadensis, Pa.) and two obtained from R & R Research (Stanwood, Wash.). Four b9 wild-type rabbits were derived from a separate R & R Research colony that originated from a pedigreed colony developed and characterized at the National Institute of Allergy and Infectious Diseases (NIAID) (McCartney-Francis et al., Proc. Natl. Acad. Sci. USA 81:1794-1798, 1984; and Popkov et al., J. Mol. Biol. 325:325-335, 2003. Spleen and bone marrow from each rabbit were collected and processed for total RNA preparation and RT-PCR amplification of rabbit Vκ, Vλ, and VH encoding sequences using established protocols (Rader, Methods Mol Biol 525:101-128, xiv, 2009. Rabbit (rb) Vκ/human (hu) Cκ/rbVH and rbVλ/huCλ/rbVH segments, respectively, were assembled in one fusion step based on 3-fragment overlap extension PCR. Note that the VL derived from b9 rabbits were also assembled with VH from NZW rabbits. The Fab-encoding fragments were digested with SfiI and ligated with SfiI-treated phage display vector pC3C (Hofer et al., J Immunol Meth 318:75-87, 2007) at 16° C. for 24 h. Subsequently, 15 μg purified pC3C-rbVκ/hCκ/rbVH ligated products were transformed into E. coli strain SR320 (a kind gift from Dr. Sachdev S. Sidhu, University of Toronto, Toronto, Ontario, Canada) by 30 separate electroporations (each using 0.5 μg DNA in 50 μl electrocompetent cells) and yielded 7.5×109 independent transformants for library κ. For library λ, 4.8×109 independent transformants were obtained using the same procedure. Using VCSM13 helper phage (Stratagene; La Jolla, Calif.), the phagemid libraries were converted to phage libraries and stored at −80° C. Phage library κ and library λ were re-amplified using XL1-Blue (Stratagene) or ER2738 (Lucigen) and mixed equally before four rounds of panning against biotinylated hFc-hROR1 or hROR1-AVI-6HIS. During the panning, 5 μg/mL antigen was pre-incubated with streptavidin coated magnetic beads (Dynabeads MyOne Streptavidin Cl; Invitrogen) at 37° C. for 30 min and then binders from the phage library were captured in the presence of 1 mg/mL unspecific polyclonal human IgG (Thermo Scientific) when hFc-ROR1 was used. Starting from the third round of panning, the input phage was negatively depleted by incubation with empty beads before selection against antigen-loaded beads. Following selection, supernatants of IPTG-induced bacterial clones were analyzed by ELISA and by flow cytometry. Repeated clones were identified by DNA fingerprinting with AluI, and the VL and VH sequences of unique clones were determined by DNA sequencing (
Construction, expression, and purification of chimeric rabbit/human Fab and IgG1: MAb XBR1-402 in chimeric rabbit/human Fab format was cloned into E. coli expression plasmid pC3C-His and expressed and purified as described in Kwong and Rader, Curr Protoc Protein Sci Chapter 6:Unit 6 10, 2009. For the expression of mAb XBR1-402 in chimeric rabbit/human IgG1 format, the previously described vector PIGG-R11 was used (Yang et al., PloS One 6:e21018, 2011). The VH encoding sequence of Fab XBR1-402 was PCR amplified using primers XBR1-402_VH_F and XBR1-402_VH_R, and cloned via ApaI/SacI into PIGG-R11. Then the light chain encoding sequence of XBR1-402 was PCR amplified using primers XBR1-402_λ_F and LEAD-B, and cloned via HindIII/XbaI into PIGG-R11 with the corresponding heavy chain encoding sequence. Note that an internal ApaI site in FR4 of VH encoding sequences of Fab XBR1-402 was removed by silent mutation in primer XBR1-402_VH_R. In addition, we changed a TAG stop codon, which was suppressed during selection in E. coli strain XL1-Blue, to CAG (glutamine) encoding the first amino acid of native VH (
