WO2018083238A1 - Nouveaux anticorps anti-py792-ddr1 - Google Patents

Nouveaux anticorps anti-py792-ddr1 Download PDF

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WO2018083238A1
WO2018083238A1 PCT/EP2017/078184 EP2017078184W WO2018083238A1 WO 2018083238 A1 WO2018083238 A1 WO 2018083238A1 EP 2017078184 W EP2017078184 W EP 2017078184W WO 2018083238 A1 WO2018083238 A1 WO 2018083238A1
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amino acid
seq
acid sequence
antibody
cdr
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PCT/EP2017/078184
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English (en)
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Michael Gerg
Michael Schraeml
Lars HILLRINGHAUS
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Roche Diagnostics Gmbh
F. Hoffmann-La Roche Ag
Chugai Seiyaku Kabushiki Kaisha
Roche Diagnostics Operations, Inc.
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Publication of WO2018083238A1 publication Critical patent/WO2018083238A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an antibody as characterized in the appended claims, wherein the antibody specifically binds to the discoidin domain receptor 1 (DDR1) which is phosphorylated at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15.
  • DDR1 discoidin domain receptor 1
  • the present invention further relates to nucleic acid molecules encoding the light chain variable region or the heavy chain variable region of the antibody of the invention, as well as vectors comprising said nucleic acid molecules.
  • the invention further relates to a host cell or non-human host comprising the vector(s) of the invention, as well as to a method for the production of an antibody according to the invention comprising culturing the host cell of the invention under suitable conditions and isolating the antibody produced.
  • the present invention relates to an antibody obtainable by the method of the invention, to a composition comprising at least one of the antibody of the invention, the nucleic acid molecule of the invention, the vector of the invention, the host cell of the invention or the antibody produced by the method of the invention.
  • the present invention also relates to the use of the antibody of the invention for determining phosphorylation of DDR1 at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15 as well as to a method of determining phosphorylation of DDR1 at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15.
  • the discoidin domain receptor DDR1 is a receptor tyrosine kinase (RTK) and a non-integrin collagen receptor that binds a number of different collagen types and plays important roles in e.g. embryo development (for a review see Leiting B. (2014), International Review of Cell and Molecular Biology, Volume 310: 39-87 and Borza CM and Pozzi A (2014), Matrix Biology 34 185-192).
  • DDR-facilitated cellular functions include cell migration, cell survival, proliferation, and differentiation, as well as remodeling of extracellular matrices (ECM) expression and activity via the control of matrix metalloproteinase (MMP).
  • ECM extracellular matrices
  • DDRl is mainly expressed in epithelial cells, but is also found in cells of the immune system, such as stimulated peripheral blood mononuclear cells and on activated T cells. DDRl has further been shown to be able to mediate cell migration of monocytic cells and T cells in three- dimensional (3D) collagen matrices. Thus, DDRl appears to be an important player also in immune responses, which depend on the effective migration of activated leukocytes into infectious or inflammatory tissue sites.
  • 3D three- dimensional
  • the human DDRl gene maps to chromosome 6 (6p21.3) at the major histocompatibility complex locus between the HLA-E and HLA-C genes.
  • the DDRl gene spans 24 kb and comprises 17 exons.
  • the extracellular domain is encoded by exons 1 to 8, the transmembrane domain by exon 9.
  • Exons 10 to 12 encode the cytosolic juxtamembrane (JM) domain, with the remaining exons predominantly coding for the catalytic domain.
  • JM cytosolic juxtamembrane
  • DDRl is a typical receptor tyrosine kinase in that it is a single span type I transmembrane protein with a C-terminal tyrosine kinase domain in the cytoplasmic region.
  • Receptor tyrosine kinases transmit signals into cells by providing docking sites for effector molecules in the form of phosphorylated cytoplasmic tyrosines, a result of ligand-induced kinase dimerization and subsequent receptor autophosphorylation.
  • the DDRs i.e. DDRl and DDR2 are unusual RTKs in that they form ligand-independent stable dimers that are non-covalently linked.
  • DDRl undergoes ligand-induced autophosphorylation like all receptor tyrosine kinases.
  • DDRl isoforms Five different DDRl isoforms have been identified, which result from alternative splicing of exons 10 to 14 of the DDRl gene.
  • Tyrosine at position 792 in the DDRl sequence is present in isoforms 1, 2, 4 and 5 and is one tyrosine in the DDRl sequence that becomes phosphorylated upon ligand binding.
  • This phosphorylation site provides a docking site for the down-stream effectors SH2-containing transforming protein A (ShcA), SH2-containing inositol polyphosphate 5-phosphatase 1/2 (SHIP 1/2) and SH2-containing protein tyrosine phosphatase 2 (SHP-2).
  • SH2-containing transforming protein A ShcA
  • SHIP 1/2 SH2-containing inositol polyphosphate 5-phosphatase 1/2
  • SHP-2 SH2-containing protein tyrosine phosphatase 2
  • the signaling pathways activated by DDRs are not completely understood and may differ depending on the context and cell-
  • Dysregulated DDR function is associated with the progression of various human diseases, including fibrosis, atherosclerosis, arthritis, and cancer.
  • artherosclerosis is a disease characterized by thickened neointimal lesions in the vessel wall to which smooth muscle cells (SMCs) contribute by increased proliferation and migration, as well as MMP and ECM synthesis.
  • SMCs smooth muscle cells
  • Mice lacking DDR1 were found to be protected from this intimal thickening after mechanical carotid injury and showed decreased SMC proliferation, MMP production, and ECM synthesis.
  • DDR1 has also been shown to be a regulator of kidney disease.
  • Dddrl -negative mice are protected against hypertension- induced kidney disease and that DDR1 mediates both inflammation and fibrosis in this pathology.
  • a similar pathophysiological role for DDR1 was also found in another kidney disorder, Alport syndrome.
  • DDR 1 -negative mice were also protected from crescentic glomerulonephritis and obstructive nephropathy. In both pathologies, DDR1 mediated inflammatory responses and fibrosis.
  • mice devoid of DDR1 were protected from bleomycin-induced lung injury, indicating that DDR1 may be important in modulating idiopathic pulmonary fibrosis, which is characterized by persistent epithelial injury.
  • DDRs play a key role in cancer progression, in part by regulating the interaction of cancer cells with collagens.
  • DDRs are overexpressed in a large number of different types of cancer, ranging from lung, breast, brain, esophagus, head and neck, liver, and prostate cancers to lymphomas and leukemias.
  • DDR1 activation in breast and colon carcinoma cell lines was found to trigger pro-survival signals.
  • DDR1 can confer resistance to chemotherapy in breast cancer and lymphoma cell lines.
