WO2014140330A1 - Anti-ddr1 internalizing antibodies and their medical use - Google Patents

Anti-ddr1 internalizing antibodies and their medical use Download PDF

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Publication number
WO2014140330A1
WO2014140330A1 PCT/EP2014/055191 EP2014055191W WO2014140330A1 WO 2014140330 A1 WO2014140330 A1 WO 2014140330A1 EP 2014055191 W EP2014055191 W EP 2014055191W WO 2014140330 A1 WO2014140330 A1 WO 2014140330A1
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antibody
cancer
region
seq
variable
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PCT/EP2014/055191
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French (fr)
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María Cristina PÉREZ
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Oryzon Genomics, S.A.
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'

Definitions

  • the present invention relates to antibodies/binding molecules that specifically bind to discoidin domain receptor 1 (DDR1).
  • DDR1 discoidin domain receptor 1
  • the use of these antibodies/binding molecules in human and veterinary medicine, for example in the treatment and diagnosis of cancer, tumorous diseases and/or proliferative disorders is also subject of the present invention. Further, compositions and kits comprising the antibodies are provided herein. Background art
  • DDR1 Discoidin domain receptor 1
  • RTK Receptor Tyrosine Kinase
  • the DDR1 protein belongs to a subfamily of tyrosine kinase receptors with a homology region to the Dictyostelium discoideum protein discoidin I in the extracellular domain (ECD).
  • DDR1 is activated by various types of collagen and its autophosphorylation is stimulated by all collagens so far tested (type I to type VI).
  • DDR1 is one of two non-integrin tyrosine kinase receptors activated by collagen.
  • the interaction of DDR1 with collagen induces receptor activation and downstream signaling which regulates cellular processes such as cell adhesion, migration, differentiation, cytokine and chemokine production (see e.g., Mihai et al. (2009) J. Mol. Biol.
  • DDR1 DDR1-dependent Xu et al. (2011) Matrix Biology, 30(1), 16-26.
  • the known functions of DDR1 include promotion of cell adhesion and migration on collagen matrices at least partially due to upregulation of matrix metallopro teases (MMPsl , 2 and 9), and modulation of cell proliferation (see Kamohara et al, 2001 FASEB J 15(14): 2724-6; Vogel et al, 2001 Mol. Cell Biol. 21(8):2906-17; Franco et al, 2010 Circ Res 106(11): 1775-83; Dejmek et al, 2003 Int J Cancer 103(3):344-51).
  • MMPsl matrix metallopro teases
  • DDRl is important for axon growth of cerebrellar granule neurons (Bhatt et al., 2000 Genes Dev. 14(17):2216-28) and for normal development of the mammary gland (Vogel et al, 2001 Mol. Cell Biol. 21(8):2906-17).
  • DDRl expression and activation are triggered by apoptotic stimuli, and promote cell survival by a mechanism dependent on activation of MAPK and induction of p53 (Ongusaha et al., 2003 EMBO J. 22(6): 1289-301).
  • DDRl is widely expressed in normal and transformed epithelial cells. In situ studies and Northern-blot analysis showed that expression of DDRl protein is restricted to epithelial cells, particularly in the kidney, lung, gastrointestinal tract, and brain.
  • DDRl is significantly over-expressed in several human tumors including endometrial cancer (WO2011/009637), as well as malignant glioma, breast, colon, ovarian, lung, esophageal, and brain cancers (see e.g., Turashvili et al. (2007) BMC Cancer. 7:55; Yamanaka et al. (2006) Oncogene 25:5994-6002); Yang et al. (2010) 24(2):311-9; Nemoto et al. (1997) Pathobiology 65(4): 195-203; Johansson et al. (2005) Oncogene 24:3896-3905; Heinzelmann- Schwartz et al. (2004) Clin Cane. Res.
  • DDRl is also reported to be overexpressed in injured arteries and has been implicated in additional diseases such as inflammation (Hachehouche et al. (2010) Mol Immunol.;47(9): 1866-9), cirrhotic liver (Song et al. (2011) Am J Pathol. 178(3): 1134-44.), pulmonary fibrosis (C Avivi-Green et al (2006) Am J Respir Crit Care Med, 174(4), 420-27) pituitary adenoma (Yoshida et al. (2007) J. Neuro-Oncol. 82:29-40), congestive heart failure (Andersson et al. (2006) Acta Physiol.
  • Anti-DDRl antibodies have been proposed in the art in the treatment of various types of cancer in WO 2010/019702 and WO2013/034933; these prior art antibodies are not reported to have the capacity to internalize into cells. This capacity is highly advantageous, as it allows the internalization of antibodies that are coupled with a therapeutic agent (like, inter alia, a toxin) into cancer cells, thereby effectively targeting and killing tumor cells.
  • a therapeutic agent like, inter alia, a toxin
  • the technical problem underlying the present invention lies in the provision of means and methods for effective medical intervention in proliferative disorders, in particular in cancer.
  • the invention seeks to solve the problem of providing antibodies against human DDRl that are suitable for use in Antibody-Drug Conjugate (ADC) therapy for the targeted delivery of cytotoxic agents into cancer cells.
  • ADC Antibody-Drug Conjugate
  • Anti-DDRl antibodies for use in ADC should exhibit high affinity and specificity for human DDRl , should be efficiently internalized into DDRl -expressing cells and exhibit in vivo antitumor activity at low doses when administered as an Antibody-Drug Conjugate (ADC).
  • the present invention relates to antibodies or functional fragments or functional derivatives thereof specifically binding to discoidin domain receptor 1 (DDRl).