All the other mAbs in chimeric rabbit/human Fab format were cloned into E. coli expression plasmid pET11a and expressed and purified as described (Yang et al., PloS One 6:e21018, 2011). For the expression of mAbs ERR1-324, ERR1-TOP43 and ERR1-TOP54 in chimeric rabbit/human IgG1 format, pCEP4 (Invitrogen) was used to clone the heavy chain and light chain separately. For heavy chain, a gBlock containing a heavy-chain signal peptide encoding sequence, VH of ERR2-302 (a mAb to hROR2) and CH1 (1-49) of human IgG1 was synthesized by IDT (San Jose, Calif.) and amplified with primers KpnI/AscI-Signal and CH1-internal/overlap-R, and fused to CH1 (50-88)-CH2-CH3 amplified from PIGG with primers CH1-internal/overlap-F and HC-CH3-R-XhoI by overlap extension PCR with primers KpnI/AscI-Signal and HC-CH3-R-XhoI, and then cloned into pCEP4 by AscI/XhoI. Note that a EheI site was introduced into CH1 at Ala12 by synonymous mutation when the gBlock was synthesized. Consequently, this construct served as vector to clone the heavy chains of other mAbs by replacing the VH using AscI/EheI: VH of ERR1-324, ERR1-TOP43 and ERR1-TOP54 were amplified with forward primer ERR1-324 HC-F, ERR1-TOP43 HC-F and ERR1-TOP54 HC-F and reverse primer VH-CH1-R-EheI separately, followed by extension PCR to add the signal peptide with primer KpnI/AscI-Signal and VH-CH1-R-EheI. Then, each VH was inserted into the vector by AscI/EheI. For light chain cloning, while lambda light chains of ERR1-TOP43 and ERR1-TOP54 were amplified with primers ERR1-TOP43 LC-F and ERR1-TOP54 LC-F separately combined with LC-R-XhoI, kappa light chains of ERR1-324 was amplified with primers ERR1-324 KC-F and KC-R-XhoI. Then, a signal peptide encoding sequence was added by extension PCR with forward primer KpnI/AscI-Signal and reverse primer LC-R-XhoI or KC-R-XhoI. Subsequently, each light chain PCR product was cloned into pCEP4 by AscI/XhoI. The resulting constructs containing heavy chain or light chain for each IgG were co-transfected transiently into HEK293F cells (Invitrogen) using 293fectin (Invitrogen), and the corresponding proteins were purified with a 1-mL recombinant Protein A HiTrap column (GE Healthcare, Piscataway, N.J.) as described (Yang et al., PloS One 6:e21018, 2011; and Yang and Rader, Methods Mol Biol 901:209-232, 2012). The quality and quantity of purified IgG1 was analyzed by SDS-PAGE and A280 absorbance, respectively.
ELISA: For ELISA (
To determine the epitopes (
Flow cytometry: Cells were stained using standard flow cytometry methodology. Briefly, for purified anti-ROR1 Fab (
Surface plasmon resonance: Surface plasmon resonance for the measurement of the affinities of all Fabs to hFc-hROR1 and for epitope mapping studies were performed on a Biacore X100 instrument using Biacore reagents and software (GE Healthcare, Piscataway, N.J.). Anti-Human IgG (Fc) antibody was immobilized on a CMS sensor chip following the instruction of Human Antibody Capture Kit (GE Healthcare, Piscataway, N.J.). Then, hFc-hROR1 fusion proteins were captured at certain density (indicated in
StrepII-tagged human ROR1-extracellular domain was produced as follows: the nucleotide sequence encoding the extracellular domain of human ROR1 (NP_005003.2) was N-terminally fused to a signal sequence (MNFGLRLIFLVLTLKGVQC) and C-terminally fused with a sequence encoding a peptide comprising a strepII-tag (WSHPQFEK). The entire nucleotide sequences with flanking 5′NotI and 3′HindIII sites were produced by total gene synthesis (GenScript, Piscataway, USA), assembled in the proprietary mammalian expression vector pEvi5 by Evitria AG (Schlieren, Switzerland) and verified by DNA sequencing.
Expression of the proteins was performed in suspension-adapted CHO K1. Supernatants from pools of transfected CHO K1 cells were harvested by centrifugation and sterile filtered (0.2 μm) before FPLC-based affinity purification using StrepTactin columns (IBA GmbH, Goettingen, Germany).