  • the molecular mechanisms underlying the roles of the DDRs in various steps of cancer progression are largely undefined (see e.g. Leiting B., International Review of Cell and Molecular Biology, Volume 310: 39-87 and Borza CM and Pozzi A (2014), Matrix Biology 34 185-192).
  • DDR1 intracellular signaling and its role in a range of pathologies is currently investigated by various research groups, there is still a need to shed light on the mechanisms of DDRl mediated signaling events, on factors that influence said signaling pathways and in particular on the role of DDRl in disease pathologies.
  • tools are required that enable a more efficient and accurate monitoring of the activation state of the DDRl receptor upon stimulation.
  • the present invention relates to an antibody that specifically binds to the discoidin domain receptor 1 (DDRl) which is phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15, wherein the antibody is characterized in that the CDRs comprise the following amino acid sequences or a variant thereof that differs in at most one amino acid substitution:
  • DDRl discoidin domain receptor 1
  • a CDR1 comprising the amino acid sequence of SEQ ID NO: l
  • a CDR2 comprising the amino acid sequence of SEQ ID NO:2
  • a CDR3 comprising the amino acid sequence of SEQ ID NO:3
  • a CDR1 comprising the amino acid sequence of SEQ ID NO:4, a CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a CDR3 comprising the amino acid sequence of SEQ ID NO:6.
  • the overall structure of antibodies is well known in the art and comprises of two heavy chains and two light chains, connected by disulfide bonds.
  • the heavy chains and the light chains each consist of one constant domain and one variable domain. Binding specificity to an antigen is provided by the variable domains of the light and heavy chains that form the antibody. More specifically, the parts of antibodies that determine their specificity and make contact with a specific ligand are referred to as the complementarity determining regions (CDRs).
  • the CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions CDR1, CDR2 and CDR3 in each variable domain, embedded into four framework regions (FW).
  • CDR-HC depicts a CDR region of a variable heavy chain and CDR-LC (or CDR(LC)) relates to a CDR region of a variable light chain.
  • FW-HC depicts a framework region of a variable heavy chain and FW-LC (or FW(LC)) relates to a framework region of a variable light chain.
  • comprising denotes that further sequences/components can be included in addition to the specifically recited sequences and/or components. However, this term also encompasses that the claimed subject-matter consists of exactly the recited sequences and/or components.
  • additional amino acids extend can be present at either the N-terminal end, or the C-terminal end, or both. Additional sequences can include e.g. sequences introduced e.g. for purification or detection, as discussed in detail herein below. Furthermore, where individual sequences "comprise” the recited sequence, they also can include additional amino acids at either the N-terminal end, or the C-terminal end, or both.
  • the antibody specifically binds to DDRl which is phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15.
  • the sequence represented in SEQ ID NO: 15 represents isoform 1 of DDRl. Accordingly, this antibody is also referred to herein as an anti-pY 792 -DDRl antibody.
  • the amino acid sequence of DDRl comprising a phosphorylated tyrosine at position 792 of the sequence represented in SEQ ID NO: 15 is also referred to herein as pY 7 9 2 - DDR1. It will be appreciated that also in the cases where the antibody of the invention comprises additional amino acids, as detailed above, said antibody necessarily has to specifically bind to pY 7 9 2 -DDRl.
  • the term “specifically binds” means that the antibody specifically binds only DDRl when DDRl is phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15, but does not or essentially does not cross-react with a different protein, in particular a different protein of similar structure such as e.g. DDR2, or with DDRl that is not phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15.
  • ligand binding assays include e.g. western blots, ELISA (including competition ELISA)-, RIA-, ECL-, IRMA-tests, as well as physiological assays, like cytotoxic assays.
  • specificity for DDR1 is determined by western blots using lysates of collagen stimulated cells, expressing high levels of phosphorylated DDR1. Unstimulated cell lysates can be used as negative control.
  • Cross-reactivity of a panel of antibodies under investigation can be tested, for example, by assessing binding of said panel of antibodies under conventional conditions to pY 79 2-DDRl as well as to a number of more or less (structurally and/or functionally) closely related proteins, and to DDR1 that is not phosphorylated at the tyrosine in position 792. Only those molecules that bind to pY 792 -DDRl but do not or do not essentially bind to any of the other proteins or to DDR1 that is not phosphorylated at the tyrosine in position 792 are considered to bind specifically.
  • a molecule that essentially does not cross-react refers to a molecule that binds to the target molecule (i.e. pY 792 -DDRl) with at least 5-times higher affinity than to a different protein of similar structure and to DDR1 that is not phosphorylated at the tyrosine in position 792, more preferably at least 10-times higher affinity, such as e.g. at least 50-times higher affinity, more preferably at least 100-times higher affinity, such as e.g. at least 250- times higher affinity.
  • target molecule i.e. pY 792 -DDRl
  • DDR1 that is not phosphorylated at the tyrosine in position 792
  • the term "antibody” relates to full immunoglobulin molecules as well as to antigen binding fragments thereof, like, Fab, Fab', F(ab') 2 , Fv. Furthermore, the term relates to modified and/or altered antibody molecules, as well as to recombinantly or synthetically generated/synthesized antibodies.
  • antibody also comprises bifunctional antibodies, trifunctional antibodies, fully-human antibodies, chimeric antibodies, and antibody constructs, like single chain Fvs (scFv) or antibody-fusion proteins.
  • a “Fab fragment” as used herein is comprised of one light chain and the C H I and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a "Fab' fragment” contains one light chain and a portion of one heavy chain that contains the V H domain and the C H I domain and also the region between the C H I and C H 2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.
  • a “F(ab') 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C H I and C H 2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab') 2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.
  • Fab/c fragment contain both Fc and Fab determinants, wherein an "Fc" region contains two heavy chain fragments comprising the C H 2 and C H 3 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C H 3 domains.
  • the “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Single-chain Fvs also abbreviated as “scFv” are antibody fragments that have, in the context of the present invention, the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • Fully-human antibody refers to an antibody which comprises human immunoglobulin protein sequences only. Nonetheless, a fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell or it may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, a fully human antibody may contain hamster carbohydrate chains if produced in a hamster, in a hamster cell, such as e.g.
  • a "mouse antibody” or “murine antibody” is an antibody that comprises mouse (murine) immunoglobulin protein sequences only
  • a "rat antibody” or a “rabbit antibody” is an antibody that comprises rat or rabbit immunoglobulin sequences, respectively, only.
  • such murine, rat or rabbit antibodies may contain carbohydrate chains from other species, if produced in such an animal or a cell of such an animal.
  • the antibodies may contain hamster carbohydrate chains if produced in a hamster cell, such as e.g. CHO cells, or in a hybridoma derived from a hamster cell.