  • DDRl discoidin domain receptor 1
  • binding molecule in accordance with this invention relates to functional fragments or functional derivatives of the herein disclosed and the herein defined antibodies. Accordingly, the present invention relates to antibodies/binding molecules specifically binding to discoidin domain receptor 1 (DDRl).
  • said antibodies specifically binding to DDRl are capable of internalizing into cells.
  • these antibodies, antibody fragments or antibody derivatives comprise the variable regions and/or CDRs as provided herein.
  • the invention also provides for binding molecules/antibodies that comprise variable regions and/or CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the variable regions and/or CDRs as provided herein and that specifically bind to discoidin domain receptor 1.
  • the inventive antibodies/binding molecules comprise variable regions and/or CDRs as comprised in a (binding) molecule obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ablpBhl under accession number DSM 25529 with the depositary institute DSMZ; or obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ab2pBhl under accession number DSM 25530 with the depositary institute DSMZ; or obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ab3pBhl under accession number DSM 25531 with the depositary institute DSMZ.
  • the invention also provides for binding molecules/antibodies that comprise CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the CDRs as comprised in these molecules encoded by the nucleic acid molecule comprised in said deposited vectors and that specifically bind to discoidin domain receptor 1 (DDRl).
  • the invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the variable regions as comprised in these molecules encoded by the nucleic acid molecule comprised in said deposited vectors and that specifically bind to discoidin domain receptor 1 (DDRl).
  • the present invention also provides for antibodies that bind to/recognize the same epitope as any of the binding molecules obtainable upon expression of the nucleic acid molecule comprised in any of the vectors as described under the designation DDR1- ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively deposited by Oryzon Genomics S.A., Spain on December 20th, 201 1 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
  • DDRl is overexpressed in a variety of cancer cells.
  • the present invention provides new anti-DDRl antibodies/binding molecules that have the unexpected property to internalize into cells and that allow therefore for an effective targeting of DDRl -expressing cells, such as DDRl -expressing epidermoid (also called squamous cell), endometrial, bladder, colon, breast, stomach, lung, pancreas, hepatic, and prostate cancer cells.
  • DDRl -expressing epidermoid also called squamous cell
  • endometrial bladder, colon, breast, stomach, lung, pancreas, hepatic, and prostate cancer cells.
  • This capacity to internalize is highly advantageous, as it allows the internalization of antibodies that are coupled with a therapeutic agent (like, inter alia, a toxin) into cancer cells, thereby effectively targeting and killing tumor cells.
  • a therapeutic agent like, inter alia, a toxin
  • DDRl is known to internalize upon collagen binding/stimulation, i.e. collagen binding is believed to be essential for internalization of DDRl .
  • the anti-DDRl antibodies provided herein are capable of inducing internalization of DDRl in the absence of collagen stimulation (see, e.g., example 7).
  • the entire DDRl -antibody complex (optionally including therapeutic or diagnostic agents coupled to the antibody) is internalized.
  • the antibodies provided herein are capable of specifically targeting DDRl -expressing cells and to effectively introduce therapeutic or diagnostic agents into such cells. Due to said specific targeting and destruction of DDR1- expressing cancer cells, a more effective cancer therapy as compared to conventional therapy is expected.
  • the antibodies/binding molecules of the present invention are expected to have therapeutic utility in diseases where DDRl expression, internalization and/or activation is implicated.
  • diseases where DDRl expression, internalization and/or activation is implicated.
  • One of such disease is cancer and/or tumorous diseases, for example epidermoid (also called squamous cell), endometrial, bladder, colon, breast, stomach, lung, pancreas, hepatic, or prostate cancer.
  • epidermoid also called squamous cell
  • endometrial bladder
  • colon colon
  • breast stomach
  • lung pancreas
  • hepatic hepatic
  • prostate cancer for example epidermoid (also called squamous cell), endometrial, bladder, colon, breast, stomach, lung, pancreas, hepatic, or prostate cancer.
  • the antibodies/binding molecules of the present invention may also be useful in certain further proliferative disorders such as inflammatory disorders or atherosclerosis.
  • the anti-DDRl antibodies as provided herein may also be useful in the medical intervention of other disorders where DDR1 has been reported to play a role, like cirrhotic liver, pulmonary fibrosis, pituitary adenoma, congestive heart failure, Alport syndrome, obstructive nephropathy and lymphangioleiomyomatosis.
  • the present invention relates to the following items:
  • DDR1 discoidin domain receptor 1
  • the variable region of the heavy chain of said antibody comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the heavy chain of said antibody comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24, or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24.
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the light chain of said antibody comprises a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO: 18, or a CDR sequence having 75% or more amino acid identity to said CDR.
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18, or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18.
  • DDR1 discoidin domain receptor 1
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • the antibody of item 1 1 is a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18; and
  • variable region of the heavy chain of said antibody comprises a CDR-Hl region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24.
  • DDRl discoidin domain receptor 1
  • said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43, or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable V H -region; or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region.
  • said antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 , or a variable V L -region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region or a variable V L -region having an amino acid sequence as shown in SEQ ID NO: 42, or a variable V L -region having an amino acid sequence which has 75% or more identity to said variable V L -region.
  • said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.
  • said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44; and wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or
  • a vector comprising a nucleic acid molecule according to item 27 or 28.
  • the vector of item 29, which further comprises a nucleic acid molecule having a regulatory sequence which is operably linked to said nucleic acid molecule according to item 27 or
  • the host of item 33 wherein said host is a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.
  • a composition comprising the antibody as defined in any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 .
  • composition of item 36 further comprising a secondary antibody that is specifically binding to the primary antibody as defined in any one of items 1 to 26, whereby said secondary antibody is conjugated to a therapeutic agent.