Recombinant human twin strep-tagged ROR2 (NP_004551.2; twin strep sequence WSHPQFEKGGGSGGGSGGSAWSHPQFEKGS) was expressed and purified in-house according to the following protocol: the EBNA expression vector pCB14b-ROR2-ECD-TwinStrep, directing expression of ROR2 extracellular domain (ECD), C-terminally tagged with a TwinStep tag, was transfected into HEK293T using Lipofectamine® LTX with PLUS™ Reagent (Thermo Fisher Scientific, 15388100). Following a 1-day incubation (37° C., 5% CO2, growth media: Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept)), cells were expanded under selection conditions (2 μg/mL of puromycin (Sigma-Aldrich, P8833-25 mg stock at 2 mg/mL)). Cells were split and further expanded (37° C., 5% CO2); once confluency was reached, tissue culture dishes were coated with 20 μg/ml poly-L-Lysine (Sigma-Aldrich, P1524) for 2 h at 37° C. and washed twice with PBS. Then, cells were trypsinized, washed with PBS and split 1:3 onto poly-L-lysine-coated plates. Again after reaching confluency, cells were washed with PBS followed by with media replacement using production media (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-03) supplemented with 1 μg/mL puromycin (Sigma-Aldrich, P8833), 100 IU/mL of Pen-Strep-Fungizone (Bioconcept, 4-02F00-H), 161 μg/mL of N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 μg/mL of L-glutathione reduced (Sigma-Aldrich, G6529). Supernatant, harvested bi-weekly and filtered (0.22 μm) to remove cells, was stored at 4° C. until purification. For purification, filtered supernatant was loaded onto a Streptactin® Superflow® high capacity cartridge (IBA, Gottingen, Germany, 2-1238-001) column; purification and elution was performed according to the manufacturer's protocol on an AEKTA pure (GE Healthcare). Fractions were analyzed for protein purity and integrity by SDS-PAGE. Protein-containing fractions were mixed and subjected to buffer exchange using Amicon filtration units (Millipore, Schaffhausen, Switzerland) to reach a dilution of ≥1:100 in PBS, and then sterile filtered using a low retention filter (0.20 μm, Carl Roth, Karlsruhe, Germany, PA49.1).
Expression vectors: Antibody variable region coding regions were produced by total gene synthesis (GenScript) using MNFGLRLIFLVLTLKGVQC as leader sequence, and were assembled with human IgH-γ1 and IgL-κ or IgL-λ constant regions, as applicable, in the expression vector pCB14. This vector, a derivative of the episomal mammalian expression vector pCEP4 (Invitrogen), carries the EBV replication origin, encodes the EBV nuclear antigen (EBNA-1) to permit extrachromosomal replication, and contains a puromycin selection marker in place of the original hygromycin B resistance gene.
Expression and purification of ROR1 antibodies: pCB14-based expression vectors were transfected into HEK293T cells using Lipofectamine® LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, Reinach, Switzerland, 15388100); following a 1-day incubation (37° C., 5% CO2, growth media: Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/L) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)), cells were expanded under selection conditions (2 μg/mL of puromycin (Sigma-Aldrich, Buchs SG, Switzerland, P8833-25 mg stock at 2 mg/mL)). Cells were split and further expanded (37° C., 5% CO2); once confluency was reached, tissue culture dishes were coated with 20 μg/ml poly-L-Lysine (Sigma-Aldrich, P1524) for 2 h at 37° C. and washed twice with PBS. Then, cells were trypsinized and split 1:3 onto poly-L-lysine-coated plates. Again after reaching confluency, cells were washed with PBS followed by media replacement to production media (DMEM/F-12, Gibco/Thermo Fisher Scientific, 31330-03) supplemented with 1 μg/mL puromycin (Sigma, P8833), 100 IU/mL of Pen-Strep-Fungizone (Bioconcept), 161 μg/mL of N-acetyl-L-cysteine (Sigma-Aldrich, A8199) and 10 μg/mL of L-glutathione reduced (Sigma-Aldrich, G6529). Supernatant, harvested bi-weekly and filtered (0.22 μm) to remove cells, was stored at 4° C. until purification.