  • Fully-human antibodies can be produced, for example, by phage display which is a widely used screening technology which enables production and screening of fully human antibodies. Also phage antibodies can be used in context of this invention. Phage display methods are described, for example, in US 5,403,484, US 5,969,108 and US 5,885,793. Another technology which enables development of fully-human antibodies involves a modification of mouse hybridoma technology. Mice are made transgenic to contain the human immunoglobulin locus in exchange for their own mouse genes (see, for example, US 5,877,397).
  • chimeric antibodies refers to antibodies that comprise a variable region of a human or non-human species fused or chimerized to an antibody region (e.g., constant region) from another species, either human or non-human (e.g., mouse, horse, rabbit, dog, cow, chicken).
  • antibody also encompasses antibody constructs, such as antibody-fusion proteins, wherein the antibody comprises (an) additional domain(s), e.g. for the isolation and/or preparation of recombinantly produced constructs, in addition to the domains defined herein by specific amino acid sequences.
  • the antibody of the present invention can be produced such that it is a recombinant antibody, for example a recombinant human antibody, a heterologous antibody or a hetero-hybrid antibody.
  • recombinant (human) antibody includes all (human sequence) antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • Recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V H and V L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V H and V L sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism that is not the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
  • hetero -hybrid antibody refers to an antibody having light and heavy chains that originate from different organisms.
  • an antibody having a human heavy chain associated with a murine light chain is a hetero-hybrid antibody.
  • hetero-hybrid antibodies include chimeric and humanized antibodies.
  • the antibody in accordance with the present invention comprises the recited combination of light chain CRDs and heavy chain CRDs.
  • the surrounding framework sequence of the respective variable domain into which the CDRs are incorporated can be chosen by the skilled person without further ado.
  • the framework sequences described further below or the specific framework sequence employed in the appended examples can be used.
  • the CDRs can comprise the specifically recited sequence or can differ therefrom in at most one amino acid substitution.
  • one amino acid in each of the CDRs can be replaced by a different amino acid. It will be appreciated that also encompassed is that an amino acid substitution is present in some, but not all CDRs of one chain or of one antibody.
  • substitution refers to the replacement of an amino acid with another amino acid. Thus, the total number of amino acids remains the same.
  • the deletion of an amino acid at a certain position and the introduction of one (or more) amino acid(s) at a different position is explicitly not encompassed by the term "substitution”.
  • substitutions in accordance with the present invention, can be conservative amino acid substitutions or non-conservative amino acid substitutions.
  • conservative amino acid substitution is well known in the art and refers to the replacement of an amino acid with a different amino acid having similar structural and/or chemical properties. Such similarities include e.g.
  • nonpolar (hydrophobic) amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, and methionine
  • polar neutral amino acids include glycine, serine, threonine, cysteine, asparagine, and glutamine
  • positively charged (basic) amino acids include arginine, lysine, and histidine
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Non-conservative amino acid substitutions can be introduced in order to introduce new reactive groups, for example, for the conjugation to other compounds, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for the formation of non- naturally occurring intermolecular disulphide linkages.
  • PEG polyethylene glycol
  • HES hydroxyethyl starch
  • cysteine can be introduced into the amino acid sequence.
  • the thiol moiety thus generated can then be used for the conjugation to other compounds, for example, in order to increase the serum half-life of the respective anti-pY79 2 -DDRl antibody.
  • the substitution is a non-conservative amino acid substitution
  • it is a substitution that introduces a cysteine.
  • the substitution in any (or all) of the CDRs is a conservative amino acid substitution. It will be appreciated that also an antibody having such substituted amino acids in one or more of the CDRs necessarily has to be an antibody that specifically binds to the discoidin domain receptor 1 (DDR1) which is phosphorylated at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15, as defined herein above.
  • DDR1 discoidin domain receptor 1
  • the CDRs comprise the sequence specifically recited above, i.e. without any amino acid variation.
  • novel anti-pY 7 9 2 -DDRl antibodies are provided that enable the specific and efficient monitoring of DDRl receptor activation.
  • DDRl has been shown to play important roles in various diseases and, accordingly, monitoring the activation of DDRl and its signaling activity is of utmost interest for understanding the involvement of DDRl in said diseases.
  • the anti-pY 7 9 2 -DDRl antibodies of the present invention provide a superior binding efficiency and specificity.
  • Antibody-binding to ⁇ 73 ⁇ 4 - DDR1 was determined in five different assays, including immunohistochemical staining of collagen stimulated (positive control) and unstimulated formalin-fixed paraffin embedded cells; western blots with lysates from collagen stimulated (positive control) and unstimulated cells; ELISA and Biacore with phosphorylated and non-phosphorylated peptides; as well as a peptide array with spotted phosphorylated and non-phosphorylated peptides spanning amino acid sequences of related phosphorylation sites from other receptor tyrosine kinases.
  • the anti-pY 7 9 2 -DDRl antibodies according to the present invention showed a surprising capability to bind to phosphorylated tyrosine at position 792 of the DDRl sequence represented in SEQ ID NO: 15, whereas DDRl that is not phosphorylated at this position was not bound. To the inventors' best knowledge, these antibodies thus represent the first monoclonal anti-pY 7 9 2 -DDRl specific antibodies available in the art.
  • the present invention also relates to the antibody of the invention, wherein the antibody comprises:
  • FWs comprise the following amino acid sequences or a variant thereof that is at least 85% identical thereto and wherein the CDRs comprise the following amino acid sequences or a variant thereof that differs in at most one amino acid substitution:
  • the primary structure shown in formula I represents the order of the components of the light chain variable domain of the antibody of the present invention from the N-terminus to the C- terminus.
  • the primary structure shown in formula II represents the order of the components of the heavy chain variable domain of the antibody of the present invention from the N-terminus to the C-terminus.
  • framework region FW 1 represents the most N-terminal part of the respective variable chain domain
  • FW 4 represents the most C-terminal part of the respective variable chain domain.
  • the respective FW and CDR sequences "comprise” the recited amino acid sequences.
  • the respective FW and CDR sequences consist of said amino acid sequences, i.e. the light chain variable domain(s) and heavy chain variable domain(s) of the anti-pY 7 9 2 -DDRl antibody of the invention consist of the FWs and CDRs as represented in formula I and formula II, respectively, wherein the respective FW and CDR sequences consist of the recited amino acid sequences.
  • the above provided definitions and specifically exemplified embodiments apply mutatis mutandis.
  • the individual FWs can comprise the, or consist of the specifically recited amino acid sequence or of an amino acid sequence at least 85% identical thereto.
  • the identity is at least 90%, more preferred at least 92.5%, more preferred at least 95%, even more preferred the identity is at least 98%, such as at least 99% and most preferably the identity is at least 99.5%.