  • the composition of item 37, wherein said therapeutic agent is a toxin.
  • the composition of item 37 or 38, wherein said primary antibody is an IgG antibody and said secondary antibody is a goat anti-human IgG secondary antibody.
  • the antibody of item 25 or the composition of item 38 or 39, wherein said toxin is Saporin.
  • composition of any one of items 36 to 40 which is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s).
  • a method for the treatment of cancer comprising the administration of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 to a subject in need of such a treatment.
  • 46. The method of item 45, wherein said subject is a human.
  • An antibody obtainable by a process comprising culturing a host transfected or transformed with the vector as defined in item 28 under conditions that provide for the production of the antibody by the host and allow for the recovering of the antibody from the culture.
  • composition of item 36 which is a diagnostic composition further comprising, optionally, means and methods for detection.
  • a method for diagnosing cancer comprising detecting or assaying DDR1 in a biological sample of an individual suspected of suffering from cancer using the antibody according to any one of items 1 to 26.
  • Kit comprising the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41 and 50.
  • DDR1 discoidin domain receptor 1
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
  • the present invention relates to the following items:
  • DDR1 discoidin domain receptor 1
  • DDR1 discoidin domain receptor 1
  • the variable region of the heavy chain of said antibody comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 , SEQ ID NO.: 24 or SEQ ID NO.: 36, or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , SEQ ID NO: 20 , or SEQ ID NO: 32 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 , SEQ ID NO: 22 , or SEQ ID NO: 34 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 , SEQ ID NO: 14 , or SEQ ID NO: 26 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , SEQ ID NO: 16 , or SEQ ID NO: 28 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO: 6 , SEQ ID NO: 18 , or SEQ ID NO: 30 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises
  • DDR1 discoidin domain receptor 1
  • variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 2 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • DDR1 discoidin domain receptor 1
  • variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
  • DDR1 discoidin domain receptor 1
  • variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 26 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. 14.
  • DDRl discoidin domain receptor 1
  • said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 , SEQ ID NO:43 , or SEQ ID NO:47 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable Vn-regions; or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 , SEQ ID NO:40 , SEQ ID NO:40
  • variable Vn-region having an amino acid sequence which has 75% or more identity to one of said variable Vn-regions.
  • said antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 , SEQ ID NO:41 , or SEQ ID NO:45 , or a variable V L -region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable V L -regions or
  • variable V L -region having an amino acid sequence as shown in SEQ ID NO:38 , SEQ ID NO:42 , or SEQ ID NO:46 or a variable V L -region having an amino acid sequence which has 75% or more identity to one of said variable V L -regions.
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region;
  • said antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 or a variable V L -region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region or a variable V L -region having an amino acid sequence as shown in SEQ ID NO:38 or a variable V L -region having an amino acid sequence which has 75% or more identity to said variable V L -region.
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region;
  • said antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable V L -region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region or a variable V L -region having an amino acid sequence as shown in SEQ ID NO:42 or a variable V L -region having an amino acid sequence which has 75% or more identity to said variable V L -region.
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:48 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region;
  • said antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region or a variable V L -region having an amino acid sequence as shown in SEQ ID NO:46 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable V L -region.
  • DDRl discoidin domain receptor 1
  • said antibody is DDRl-abl as encoded by a nucleic acid molecule comprised in vector DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 201 1; or wherein said antibody is DDRl-ab2 as encoded by a nucleic acid molecule comprised in vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1; or
  • said antibody is DDRl-ab3 as encoded by a nucleic acid molecule comprised in vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 201 1.
  • said antibody wherein said antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody.
  • the antibody of item 24, wherein the therapeutic agent is a toxin.
  • the antibody of item 24, wherein the therapeutic agent is an anticancer agent.
  • the vector of item 29 or 30, wherein the vector is an expression vector.
  • said vector is DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 2011 ; or
  • said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 ; or
  • vector is DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011. 33.
  • the host of item 33 wherein said host is a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.
  • 35 A process for the production of the antibody as defined in any one of items 1 to 26, said process comprising culturing a host of item or 34 under conditions allowing the expression of the antibody and recovering the produced antibody from the culture.
  • composition comprising the antibody as defined in any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34.
  • composition of item 36 further comprising a secondary antibody that is specifically binding to the primary antibody as defined in any one of items 1 to 26, whereby said secondary antibody is conjugated to a therapeutic agent.
  • composition of item 37 or 38 wherein said primary antibody is an IgG antibody and said secondary antibody is a goat anti-human IgG secondary antibody.
  • the composition of any one of items 36 to 40 which is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s).
  • any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 for the preparation of a pharmaceutical composition for the treatment of cancer.
  • a method for the treatment of cancer comprising the administration of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 to a subject in need of such a treatment.
  • An antibody obtainable by a process comprising culturing a host transfected or transformed with any one of the vectors as defined in item 32 under conditions that provide for the production of the antibody by the host and allow for the recovering of the antibody from the culture.
  • composition of item 36 which is a diagnostic composition further comprising, optionally, means and methods for detection.
  • a method for diagnosing cancer comprising detecting or assaying DDR1 in a biological sample of an individual suspected of suffering from cancer using the antibody according to any one of items 1 to 26.
  • Kit comprising the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41.
  • DDRl discoidin domain receptor 1
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 67 and a 3 '-primer as shown in SEQ ID NO: 68 for amplification of a first binding domain; and/ or
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 69 and a 3 '-primer as shown in SEQ ID NO: 70 for amplification of a second binding domain.