For purification, filtered supernatant was loaded onto a PBS-equilibrated Protein A HiTrap column (GE Healthcare, Frankfurt am Main, Germany, 17-0405-01) or a JSR Amsphere™ Protein A column (JSR Life Sciences, Leuven, Belgium, JWT203CE) and washed with PBS; elution was performed using 0.1M glycine (pH 2.5) on an AEKTA pure (GE Healthcare). Fractions were immediately neutralized with 1M Tris-HCl buffer (pH 8.0), and analyzed for protein purity and integrity by SDS-PAGE. Protein-containing fractions were mixed and subjected to buffer exchange using Amicon filtration units (Millipore, Schaffhausen, Switzerland, UFC901008) to reach a dilution of 1:100 in PBS, and then sterile filtered using a low retention filter (0.20 μm, Carl Roth, Karlsruhe, Germany, PA49.1).
Isotype control antibodies were transiently expressed in CHO cells by methods known in the art and recombinant antibodies were purified by standard protein A purification from CHO cell supernatants, as known in the art. The purity and the integrity of the recombinant antibodies were analyzed by SDS-PAGE.
Each well of a 96-well plate was coated with 100 μL of 2 μg/mL strep-tagged human ROR1 or ROR2 (from Example 5) in 0.1 M bicarbonate coating buffer (pH 9.6), and incubated for 12 h at 4° C.
After blocking with 150 μL of 3% (w/v) bovine serum albumin (BSA)/TBS for 1 h at 37° C., the following antibodies were added to a well within each plate at a concentration of 0.5 μg/mL, and serially diluted (dilution factor 4) with 1% (w/v) BSA/TBS, before incubation for 1 h at 37° C.: ERR1-301 (mAb027), XBR1-402 (mAb031), ERR1-306 (mAb033), ERR1-324 (mAb034), ERR1-403 (mAb035) and ERR1-Top43 (mAb036). HRP-conjugated F(ab′)2 anti-human FC-gamma (Jackson Immunoresearch, 109-036-008) was then added at a 1:20′000 dilution, 100 μl per well, and incubated for 1 h at 37° C. prior to detection using an Spark 10M plate reader (Tecan). As shown in
5×105 of each cell type were added per well to 96-well plates. Plates were centrifuged (3 min, 1300 rpm) with re-suspension in buffer (PBS supplemented with 2% (v/v) of FCS). 2A2 (mAb066) was added to each well to reach a concentration of 2 μg/mL. Plates were then incubated on ice for 30 min and washed with 2004 of buffer prior to resuspension in 2004 of buffer supplemented with anti-human IgG antibody (Fc gamma-specific) PE (eBioscience 12-4998-82) at a 1:250 dilution. Following 30 min incubation on ice and one washing, cells were analyzed using a FACSCalibur instrument (BD Biosciences) and data was analyzed using FlowJo analytical software (Tree Star, Ashland, Oreg.).
Sortase A. Recombinant and affinity purified Sortase A enzyme from Staphylococcus aureus was produced in E. coli as disclosed in WO2014140317A1.
Generation of glycine-modified toxins. In order to generate SMAC-Technology™ conjugated ADCs with pentaglycine-modified EDA-anthracycline derivative (G5-PNU) was manufactured by Concortis (
Sortase-mediated antibody conjugation. The above-mentioned toxin was conjugated to anti-ROR1 antibodies as per Table 3 by incubating LPETG-tagged mAbs [10 μM] with glycine modified toxin [200 μM] and 3 μM Sortase A in the listed conjugation buffer for 3.5 h at 25° C. The reaction was stopped by passing it through an rProtein A GraviTrap column (BioRad). Bound conjugate was eluted with 5 column volumes of elution buffer (0.1 M glycine pH 2.5, 50 nM NaCl), with 1 column volume fractions collected into tubes containing 25% v/v 1M HEPES pH 8 to neutralise the acid. Protein containing fractions were pooled and formulated in the formulation buffer of Table 3 using a ZebaSpin desalting column.