  • a different degree of sequence identity may be allowable, depending on the actual sequence and e.g. the length of the respective FW sequence, as well as its location within the respective variable chain domain.
  • % sequence identity describes the number of matches ("hits") of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences (or the overall compared part thereof). Percent identity is determined by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the percentage of amino acid residues that are the same may be determined for two or more sequences or sub-sequences when these (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected.
  • NCBI BLAST algorithm Altschul, S.F. et al. [1997] Nucleic Acids Res. 25:3389-3402
  • CLUSTALW computer program Tompson, J.D. et al. [1994] Nucleic Acids Res. 22:4673-4680
  • FASTA FASTA
  • the NCBI BLAST algorithm is employed in accordance with this invention.
  • the BLASTP program uses as default a word length (W) of 3, and an expectation (E) of 10.
  • the above described degree of variation in the framework regions as compared to the respective specifically recited amino acid sequence can be due to the substitution, insertion, addition, or deletion of (an) amino acid(s).
  • substitution has been defined herein above. In those cases where more than one amino acid is to be substituted, each amino acid is independently replaced with another amino acid, i.e. for each amino acid that is removed a different amino acid is introduced at the same position.
  • insertion refers to the addition of one or more amino acids to the specifically recited amino acid sequence, wherein the addition is not to the N- or C-terminal end of the polypeptide.
  • addition in accordance with the present invention, refers to the addition of one or more amino acids to the specifically recited amino acid sequence, either to the N- or C-terminal end of the polypeptide, or to both.
  • deletion refers to the loss of one or more amino acids from the specifically recited amino acid sequence.
  • the variation in the amino acid sequences of the framework regions is due to the substitution of (an) amino acid(s).
  • Substitutions as defined herein above, can be conservative amino acid substitutions or non-conservative amino acid substitutions.
  • substitutions in the framework regions are conservative amino acid substitutions.
  • the antibody comprises a light chain variable domain consisting of framework regions (FW) and CDRs as represented in formula I above, and a heavy chain variable domain consisting of FWs and CDRs as represented in formula II above, wherein the FWs comprise the following amino acid sequences or a variant thereof that is at least 85% identical thereto and wherein the CDRs comprise the following amino acid sequences:
  • the total amount of all variations present in framework regions 1 to 4 of the light chain variable domain taken together is in one embodiment at most 16 amino acid substitutions and the total amount of all variations present in framework regions 1 to 4 of the heavy chain variable domain taken together is in said embodiment at most 18 amino acid substitutions.
  • the CDRs consist of the above recited specific sequences (i.e. without any variations) and the above recited framework regions comprise at most the following amount of amino acid variations within the above recited specific sequences:
  • FW(HC)3 at most 4 amino acid variations; and FW(HC)4 at most 1 amino acid variation.
  • the amino acid variations are substitutions.
  • the total amount of variations present in the light or heavy chain variable domain framework regions is at most 9 amino acid substitutions, such as e.g. at most 8 amino acid substitutions, e.g. at most 6 amino acids substitutions, such as at most 4 amino acids substitutions, e.g. at most 3 amino acids substitutions, such as at most 2 amino acids substitutions.
  • FWs are amino acid sequences that form part of the frame or scaffold of the variable chain regions
  • substitution within said sequences in particular in form of conservative amino acid substitutions, will in many cases not affect the binding capability of the anti-pY79 2 -DDRl antibody.
  • these amino acids typically are not directly involved in the binding to pY 792 -DDRl, and their substitution for suitable alternative amino acids can be designed such that no alteration in the three- dimensional structure and folding of the protein occurs.
  • substitutions can provide numerous beneficial effects such as for improved expression in certain hosts or for stabilization of the protein by introduction of e.g. additional disulphide bridges.
  • the present invention further relates to an antibody comprising a light chain variable domain consisting of an amino acid sequence that is at least 85% identical to the light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 16, and a heavy chain variable domain consisting of an amino acid sequence that is at least 85% identical to the heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 17; wherein the antibody specifically binds to DDRl which is phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15.
  • the antibody of the invention binds to pY 792 -DDRl with an affinity that is at least the same or essentially the same as an antibody that comprises as light chain variable domain the amino acid sequence of SEQ ID NO: 16 and as heavy chain variable domain the amino acid sequence of SEQ ID NO: 17 (i.e. the variable chain regions designated as 15F10 in the appended examples).
  • the binding affinity of the antibody 15F10 is provided in Table 3 herein below. Accordingly, in one embodiment in accordance with this aspect of the invention, the antibody of the invention binds to pY 792 -DDRl with a K D of 1.8E-10 M or lower.
  • K D refers to the equilibrium dissociation constant (the reciprocal of the equilibrium binding constant) and is used herein according to the definitions provided in the art. Means and methods for determined the K D value are as described above.
  • the binding affinity to pYs 2 o-DDRl is considered to be essentially retained if the difference or the ratio between the K D of the antibody comprising the specifically recited light and heavy chain variable domains and the K D of the same antibody with amino acid modifications is within two orders of magnitude, more preferably within one order of magnitude.
  • the binding affinity is fully retained, i.e. the K D of the antibody comprising light and heavy chain variable domains with amino acid modifications is equal or lower than the K D of the same antibody comprising the specifically recited light and heavy chain variable domains.
  • a lower K D value corresponds to a higher or better affinity as is well known in the art. Therefore, the invention also encompasses antibodies comprising modified amino acids that have an increased binding affinity compared to the antibody without such amino acid modifications.
  • the anti-pY 792 -DDRl antibodies of the invention bind to pY 792 -DDRl with a K D of 2E-10 M or lower, such as e.g. 1.5E-10 M or lower, or even lE-10 M or lower.
  • the antibody comprises as light chain variable domain the amino acid sequence of SEQ ID NO: 16 and as heavy chain variable domain the amino acid sequence of SEQ ID NO: 17 (i.e. corresponding to the variable chain regions designated as 15F10 in the appended examples).
  • the above recited sequences for the variable light and heavy chain regions are the amino acid sequences that have been employed in the appended examples.
  • Full-length amino acid sequences for the light and heavy chains present in the antibodies employed in the examples (that comprise said variable domains) are represented in SEQ ID NOs: 18 and 19.
  • this anti-pY 7 9 2 -DDRl antibody specifically recognizes phosphorylated tyrosine at position 792 of DDRl when used in immunohistochemical staining of cells and/or tissues and is therefore suitable for the determination of the presence or absence of a phosphorylation at tyrosine 792 of DDRl.
  • the present invention further relates to a nucleic acid molecule encoding a light chain variable region of any one of the antibodies of the invention defined herein above.
  • This nucleic acid molecule is referred to herein as the first nucleic acid molecule of the invention.