  • DDRl discoidin domain receptor 1
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
  • An antibody that specifically binds to discoidin domain receptor 1 (DDRl), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011 , using DDRl discoidin domain receptor 1
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 75 and a 3 '-primer as shown in SEQ ID NO: 76 for amplification of a first binding domain; and/ or
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 77 and a 3 '-primer as shown in SEQ ID NO: 78 for amplification of a second binding domain.
  • anti-DDRl antibody As used herein, the term “anti-DDRl antibody”, “antiDDRl antibody”, “antibody to DDR1”, “DDR1 antibody” and “DDR1 binding molecule” are used interchangeably to refer to an antibody or a functional fragment or a functional derivative thereof specifically binding to DDR1 according to the invention.
  • the “anti-DDRl antibodies or functional fragments or functional derivatives thereof bind specifically to the extracellular domain of DDR1.
  • antibodies/binding molecules/antibody fragments and/or antibody derivatives that are functional in this binding capacity, whereby said molecules bind to the same epitope(s) as any of the binding molecules obtainable by the expression of the nucleic acid molecule comprised in a vector as deposited under the designation DDR-ablpBhl, DDR1- ab2pBhl and DDRl-ab3pBhl, respectively by Oryzon Genomics S.A., Spain on December 20 th , 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • antibody is not limited to immunoglobulins, but also comprises, inter alia, antibody fragments, artificial antibodies, antibody derivatives, single chain antibodies (also bispeciflc single chain antibodies), diabodies, triabodies, etc.
  • antibody also relates to binding molecules that comprise CDRs or binding portions of the antibodies described herein.
  • antibodies of this invention are, inter alia, capable of internalizing into cells.
  • the term "antibody is capable of internalizing into cells” means, in context of this invention, that the antibody has the capacity to induce internalization of the DDR1 protein.
  • Antibodies, antibody fragments, antibody derivatives and/or corresponding binding molecules of this invention preferably have the functional feature being capable of "internalization into cells” as defined herein.
  • the antibodies provided herein are "capable of internalizing upon binding to DDR1". This term means that the antibodies first bind to DDR1 and induce in a second step internalization.
  • the antibodies activate the internalizing capacity of DDRl .
  • the present invention provides antibodies/antibody fragments/antibody derivatives specifically binding to DDRl , whereby the antibodies are capable of being internalized.
  • "Internalization” as used herein may refer to (i) the internalization of only DDRl (for example, if the DDRl -antibody-binding is dissolved after induction of internalization; this is thought to occur only in rare cases) or (ii) the internalization of the antibody-DDRl complex (for example, if the anti-DDRl -antibody remains bound to DDRl after induction of internalization).
  • the antibodies of the present invention comprise antibodies that are capable of inducing the internalization of the antibody- DDRl complex , i.e. the antibodies have the capacity to penetrate inside a DDRl -expressing cell upon binding to DDRl . Therefore, the present invention relates to an antibody specifically binding to DDRl, whereby the antibody-DDRl complex is internalized.
  • Such an antibody- DDRl complex may include further compounds or agents, such as therapeutic or diagnostic agents coupled to the antibody.
  • the antibody of the invention is a DDRl internalizing antibody.
  • a DDRl internalizing antibody can bind cell surface DDRl and be internalized into the cell.
  • the antibodies of the present invention are advantageous as they do not necessitate the use of a ligand (like collagen) for the stimulation or induction of internalization. Nonetheless, internalization may be enhanced by the use of such ligands. Accordingly, the herein provided antibodies may internalize by or upon ligand stimulation (such as collagen) or the internalization may additionally be stimulated by a ligand (such as collagen).
  • ligand refers in particular to compounds that differ from the antibodies of the present invention and are capable of inducing internalization as defined above. Preferably, such ligands induce or enhance internalization by binding or upon binding to DDRl .
  • Exemplary ligands known in the art are type I, type II, type III, type IV, type V and/or type VI collagen.
  • the ability of an antibody to internalize can be determined by a variety of standard assays. For example, upon a signal that induces protein internalization, comparison between the protein level detected on the cell surface of unstimulated cells versus stimulated cells can be done in fixed cells or in vivo cells. The localization of the protein can be detected with a primary antibody which specifically binds the protein of interest and a secondary antibody coupled to a chromophore that recognises the primary antibody. The chromophore can then be detected for example by fluorescent microscopy or flow cytometry techniques.
  • the ability of an antibody to internalize can also be determined for example using the assays disclosed in Examples 6, 7 and 8 below.
  • the herein provided antibodies specifically bind to discoidin domain receptor 1 (DDRl), preferably to DDRl of a mammal, most preferably to DDRl of a human.
  • DDRl discoidin domain receptor 1
  • several discoidin domain receptor 1 (DDRl) isoforms are known.
  • DDRl discoidin domain receptor 1
  • the antibodies of this invention bind to said extracellular domain of the DDRl receptor/polypeptide/protein as defined herein.
  • non-DDRl protein is to be understood as a protein that does not present any domain that is included in the DDRl extracellular region (for example the discoidin domain). Specificity can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • antibody specifically binding to DDRl refers to an antibody or a functional fragment thereof that specifically binds to a DDRl polypeptide (or a fragment or epitope of a DDRl polypeptide) and that does not specifically bind to other non-DDRl polypeptides.
  • antibodies (or functional fragments thereof) binding specifically to a DDRl polypeptide or fragment thereof do not non- specifically cross-react with other antigens (e.g., binding cannot be competed away with a non- DDRl polypeptide/protein, e.g., BSA in an appropriate immunoassay).
  • Antibodies or functional fragments that specifically (or immunospecifically) bind to a DDRl polypeptide/protein can be identified, for example, by immunoassays or other techniques known to those of skill in the art.