ADC analytics. DAR was assessed by Reverse Phase Chromatography performed on a Polymer Labs PLRP 2.1 mm×5 cm, 5 μm column run at 1 mL/min/80° C. with a 25 minute linear gradient between 0.05 and 0.1% TFA/H2O and 0.04 to 0.1% TFA/CH3CN. Samples were first reduced by incubation with DTT at pH 8.0 at 37° C. for 15 minutes. The DAR determined by Reverse Phase Chromatography is summarized in Table 3 below.
From these analyses it can be concluded that the SMAC-Technology™ conjugation has proceeded at high efficiency resulting in overall average DARs in the range of ca. 3.5 to 4.0 for IgG-format anti-ROR1 antibody-toxin combinations.
Cytotoxicity of 50:50 (by weight) mixtures of ERR1-324-G5-PNU with further anti-ROR1 ADCs was investigated using human cell line 697, and compared to the cytotoxicity of the individual ADCs.
For this, 2.5×104 697 cells per well were plated on 96-well plates (excluding edge wells, which contained water) in 754 RPMI supplemented with 10% by vol. FCS, 100 IU/ml Pen-Strep-Fungizone and 2 mM L-Glutamine and were grown at 37° C. in a humidified incubator at 7.5% CO2 atmosphere. After 1-day incubation, each ADC or ADC mixture was added to respective wells in an amount of 254 of 3.5-fold serial dilutions in growth medium (resulting in final ADC or ADC mixture concentrations from 20 μg/mL to 0.88 ng/ml). After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 504 was removed from each well, and then 504 of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9423) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Tecan Infinity F200 plate reader with an integration time of 1 s per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software. The IC50 values were determined using the built-in “log(inhibitor) vs. response—Variable slope (four parameters)” IC50 determination function of Prism Software.
Cytotoxicity of anti-ROR1 scFv-Fc-based bi-epitope reactive ADCs (BETR-ADCs™) and ADC mixtures was investigated using human cell line 697. The same protocol as Example 10 was applied to the ADCs of Table 9.
Cytotoxicity of DVD-Ig-based bi-epitope reactive anti-ROR1 ADCs (BETR-ADCs™) and individual anti-ROR1 ADCs was investigated using human cell line 697. The same protocol as Example 10 was applied to the ADCs of Table 10.
Cytotoxicity of 50:50 mixtures of anti-ROR1 ADCs was investigated using human cell lines: HT-29, MDA-MB-468, A549, HS 578T. For this, the following cells per well were plated on 96-well plates (excluding edge wells, which contained water) and were grown at 37° C. in a humidified incubator at 7.5% CO2 atmosphere in growth medium (DMEM supplemented with 10% by vol. FCS, 100 IU/ml Pen-Strep-Fungizone and 2 mM L-Glutamine).
After 1-day incubation, each ADC or ADC mixture was added to respective wells in an amount of 254 of 3.5-fold serial dilutions in growth medium (resulting in final ADC or ADC mixture concentrations from 20 μg/mL to 0.88 ng/ml). After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 μL was removed from each well, and then 504 of CellTiter-Glo® 2.0 Luminescent Solution (Promega, G9423) was added to each well. After shaking the plates at 750 rpm for 5 min followed by 20 min incubation without shaking, luminescence was measured on a Tecan Infinity F200 plate reader with an integration time of 1 s per well. Curves of luminescence versus ADC concentration (ng/mL) were fitted with Graphpad Prism Software. The IC50 values, determined using the built-in “log(inhibitor) vs. response—Variable slope (four parameters)” IC50 determination function of Prism Software, are reported in Table 12.