  • the present invention also relates to a nucleic acid molecule encoding a heavy chain variable region of any one of the antibodies of the invention defined herein above.
  • This nucleic acid molecule is referred to herein as the second nucleic acid molecule of the invention.
  • nucleic acid molecule also referred to as nucleic acid sequence or polynucleotide herein, includes DNA, such as cDNA or genomic DNA.
  • the nucleic acid molecules of the invention can e.g. be synthesized by standard chemical synthesis methods and/or recombinant methods, or produced semi-synthetically, e.g. by combining chemical synthesis and recombinant methods.
  • Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods, such as restriction digests, ligations and molecular cloning.
  • the first nucleic acid molecule of the invention encodes a light chain variable region:
  • the second nucleic acid molecule of the invention encodes a heavy chain variable region:
  • the present invention further relates to a vector comprising the first nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a light chain variable region of any one of the antibodies of the invention defined herein above.
  • the present invention further relates to a vector comprising the second nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a heavy chain variable region of any one of the antibodies of the invention defined herein above.
  • Such vectors are also referred to herein as the "individual vector(s) of the invention”.
  • vectors are known to those skilled in molecular biology, the choice of which depends on the desired function.
  • Non-limiting examples of vectors include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in e.g. genetic engineering. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook et al. (loc cit.) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994).
  • the vector is an expression vector.
  • An expression vector according to this invention is capable of directing the replication and the expression of the nucleic acid molecule of the invention in a host and, accordingly, provides for the expression of the variable chain domains of the anti-pY 7 9 2 -DDRl antibodies of the present invention encoded thereby in the selected host.
  • the vector(s) comprise(s) further sequences to ensure that not only said variable chain domains of the invention are expressed, but also the full- length IgG antibodies comprising said variable chain domains of the invention.
  • Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule, for example into a translatable mRNA.
  • the vector is a eukaryotic expression plasmid for the transient recombinant expression of the heavy chain and/or the light chain of monoclonal rabbit antibodies.
  • Such vectors have been specifically developed for antibody expression but also antibody production by e.g. transient transfection of eukaryotic cells e.g. HEK 293 or derivatives thereof or CHO cells.
  • Non-limiting examples of vectors include pQE-12, the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen), lambda gtl l, pJOE, the pBBRl-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACTl, pTRE, pCAL-n- EK, pESP-1, pOP13CAT, the E-027 pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, p
  • Non-limiting examples for plasmid vectors suitable for Pichia pastoris comprise e.g. the plasmids pA0815, pPIC9K and pPIC3.5K (all Invitrogen).
  • Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos as well as a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.
  • vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • the coding sequences comprised in the vector can be ligated to transcriptional regulatory elements and/or to other amino acid encoding sequences using established methods.
  • regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) (Owens, G.C. et al. [2001] Proc. Natl. Acad. Sci. U.S.A.
  • regulatory elements ensuring the initiation of transcription comprise promoters, a translation initiation codon, enhancers, insulators and/or regulatory elements ensuring transcription termination, which are to be included downstream of the nucleic acid molecules of the invention.
  • regulatory elements ensuring the initiation of transcription comprise promoters, a translation initiation codon, enhancers, insulators and/or regulatory elements ensuring transcription termination, which are to be included downstream of the nucleic acid molecules of the invention.
  • Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals or, depending on the expression system used, signal sequences capable of directing the expressed protein to a cellular compartment or to the culture medium.
  • the vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding.
  • suitable origins of replication include, for example, the full length ColEl, a truncated ColEI, the SV40 viral and the Ml 3 origins of replication
  • suitable promoters include, without being limiting, the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, the tetracycline promoter/operator (tet p/o ), chicken ⁇ -actin promoter, CAG-promoter (a combination of chicken ⁇ -actin promoter and cytomegalovirus immediate-early enhancer), the gailO promoter, human elongation factor la-promoter, AOX1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the T7 or T5 promoter, the lacUV5 promoter, the Autographa californica multiple nuclear polyhedrosis virus (A)
  • CMV
  • an enhancer is e.g. the SV40-enhancer.
  • regulatory elements ensuring transcription termination include the SV40-poly-A site, the tk-poly-A site, the rho-independent lpp terminator or the AcMNPV polyhedral polyadenylation signals.
  • selectable markers include dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149), npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J.
  • hygro which confers resistance to hygromycin
  • Additional selectable genes namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl. Acad. Sci.
  • mannose-6- phosphate isomerase which allows cells to utilize mannose
  • ODC ornithine decarboxylase
  • DFMO ornithine decarboxylase
  • ornithine decarboxylase inhibitor 2- (difluoromethyl)-DL-ornithine
  • DFMO McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.
  • deaminase from Aspergillus terreus which confers resistance to blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338).
  • the vector is a eukaryotic expression plasmid containing an expression cassette consisting of a 5' CMV promoter including Intron A, and a BGH polyadenylation sequence.
  • the plasmid can contain a pUC18-derived origin of replication and a beta-lactamase gene conferring ampicillin resistance for plasmid amplification in E. coli.
  • an eukaryotic leader sequence can be cloned 5' of the antibody gene.
  • Suitable bacterial expression hosts comprise e. g. strains derived from JM83, W3110, KS272, TGI, K12, BL21 (such as BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE) or Rosettaa.
  • strains derived from JM83, W3110, KS272, TGI, K12, BL21 such as BL21(DE3), BL21(DE3)PlysS, BL21(DE3)RIL, BL21(DE3)PRARE
  • Rosettaa Rosettaa.
  • the nucleic acid molecules and/or vectors of the invention can be designed for introduction into cells by e.g. chemical based methods (polyethylenimine, calcium phosphate, liposomes, DEAE-dextrane, nucleofection), non chemical methods (electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery or naturally occurring transformation upon contacting cells with the nucleic acid molecule of the invention), particle-based methods (gene gun, magnetofection, impalefection) phage vector-based methods and viral methods.
  • chemical based methods polyethylenimine, calcium phosphate, liposomes, DEAE-dextrane, nucleofection
  • non chemical methods electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery or naturally occurring transformation upon contacting cells with the nucleic acid molecule of the invention
  • particle-based methods gene gun, magnetofection, impalefection
  • expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, Semliki Forest Virus or bovine papilloma virus, may be used for delivery of the nucleic acid molecules into targeted cell population.
  • viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, Semliki Forest Virus or bovine papilloma virus
  • baculoviral systems can also be used as vector in eukaryotic expression system for the nucleic acid molecules of the invention.
  • the nucleic acid molecules and/or vectors of the invention are designed for transformation of chemical competent E. coli by calcium phosphate and/or for transient transfection of HEK293 and CHO by polyethylenimine- or lipofectamine-transfection.