  • DDRl refers preferably to a DDRl protein/polypeptide or a fragment or an epitope of a DDRl polypeptide.
  • DDRl protein or “DDRl polypeptide” (or short “DDRl” which is used interchangeably herein) can refer to and include polymorphic variants, alleles, mutants, and interspecies homologs that (i) are encoded by a nucleotide sequence having substantial nucleotide sequence identity (for example, at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, still more preferably at least 90% identity and even more preferably at least 95% identity) with the nucleotide sequence indicated in the respective database for the indicated ID number (an exemplary sequence can be retrieved from NCBI under accession number NM 001954.4 and is shown in SEQ ID NO: 49); or (ii) have substantial amino acid sequence identity (for example, at least 60% identity, preferably at least 70% identity, more preferably at least
  • DDR1 refers to a mammal DDR1, most preferred to a human DDR1 polypeptide/protein or a fragment or an epitope of a human DDR1 polypeptide.
  • the antibodies/binding molecules to be employed in context of this invention and as disclosed herein bind to the extracellular domain (or an epitope or a fragment thereof) of DDR1.
  • these terms refer to the entire gene sequence, mRNA sequence, and/or protein sequence as well as fragments of these sequences. In a more specific definition, these terms refer to the minimal amount of nucleic acid or amino acid sequence that can be used to identify such sequences in a specific manner.
  • the skilled artisan recognizes that the DDR1 genes can have numerous splice forms and variants. When referring to a specific DDR1 gene or locus by a reference number (e.g., NCBI accession number), all splices forms and variants are included in the various embodiments of the invention.
  • the gene can also comprise a regulatory element.
  • the exemplary sequences provided herein may only be representative of one particular individual in a population (in particular a human population). Individuals (in particular humans) vary from one to another in their gene sequences. These variations are very minimal, sometimes occurring at a frequency of about 1 to 10 nucleotides per gene. Nonetheless, different forms of any particular DDR1 gene exist within a population, such as the human population. These different forms are called allelic variants. Allelic variants often do not change the amino acid sequence of the encoded protein; such variants are termed synonymous. Even if they do change the encoded amino acid (non-synonymous), the function of the protein is typically not affected. Such changes are evolutionarily or functionally neutral.
  • allelic variants are intended to be encompassed by the term.
  • the gene ID sequences given for a DDR1 gene are provided merely as representative examples of a wild-type sequence, in particular a wild-type human sequence.
  • the invention is therefore not limited to a single allelic form of the amplified genes or regions (and proteins they encode).
  • the antibody, antibody fragment thereof or antibody derivatives of this invention bind selectively or specifically to a DDR1 epitope.
  • the peptide scan (pepspot assay) is routinely employed to map linear epitopes in a polypeptide antigen.
  • the primary sequence of the polypeptide is synthesized successively on activated cellulose with peptides overlapping one another.
  • the recognition of certain peptides by the antibody to be tested for its ability to detect or recognize a specific antigen/epitope is scored by routine colour development (secondary antibody with horseradish peroxidase and 4-chloronaphthol and hydrogenperoxide), by a chemoluminescence reaction or similar means known in the art. In the case of, inter alia, chemoluminescence reactions, the reaction can be quantified. If the antibody reacts with a certain set of overlapping peptides one can deduce the minimum sequence of amino acids that are necessary for reaction.
  • the same assay can reveal two distant clusters of reactive peptides, which indicate the recognition of a discontinuous, i. e. conformational epitope in the antigenic polypeptide (Geysen (1986), Mol. Immunol. 23, 709-715).
  • standard ELISA assay can be carried out. Small hexapeptides may be coupled to a protein and coated to an immunoplate and reacted with antibodies to be tested. The scoring may be carried out by standard colour development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogenperoxide).
  • the antibody of the present invention is directed against and binds specifically to a DDR1 polypeptide/protein/receptor, a fragment thereof or an epitope of a DDR1 polypeptide/protein/receptor, preferably to the extracellular region of said DDR1 polypeptide/protein/receptor (or an extracellular DDR1 epitope or a extracellular DDR1 fragment)
  • the DDR1 receptor is known to exist in vivo in dimeric and monomeric form, see, inter alia, Noordeen et al. J Biol Chem (2006) 281(32):22744-51; Mihai et al. (2009) J Mol Biol. 385(2):432-45; Leitinger et al. (2003) J. Biol. Chem.
  • the antibody of this invention binds to or can be generated against a polypeptide having the full length sequence of a DDR1 protein.
  • the antibody only recognizes or binds the DDR1 protein when the protein is present in the plasma membrane as a dimer.
  • the antibody only recognises or binds the DDR1 protein when it is present in the plasma membrane as a monomer.
  • accession numbers for the nucleic acid and amino acid sequences for the various known human isoforms of DDRl are listed below (see example 1) :
  • the binding site or epitope for the herein provided antibodies is located on an extracellular domain of the DDRl protein.
  • the amino acid sequence of an exemplary extracellular domain is shown in SEQ ID NO:63 (extracellular domain of human DDRl protein, which is shared by all human isoforms of DDRl known to date).
  • the antibodies provided herein specifically bind to discoidin domain receptor 1 (DDRl) and are capable of internalizing into cells.
  • DDRl discoidin domain receptor 1
  • the antibodies provided and to be used in accordance with the present invention may comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of the specific CDR sequences provided and disclosed herein. It is understood that the identity is assessed/determined over the full length of the CDR sequence.
  • variable region of the heavy chain of the antibody of this invention comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 [CDR-H3 of DDRl -abl ], preferably SEQ ID NO.: 24 [CDR- H3 of DDRl -ab2], or SEQ ID NO.: 36 [CDR-H3 of DDRl -ab3] .