Cell line engineering for ectopic expression of hROR1 in the EMT-6 murine breast cancer cell line: Murine EMT-6 breast cancer cells were cultured in DMEM complete (Dulbecco's Modified Eagle Medium (DMEM) High Glucose (4.5 g/1) with L-Glutamine with 10% (v/v) Fetal Calf Serum (FCS), 100 IU/mL of Pen-Strep-Fungizone and 2 mM L-glutamine (all Bioconcept, Allschwil, Switzerland)) at 37° C. and 5% CO2. Cells were engineered to overexpress ROR1 by transposition as follows: cells were centrifuged (6 min, 1200 rpm, 4° C.) and resuspended in RPMI-1640 media (5×106 cells/mL). 400 μL of this cell suspension was then added to 400 μL of RPMI containing 13.3 μg of transposable vector pPB-PGK-Puro-ROR1, directing co-expression of full-length ROR1 (NP_005003.2) and the puromycin-resistance gene, and 6.6 μg of transposase-containing vector pcDNA3.1_hy_mPB. The DNA/EMT-6 cell mixture was transferred to electroporation cuvettes (0.4 cm-gap, 165-2088, BioRad, Cressier, Switzerland) and electroporated using the Biorad Gene Pulser II with capacitance extender at 300V and 950 μf. Then, cells were incubated for 5-10 min at room-temperature. Following the incubation, cells were centrifuged at 1200 rpm for 6 min, washed once and subsequently resuspended in DMEM complete prior to incubation at 37° C. in a humidified incubator at 5% CO2 atmosphere. One day after electroporation, cell pools stably expressing human ROR1 were selected by adding 3 μg/mL puromycin (Sigma-Aldrich, P8833).
ROR1 expression on selected EMT-6-ROR1 cells was confirmed by flow cytometry (not shown). To isolate ROR1-expressing EMT-6 cell clones, following trypsinization, 106 cells were centrifuged in FACS tubes; obtained pellets were resuspended in buffer (PBS with 2% (v/v) FCS). Cells were then incubated with 2A2 (mAb066, Baskar et al., 2012); 30 min, 4° C., final concentration 2 μg/mL), followed by centrifugation and washing. Cells were then resuspended as previously and incubated with anti-human IgG antibody (Fc gamma-specific) PE (eBioscience, Vienna, Austria, 12-4998-82) with a 1:250 dilution in the dark (30 min, 4° C.), washed once in buffer and kept on ice until FACS sorting.
Using a FACS Aria II, cells were single cell sorted into a 96-well flat-bottom plate containing 200 μL of DMEM complete per well. This plate was incubated at 37° C. and clones were expanded to 6-well plates before analysis of ROR1-expression by flow cytometry as outlined above, using a FACSCalibur instrument (BD Biosciences) and FlowJo analytical software (Tree Star, Ashland, Oreg.) for analysis.
Cytotoxicity. Cytotoxicity of an scFv-Ig-based bi-epitope reactive anti-ROR1 ADC (BETR-ADC′) and individual anti-ROR1 ADCs was investigated using the above engineered EMT-6 cells (clone 14). The same protocol as in Example 10 was applied to the ADCs of Table 13, plating 1000 EMT-6 cells per well.
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.
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTV
APTECS
YEPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVE
KTVAPTECS
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAP
TECS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTV
APTECS
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC
LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
VKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTV
APTECS
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGK
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGK
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK
TIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQ
ADDEADYYCGADYIGGYVEGGGTQLTVTGQPKAAPSVTLEPPSSEELQANKA
TLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTP
EQWKSHKSYSCQVTHEGSTVEKTVAPTECS
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRT
PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVE
KTVAPTECS
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN
TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK
PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP
EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
FNRGEC
Staphylococcus aureus sortase
Staphylococcus aureus sortase
Staphylococcus aureus sortase
Staphylococcus aureus, with
Streptococcus pyogenes sortase
Staphylococcus aureus sortase
Staphylococcus aureus sortase
Staphylococcus aureus sortase
aureus sortase A or engineered
Staphylococcus aureus, with X
aureus, with X being any amino
Streptococcus pyogenes sortase
Staphylococcus aureus sortase
| Number | Date | Country | Kind |
|---|---|---|---|
| 17182420.4 | Jul 2017 | EP | regional |
This application is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/EP2018/069798, filed Jul. 20, 2018, which claims priority to European Patent Application No. 17182420.4, filed Jul. 20, 2017, the entire disclosures of which are hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/069798 | 7/20/2018 | WO | 00 |