  • the present invention further relates to a vector comprising: a nucleic acid molecule encoding a light chain variable domain as defined herein above and a heavy chain variable domain as defined herein above.
  • the vector is an expression vector.
  • This second type of vector relates to a vector comprising at least two nucleic acid molecules, namely one encoding a light chain variable domain and one encoding a heavy chain variable domain. As is evident from the above combination, the light chain variable domain and heavy chain variable domain are combined in the vector such that the expression of a functional anti-pY79 2 -DDRl antibody of the invention is enabled.
  • This second type of vector is also referred to herein as the "combination vector of the invention”.
  • the present invention further relates to a host cell or non-human host comprising:
  • the individual vector of the invention comprising the first nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a light chain variable region in accordance with the invention and the individual vector of the invention comprising the second nucleic acid molecule of the invention, i.e. a nucleic acid molecule encoding a heavy chain variable region of the invention, wherein these two vectors comprise the nucleic acid molecules encoding for matching light chain and heavy chain variable regions as defined above.
  • the host cell can be any prokaryotic or eukaryotic cell.
  • prokaryote is meant to include all bacteria which can be transformed, transduced or transfected with DNA or DNA or RNA molecules for the expression of a protein of the invention.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella and Bacillus subtilis.
  • eukaryotic is meant to include yeast, higher plant, insect and mammalian cells.
  • Typical mammalian host cells include, Hela, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS/0, SP2/0 and C127 cells, COS cells, e.g. COS 1 or COS 7, CV1, quail QCl-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.
  • COS cells e.g. COS 1 or COS 7, CV1, quail QCl-3 cells
  • mouse L cells mouse sarcoma cells
  • Bowes melanoma cells e.g., Chinese hamster ovary (CHO) cells.
  • Exemplary mammalian host cells in accordance with the present invention are CHO cells.
  • Other suitable eukaryotic host cells include, without being limiting, chicken cells, such as e.g.
  • DT40 cells or yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe and Kluyveromyces lactis.
  • Insect cells suitable for expression are e.g. Drosophila S2, Drosophila Kc, Spodoptera Sf9 and Sf21 or Trichoplusia Hi5 cells.
  • Suitable zebrafish cell lines include, without being limiting, ZFL, SJD or ZF4.
  • the described vector(s) can either integrate into the genome of the host or can be maintained extrachromosomally. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleic acid molecules, and as desired, the collection and purification of the antibody of the invention may follow. Appropriate culture media and conditions for the above described host cells are known in the art.
  • the recited host is a mammalian cell, such as a human cell or human cell line.
  • the host cell transformed with the vector(s) of the invention is HEK293 or CHO.
  • the host cell transformed with the vector(s) of the invention is CHO.
  • vector comprising in accordance with the present invention it is understood that further nucleic acid sequences are present in the vectors that are necessary and/or sufficient for the host cell to produce an anti-pY79 2 -DDRl antibody of the invention.
  • Such further nucleic acid sequences are e.g. nucleic acid sequences encoding the remainder of the light chain as well as nucleic acid sequences encoding the remainder of the heavy chain.
  • the host cell or non-human host in accordance with the present invention, comprises either one vector encoding both the light chain and heavy chain variable regions as defined herein above or it comprises two separate vectors, wherein one vector carries a nucleic acid molecule encoding a light chain variable region in accordance with the present invention and the second vector carries a nucleic acid molecule encoding a matching heavy chain variable region in accordance with the present invention. Accordingly, in each case, expression of those nucleic acid molecules is linked to each other that are required to be present within one antibody molecule to ensure the production of an anti-pY792-DDRl antibody of the invention having the binding capabilities described herein above.
  • the host cells in accordance with this embodiment may e.g. be employed to produce large amounts of the anti-pY79 2 -DDRl antibodies of the present invention.
  • Said host cells are produced by introducing the above described vector(s) into the host. The presence of said vector(s) in the host then mediates the expression of the nucleic acid molecules encoding the above described light chain variable domains and heavy chain variable domains of the anti- PY792-DDRI antibodies of the invention.
  • the vector(s) of the invention can comprise further sequences enabling the expression of full length IgG antibodies, thereby resulting in the production of full length IgG antibodies by the host cells, wherein said antibodies are characterized by the presence of the variable light and/or heavy chain domains in accordance with the present invention.
  • the present invention further relates to a method for the production of an antibody that specifically binds to DDR1 which is phosphorylated at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15, the method comprising culturing the host cell of the invention under suitable conditions and isolating the antibody produced.
  • the vector(s) present in the host of the invention is/are either (an) expression vector(s), or the vector(s) mediate(s) the stable integration of the nucleic acid molecule(s) of present invention into the genome of the host cell in such a manner that expression thereof is ensured.
  • Means and methods for selection a host cell in which the nucleic acid molecules encoding the respective light and heavy chain domains of the anti-pY79 2 -DDRl antibody of the present invention have been successfully introduced such that expression of the antibody is ensured are well known in the art and have been described (Browne, S.M. & Al- Rubeai, M. [2007] Trends Biotechnol.
  • Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to the person skilled in the art.
  • bacteria such as e.g. E. coli can be cultured under aeration in Luria Bertani (LB) medium, typically at a temperature from 4 to about 37°C.
  • LB Luria Bertani
  • the medium can be buffered or supplemented with suitable additives known to enhance or facilitate both.
  • expression of the polypeptide can be induced by addition of an appropriate inducing agent, such as e.g. anhydro tetracycline.
  • mammalian cell culture can e.g. be carried out in RPMI, Williams' E or DMEM medium containing 10% (v/v) FCS, 2 mM L- glutamine and 100 U/ml penicillin/streptomycin.
  • the cells can be kept e.g. at 37°C or at 41°C for DT40 chicken cells, in a 5% C0 2 , water- saturated atmosphere.
  • a suitable medium for insect cell culture is e.g. TNM + 10% FCS, SF900 or HyClone SFX- Insect medium. Insect cells are usually grown at 27 °C as adhesion or suspension cultures.
  • Suitable expression protocols for eukaryotic or vertebrate cells are well known to the skilled person and can be retrieved e.g. from Sambrook, J & Russel, D.W. [2001] (Cold Spring Harbor Laboratory, NY).
  • the method is carried out using mammalian cells, such as e.g. CHO or HEK293 cells. In a further embodiment, the method is carried out using CHO cells.
  • the antibody expressed may be glycosylated or may be non-glycosylated.
  • a plasmid or a virus is used containing the coding sequence of the antibody of the invention and genetically fused thereto an N-terminal FLAG-tag and/or C-terminal His-tag.
  • the length of said FLAG-tag is about 4 to 8 amino acids, such as e.g. exactly 8 amino acids.
  • An above described vector can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art.
  • methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, loc cit).