  • the antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • CDR as employed herein relates to "complementary determining region", which is well known in the art.
  • the CDRs are parts of immunoglobulins and T cell receptors that determine the specificity of said molecules and make contact with specific ligand.
  • the CDRs are the most variable part of the molecule and contribute to the diversity of these molecules.
  • CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. H means the variable heavy chain and L means the variable light chain.
  • the CDR regions of an Ig- derived region may be determined as described in Kabat (1991), Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia (1987), J. Mol. Biol. 196, 901-917; and Chothia (1989) Nature, 342, 877-883.
  • Each CDR region of a variable heavy chain is herein interchangeably designated as CDR-H 1 or VH-CDRl , CDR-H2 or VH-CDR2, and CDR-H3 or VH-CDR3, respectively.
  • each CDR region of a variable light chain is designated herein CDR-L1 or VL-CDR1 , CDR-L2 or VL-CDR2, and CDR-L3 or VL-CDR3, respectively.
  • variable region of the heavy chain of the antibody of this invention comprises a CDR-H 1 region having an amino acid sequence as depicted in SEQ ID NO: 8 [CDR-H1 of DDRl-abl], preferably SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], or SEQ ID NO: 32 [CDR-H1 of DDRl-ab3].
  • the antibodies may also comprise a CDR-H1 sequence having 75% or more (e.g. more preferably 80 %, 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the heavy chain of the antibody of this invention comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 [CDR-H2 of DDRl-abl], preferably SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], or SEQ ID NO: 34 [CDR-H2 of DDRl-ab3].
  • the antibodies may also comprise a CDR-H2 sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises
  • (b) preferably a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2]; or
  • the antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2].
  • the antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 [CDR-L1 of DDRl-abl], preferably SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], or SEQ ID NO: 26 [CDR-L1 of DDR1- ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 [CDR-L2 of DDRl-abl], preferably SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], or SEQ ID NO: 28 [CDR-L2 of DDR1- ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of said antibody comprises a CDR- L3 region having an amino acid sequence as depicted in SEQ ID NO: 6 [CDR-L3 of DDRl- abl], preferably SEQ ID NO: 18 [CDR-L3 of DDRl-ab2], or SEQ ID NO: 30 [CDR-L3 of DDRl-ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of the antibody of this invention comprises
  • (b) preferably a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2]; or
  • the antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of the antibody of this invention comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2].
  • the antibodies may also comprise a CDR sequence having 75% or more (e.g.
  • the antibodies/binding molecules etc. of the present invention may be characterized by at least one CDR sequence as described above.
  • the antibody comprises 2 or more CDRs. More preferably, the antibody comprises 3, 4, 5 or more CDRs. Still even more preferably, said antibody comprises 6 CDRs. Yet, even more preferably the antibody comprises a set of 6 CDRs: 3 CDRs in the variable region of the light chain of the antibody and 3 CDRs in the variable region of the heavy chain of the antibody.
  • variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 [CDR-L1 of DDRl-abl], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 [CDR-L2 of DDRl-abl], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 [CDR-L3 of DDRl-abl] and the variable region of the heavy chain of the antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 [CDR-H1 of DDRl-abl], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 [CDR-H2 of DDRl-abl], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 [CDR-H3 of
  • the invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 [CDR-L1 of DDRl-ab3], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 [CDR-L2 of DDRl-ab3], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 [CDR-L3 of DDRl-ab3] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 [CDR-H1 of DDRl-ab3], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 [CDR-H2 of DDRl-ab3], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36
  • the invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.:
  • the invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
  • the antibodies of the present invention may also comprise entire variable regions of the light chain of the antibody and/or entire variable regions of the heavy chain of the antibody as disclosed herein. Accordingly, the antibody comprises in one embodiment a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 [Vn-region of DDRl-abl], preferably SEQ ID NO:43 [V H -region of DDRl-ab2], or SEQ ID NO:47 [V H -region of DDRl-ab3], or it comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 40 [Vn-region of DDRl-abl], preferably SEQ ID NO:44 [V H -region of DDRl-ab2], or SEQ ID NO:48 [Vn-region of DDRl -ab3].
  • the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 [VL-region of DDRl-abl], preferably SEQ ID NO:41 [VL-region of DDRl-ab2], or SEQ ID NO:45 [VL-region of DDRl-ab3], or a variable VL- region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl], preferably SEQ ID NO:42 [VL-region of DDRl-ab2], or SEQ ID NO:46 [VL-region of DDRl - ab3].
  • the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 [VH-region of DDRl-abl], or a variable VH- region having an amino acid sequence as shown in SEQ ID NO:40 [VH-region of DDRl-abl]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 [VL-region of DDRl-abl] or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl].
  • the antibody comprises a variable VH-region having an amino acid sequence as shown in SEQ ID NO:40 [VH-region of DDRl-abl] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl].
  • the antibody comprises a variable VH- region as encoded by a nucleic acid molecule as shown in SEQ ID NO:47 [VH-region of DDR1- ab3], or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:48 [VH- region of DDRl-ab3]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45 [VL-region of DDRl-ab3], or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:46 [VL-region of DDRl-ab3].
  • the antibody comprises a variable VH-region having an amino acid sequence as shown in SEQ ID NO:48 [VH-region of DDRl-ab3] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:46 [VL-region of DDRl-ab3].
  • the antibody comprises a variable V R - region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 [Vn-region of DDR1- ab2] or a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44 [Vn- region of DDRl-ab2]; and the antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 [V L -region of DDRl-ab2] or a variable V L -region having an amino acid sequence as shown in SEQ ID NO:42 [V L -region of DDRl-ab2].
  • the antibody comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 [Vn-region of DDRl-ab2] and a variable V L -region having an amino acid sequence as shown in SEQ ID NO:42 [V L -region of DDRl-ab2].
  • the herein provided antibodies can comprise one or more of the heavy or light chain variable sequences above or a sequence at least 75%, 80%, more preferably at least 85 %, 90 %, even more preferably at least 95 %, 96 %, 97 %, 98 %, or most preferably 99 % identical thereto.
  • the variation in the sequences occurs in the framework regions, i.e. outside of the CDR sequences.
  • the antibody comprises:
  • variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn- region;
  • variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or
  • variable VL-region having an amino acid sequence as shown in SEQ ID NO: 38 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL- region;
  • said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6;
  • said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12.
  • the antibody comprises:
  • variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:47 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:48 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable VH- region;
  • variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45, or a variable V H -region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region, or a variable V L -region having an amino acid sequence as shown in SEQ ID NO: 46 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable V L - region;
  • said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30; and/or
  • said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 .
  • the antibody comprises
  • variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
  • variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn- region;
  • variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable V L -region as encoded by a nucleic acid molecule having 75% or more identity to said variable V L -region, or
  • variable V L -region having an amino acid sequence as shown in SEQ ID NO: 42 or a variable V L -region having an amino acid sequence which has 75% or more identity to said variable V L - region;
  • said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18; and/or
  • said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24.
  • the variation in the sequences occurs in the framework regions, i.e. outside of the CDR sequences.
  • the antibodies of these embodiments contain specific CDR regions as defined above that are not subject to variation.
  • the framework region of these antibodies can show a variation/identity of 75 % or more (or 80%, more preferably at least 85 %, 90 %, even more preferably at least 95 %, 96 %, 97 %, 98 %, or most preferably 99 %) to the framework region of the specific variable VL-region(s) and variable Vn-region(s) as defined above.
  • the framework region(s) can be identified by methods known in the art.
  • the term "framework region” refers to the sequence of the variable VL-region(s) and/or the variable VR- region(s) that is outside of the CDR sequences.
  • a particularly preferred antibody according to the invention is the antibody designated herein as DDRl-ab2.
  • This antibody has been found to exhibit high affinity and specificity for human DDR1 , is able to internalize into cells quickly and kill cancer cells when conjugated to a drug such as a toxin and has shown antitumor activity in in vivo xenograft models of epidermoid (squamous cell) cancer and endometrial cancer when conjugated to a toxin when administered at doses below 500 ⁇ g/kg of ADC.
  • DDRl-ab2 has been shown to be devoid of toxicity in a battery of in vitro and in vivo toxicity studies, as described in more detail in the appended Examples.
  • Antibody DDRl-ab2 is thus an excellent candidate for drug development, in particular for use in antibody-drug conjugate (ADC) therapy for the treatment of cancer.
  • ADC antibody-drug conjugate
  • the rate/speed of internalization of an antibody can be determined by time course experiments in which the disappearance of the antibody from the cell surface or the intracellular appearance of the antibody is analyzed as a function of time. Suitable methods to determine the rate of internalization include in particular in vitro methods, inter alia, FACS analysis, real time quantitative confocal fluorescence analysis of fluorescently labeled anti-DDRl antibody, time series assessment of internalized antibody detected directly or with a secondary antibody, or time series assessment of the incorporation of radioactively labeled anti-DDRl antibody inside the cell, among others. Examples of cells suitable for testing anti-DDRl antibodies are, for example, Clone 44 cells (see Example 4), which over-express DDR1 , or other cells capable of expressing and internalizing DDR1.
  • the invention provides an antibody that specifically binds to DDR1 , wherein the variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H
  • the invention also provides for binding molecules/antibodies that comprise CDR sequences that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to the CDRs as provided herein and that specifically bind to discoidin domain receptor 1.
  • CDR sequences that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to the CDRs as provided herein and that specifically bind to discoidin domain receptor 1.
  • the invention provides an antibody that specifically binds to DDR1, wherein the antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 [Vn-region of DDRl-ab2] or a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 [Vn-region of DDRl-ab2]; and the antibody comprises a variable V L -region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 [V L -region of DDRl-ab2] or a variable V L -region having an amino acid sequence as shown in SEQ ID NO:42 [V L -region of DDRl-ab2].
  • the antibody comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44 [Vn-region of DDRl-ab2] and a variable V L -region having an amino acid sequence as shown in SEQ ID NO:42 [V L -region of DDRl-ab2].
  • the invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to the variable regions as provided herein and that specifically bind to discoidin domain receptor 1.
  • the invention provides antibodies/binding molecules that specifically bind to DDR1, wherein said antibodies/binding molecules comprise variable regions and/or CDRs as comprised in a (binding) molecule obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDR1- ab2pBhl under accession number DSM 25530 with the depositary institute DSMZ by Oryzon Genomics S.A., with address at Sant Ferran 74, 08940 Cornelia de Llobregat, Spain.
  • a vector nucleic acid molecule
  • the invention also provides for binding molecules/antibodies that comprise CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the CDRs as comprised in the molecule encoded by the nucleic acid molecule comprised in said deposited vector and that specifically bind to discoidin domain receptor 1 (DDR1).
  • the invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical (e.g.
  • variable regions as comprised in the molecule encoded by the nucleic acid molecule comprised in said deposited vector and that specifically bind to discoidin domain receptor 1 (DDR1).