  • the transformed hosts can be grown in bioreactors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the antibody of the invention can then be isolated from the growth medium.
  • the isolation and purification of the, e.g., microbially expressed antibodies of the invention may be by any conventional means such as, e.g., affinity chromatography (for example using a fusion-tag such as the Strep-tag II or the His 6 tag), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC or immunoprecipitation.
  • affinity chromatography for example using a fusion-tag such as the Strep-tag II or the His 6 tag
  • gel filtration size exclusion chromatography
  • anion exchange chromatography e.g., cation exchange chromatography
  • hydrophobic interaction chromatography e.g. in Sambrook, J & Russel, D
  • the term "isolating the antibody produced” refers to the isolation of the anti-pY79 2 -DDRl antibody of the present invention.
  • the present invention relates to an antibody that specifically binds to DDR1 which is phosphorylated at the tyrosine in position 792 of the DDR1 sequence represented in SEQ ID NO: 15, wherein the antibody is obtainable or obtained by the method of the invention.
  • the present invention further relates to a composition comprising at least one of:
  • composition relates to a composition which comprises at least one of the recited compounds. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and/or enhancing their function.
  • the composition may be in solid or liquid form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s) or (a) solution(s).
  • the components of the composition can be packaged in a container or a plurality of containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a lyophilized formulation 10-ml vials are filled with 5 ml of 1% (w/v) or 10% (w/v) aqueous solution, and the resulting mixture is lyophilized.
  • a solution for use is prepared by reconstituting the lyophilized compound(s) using either e.g. water-for-injection for therapeutic uses or another desired solvent, e.g. a buffer, for diagnostic purposes.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions may be packaged as a kit with instructions for use.
  • the composition of the invention is a composition enabling the skilled person to carry out in vivo as well as in in vitro or ex vivo methods well known in the art.
  • methods such as e.g. immunohistochemical staining of tissues or cells obtained from a patient or measuring the amount of anti-pY79 2 -DDRl antibody in a particular tissue can be of value.
  • the anti-pY 792 -DDRl antibodies provided herein are also suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. Examples of immunoassays which can utilize the antibodies of the invention are immunoassays in either a direct or indirect format.
  • immunoassays examples include the enzyme linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), the Western blot assay, or immuno assays based on detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.
  • ELISA enzyme linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • Western blot assay or immuno assays based on detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.
  • the present invention further relates to the use of the antibody of the invention for determining phosphorylation of DDRl at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15.
  • the anti-pY 7 9 2 -DDRl antibody of the present invention specifically binds to DDRl when it is phosphorylated at the tyrosine in position 792, but does not or essentially does not cross-react with a different protein, in particular a different protein of similar structure, or with DDRl that is not phosphorylated at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15. Accordingly, said antibody is suitable for determining the phosphorylation of DDRl at said tyrosine at position 792.
  • Non-limiting examples of suitable methods of determining the phosphorylation of DDRl at said tyrosine at position 792 by employing the anti-pY 7 9 2 -DDRl antibody of the invention include, without being limiting, immunohistochemical and immunocytochemical methods, Western blotting, ELISA, and immuno assays based on detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.
  • the determination of phosphorylation of DDRl is by immunohistochemistry.
  • the present invention relates to a method of determining phosphorylation of DDRl at the tyrosine in position 792 of the DDRl sequence represented in SEQ ID NO: 15, the method comprising detecting the binding of the antibody of the invention to DDRl.
  • the anti-pY 7 9 2 -DDRl antibody of the invention for determining the phosphorylation of DDRl at the tyrosine in position 792, it can be employed in a method of determining said phosphorylation, as discussed above.
  • a sample containing DDRl is brought in contact with the antibody of the invention and the binding of the anti-pY 792 - DDR1 antibody to DDRl is detected. If the antibody binds to DDRl, then said DDRl is phosphorylated. If no binding occurs, then the DDRl is not phosphorylated at the tyrosine in position 792.
  • the method includes the use of suitable controls to ensure that any positive (i.e.
  • phosphorylation is detected
  • negative result i.e. no phosphorylation is detected
  • Suitable positive as well as negative controls can be designed and included in the experimental setup by a skilled person without further ado and include e.g. DDRl preparations known to have a phosphorylation at tyrosine 792, for example due to the application of a previous phosphorylation step, as positive control, as well as DDRl preparations known to not have a phosphorylation at tyrosine 792, for example due to the application of a previous de -phosphorylation step, as negative control.
  • each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from.
  • a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I
  • the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A,
  • FIG. 1 Detection of pDDRl in IHC FFPE control cell lines. Phosphorylation was induced by stimulation of the U20S cells with collagen. Subsequent to stimulation the cells were fixed and paraffin embedded. It is shown that the selected clone can detect phosphorylated DDR1 (lower panel), whereas no signal is obvious in the unstimulated control cells (upper panel). Membrane staining of phosphorylated DDR1 is highlighted with black arrows (lower panel).
  • Figure 2 Detection of DDR1 and pDDRl, respectively, by Western Blotting.
  • U20S cells were treated with collagen to stimulate the cells and to induce DDR1 phosphorylation.
  • untreated U20S cells were used.
  • Subsequent to stimulation the cells were lyzed, subjected to SDS-PAGE, transferred to nitrocellulose and analyzed for presence of DDR1 and pDDRl, respectively by probing with the antibodies depicted in the Figure.
  • DNA sequences were determined by double strand sequencing performed at Microsynth AG (Balgach, Switzerland).
  • Peptides were synthesized by means of fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis on a multiple peptide synthesizer e.g. from Protein Technologies, Inc. For this, 4.0 equivalents of each amino acid derivative were used. Amino acid derivatives were dissolved in dimethylformamide containg 1 equivalent of l-Hydroxy-7-azabenzotriazol. Peptides were synthesized on Tentagel R resin. Coupling reactions were carried out for 5 minutes in dimethylformamide as a reaction medium with 4 equivalents HATU and 8 equivalents of ⁇ , ⁇ -Diisopropylethylamine relative to resin loading.
  • Fmoc fluorenylmethyloxycarbonyl
  • the Fmoc group was cleaved in 8 minutes after each synthesis step using 25% piperidine in dimethyl formamide. Release of the peptide from the synthesis resin and the cleavage of the acid-labile protecting groups was achieved in 3 hours at room temperature with 9,5 ml trifluoroacetic acid, 0.25 ml triisopropylsilane, and 0,25 ml water. The reaction solution was subsequently mixed with cooled diisopropyl ether to precipitate the peptide. The precipitate was filtered, washed again with diisopropyl ether, dissolved in a small amount of aqueous acetic acid and lyophilized.
  • the crude material obtained was purified by preparative RP-HPLC using a gradient of acetonitrile/water containing 0.1% trifluoroacetic acid. The identity of the purified material was checked by means of ion spray mass spectrometry. Cysteine-containing peptides were conjugated to maleimide-activated keyhole limpet hemocyanin.
  • a biotinylated variant of the phosphorylated screening peptide DDRl_HUMAN(788-808) [pY792] or of the non-phosphorylated screening peptide DDRl_HUMAN(788-808) was coupled to Streptavidin coated 96-well plates. A small amount of serum of each rabbit was collected on day 45 and day 105 after the start of the immunization campaign. The biotinylated peptides were immobilized on plate surfaces at a concentration of 15 ng/mL.
  • the sera from each rabbit were diluted in PBS with 1% BSA and the dilutions were added to the plates.
  • the sera were tested at dilutions 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700 and 1:656100.
  • Bound antibody was detected with a HRP-labeled F(ab ) 2 goat anti-rabbit Fey (Dianova) and ABTS (Roche) as a substrate.
  • the titer of the analyzed animals was set by 50% signal decrease of the dilution curve.
  • Table 1 The titers against the different screening reagent are shown. The titer levels indicate that the sera show specificity for the phosphorylated forms of the screening reagents.
  • the antibodies against the peptide immunogens DDRl_HUMAN(788-808) [pY792] were obtained using the B-cell PCR method, as described in Seeber et al. (2014), PLoS One. 2014 Feb 4;9(2).
  • PBMCs and B-cells for the single cell deposition were prepared from the rabbits at different time points.
  • the individual pY792-DDRl rabbit antibodies were recombinantly expressed in HEK293 cells.
  • the heavy and light chain coding plasmids derived from the recombinant IgG cloning process were used for transient transfection of HEK293 cells.
  • HEK293 cells were grown in a shaking device at 125 rpm in F17-medium (Gibco) at 37°C in an atmosphere containing 8% C0 2 . Cells were split the day before transfection and seeded at a density of 0.7 - 0.8xl0 6 cells/ml. On the day of transfection, 1 - 1.5xl0 6 HEK293 cells in a volume of 2 ml were transfected with 0.5 mg heavy chain plasmid plus 0.5 mg light chain plasmid, suspended in 80 ml OptiMEMH medium (Gibco) and supplemented with 1 ml PEIpro transfection reagent (Polyplus-Transfection) in 48-well deep well plates.
  • OptiMEMH medium Gibco
  • PEIpro transfection reagent Polyplus-Transfection
  • Table 2 Detection of the different screening peptides by the developed antibody. The OD values (OD 405) are shown. From the results it is evident that the clone is specific for the phosphorylated forms of the screening peptides.
  • the slides were dewaxed and antigen retrieval was performed by applying heat for 1 hour.
  • the Ventana buffer CCl was used for antigen retrieval.
  • Appropriate concentrations of antibodies were manually applied on the slides and incubated for 32min at 37°C.
  • the linker and the secondary antibody were applied by the device and incubated for 8min at RT each.
  • Slides were counterstained with hematoxillin II and bluing reagent for 8min. After the staining procedure silicone oil was removed by washing the slides with water containing detergent. Slides were dehydrated in an ascending ethanol row and xylene and mounted with Eukitt. The results are depicted in Figure 1, which shows a specific staining of phosphorylated DDR1 with the antibody of the invention, whereas no signal is obtained in the unstimulated control cells.
  • a Biacore T200 instrument (GE Healthcare) was used to kinetically assess the binding behavior of rabbit monoclonal antibodies towards the respective phosphorylated peptide analyte:
  • the Chi2-test shows information about the quality of the fitting model. For the interpretation of Chi2 data of real time SPR experiments refer to Onell, A. and Andersson, K.; Kinetic determinations of molecular interactions using Biacore - minimum data requirements for efficient experimental design.
  • the Molar Ratio (MR) values indicate a fully functional 1:2 antibody to analyte binding ratio.
  • the antibody shows a subnanomolar high affinity K D with a fast association rate k a and a slow dissociation rate k d at 25 °C.
  • Example 8 Western blot analysis of the pY792-DDRl antibody
  • the gels were run for 15 min at 100 V and 60 min at 150 V in MOPS- running buffer (Invitrogen), using the XCell SureLock Elektrophoresis Cell (Invitrogen Novex Mini Cell System). Proteins were transferred to nitrocellulose-membranes for 8 min at 20V using the iBlot System (Invitrogen Novex).
  • the membranes were incubated in blocking buffer (1 x TBST, 5 % BSA) for lh at RT. After removing the blocking buffer, membranes were incubated for lh at RT in 1 x TBST, 2.5 % BSA supplemented with an appropriate amount of primary antibody.
  • Membranes were washed six times for 10 min in 1 x TBST and incubated for 1 h at room temperature in 1 x TBST, 2.5 % BSA containing secondary antibody (1: 1000). The membranes were washed three times for 10 min with 1 x TBST. For the detection a horse radish peroxidase (HRP) coupled secondary antibody was used and detection was done with Luminol Western blotting detection kit (Roche) by 5 min incubation at RT.
  • HRP horse radish peroxidase

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Abstract

La présente invention concerne un anticorps selon les revendications jointes, l'anticorps se liant spécifiquement au récepteur 1 du domaine discoïdine (DDR1) qui est phosphorylé au niveau de la tyrosine en position 792 de la séquence DDR1 représentée dans SEQ ID NO : 15. La présente invention concerne en outre des molécules d'acide nucléique codant pour la région variable de chaîne légère ou la région variable de chaîne lourde de l'anticorps selon l'invention, ainsi que des vecteurs comprenant lesdites molécules d'acide nucléique. L'invention concerne en outre une cellule hôte ou un hôte non humain comprenant le(les) vecteur(s) de l'invention, ainsi qu'un procédé de production d'un anticorps selon l'invention comprenant la culture de la cellule hôte de l'invention dans des conditions appropriées et l'isolement de l'anticorps produit. En outre, la présente invention concerne un anticorps pouvant être obtenu par le procédé de l'invention, une composition comprenant au moins un anticorps de l'invention, la molécule d'acide nucléique de l'invention, le vecteur de l'invention, la cellule hôte de l'invention ou l'anticorps produit par le procédé de l'invention. La présente invention concerne également l'utilisation de l'anticorps de l'invention pour déterminer la phosphorylation de DDR1 au niveau de la tyrosine en position 792 de la séquence DDR1 représentée dans SEQ ID NO : 15, ainsi qu'un procédé de détermination de la phosphorylation de DDR1 au niveau de la tyrosine en position 792 de la séquence DDR1 représentée dans SEQ ID NO : 15.
PCT/EP2017/078184 2016-11-03 2017-11-03 Nouveaux anticorps anti-py792-ddr1 WO2018083238A1 (fr)

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