  • DDR1 discoidin domain receptor 1
  • the invention provides antibodies/binding molecules that specifically bind to DDR1 , wherein said antibodies/binding molecules bind to or recognize the same epitope as any of the binding molecules obtainable upon expression of the nucleic acid molecule comprised in the vector as described under the designation DDRl-ab2pBhl deposited by Oryzon Genomics S.A., Spain on December 20 th , 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
  • DDRl-ab2pBhl deposited by Oryzon Genomics S.A., Spain on December 20 th , 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
  • the antibodies/binding molecules of the invention include the antibodies having the CDRs and/or variable sequences as disclosed herein as well as variants thereof having 75% or more (for example 80%, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, or 99 %) sequence identity.
  • sequence identity As used herein, the terms “identity”, “sequence identity”, “homology” or “sequence homology” (the terms are used interchangeably herein) are used to describe the sequence relationships between two or more nucleic acids, polynucleotides, proteins, or polypeptides, and is understood in the context of and in conjunction with the terms including: (a) reference sequence, (b) comparison window, (c) sequence identity, (d) percentage of sequence identity, and (e) substantial identity or “homologous”.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence.
  • a “comparison window” includes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the polynucleotide or polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences.
  • the comparison window may be at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
  • a gap penalty is typically introduced and is subtracted from the number of matches.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
  • the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
  • sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters. Altschul et al. (1997) Nucleic Acids Res, 2:3389-3402. It is to be understood that default settings of these parameters can be readily changed as needed in the future.
  • BLAST searches assume that proteins or nucleic acids can be modeled as random sequences. However, many real proteins and nucleic acids comprise regions of nonrandom sequences which may be homopolymeric tracts, short- period repeats, or regions enriched in one or more amino acids or nucleic acids. Such low- complexity regions may be aligned between unrelated proteins even though other regions of the protein or nucleic acid are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten et al. (1993) Comput. Chem. 17: 149-163) and XNU (Claverie et al. (1993) Comput. Chem. 17: 191-1) low-complexity filters can be employed alone or in combination.
  • Sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions.
  • percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity.
  • Percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or nucleic acid sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical or “homologous” in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 75% sequence identity, preferably at least 80%, more preferably at least 85 %, still more preferably at least 90% and even more preferably at least 95%, 96 %, 97 %, 98 % or 99 %, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • a desired identity for example, at least 75% sequence identity, preferably at least 80%, more preferably at least 85 %, still more preferably at least 90% and even more preferably at least 95%, 96 %, 97 %, 98 % or 99 %, compared to a reference sequence using one of the alignment programs described using standard parameters.
  • a desired identity for example, at least 75% sequence identity, preferably at least 80%, more preferably at least 85 %, still more preferably at least
  • the present invention provides for binding molecules/antibodies etc specifically binding to DDR1 which comprise CDRs and/or variable regions that have at least 75% sequence identity, more preferably at least 80%, even more preferably at least 85 %, still more preferably at least 90% and most preferably at least 95%, 96 %, 97 %, 98 % or 99 % sequence identity with either the encoding nucleic acid molecule or the expressed amino acid molecule of an antibody molecule (or variable regions or CDRs thereof) as obtainable from the vector deposits deposited under the designation DDRl-ablpBhl, preferably DDRl-ab2pBhl, or DDRl-ab3pBhl on December 20, 201 1 with the DSMZ Braunschweig/ Germany.
  • DDRl-ablpBhl preferably DDRl-ab2pBhl, or DDRl-ab3pBhl on December 20, 201 1 with the DSMZ Braunschweig/ Germany.
  • nucleotide sequences are substantially identical if two molecules hybridize to each other under stringent conditions.
  • stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x SSC, 0.1% SDS at 65°C or 2 x SSC, 60°C, 0.1 % SDS.
  • Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 55°C or 60°C.
  • nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.
  • substantially identical or “homologous” in their various grammatical forms in the context of peptides indicates that a peptide comprises a sequence that has a desired identity, for example, at least 75% sequence identity to a reference sequence, preferably at least 80% sequence identity to a reference sequence, more preferably 85%, even more preferably at least 90% or 95% or even 96 %, 97 %, 98 % or 99 % sequence identity to the reference sequence over a specified comparison window.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol., 48:443.
  • peptide sequences are substantially identical.
  • a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
  • Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • Arginine Arg (R) Polar, hydrophilic, charge (+) Lys, His, Gin, Asn Lys
  • Glycine Gly (G) Aliphatic, neutral Pro, Ala Ala
  • Leucine Leu (L) Aliphatic, hydrophobic, He, Val, Met, Phe, He neutral Ala
  • Lysine Lys (K) polar, hydrophilic, charge (+) Arg, Gin, Asn, His Arg
  • Methionine Met hydrophobic, neutral Leu, He, Phe Leu
  • Phenylalanine Phe (F) Aromatic, hydrophobic, Leu, He, Val, Ala, Leu neutral Tyr
  • Serine Ser (S) Polar, hydrophilic, neutral Thr, Ala, Cys Thr
  • Threonine Thr (T) Polar, hydrophilic, neutral Ser Ser
  • Tyrosine Tyr (Y) Aromatic, polar, hydrophobic Trp, Phe, Thr, Ser Phe
  • Valine Val Aliphatic, hydrophobic, He, Met, Leu, Phe, Leu neutral Ala,
  • Glutamic Acid Glu (E) Polar, hydrophilic, charge (-) Asp, Gin Asp
  • the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences and/or variable sequences as disclosed herein having 1 or more amino acid substitutions, deletions or additions.
  • the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences as disclosed herein having 1 or more, preferably 1 , 2 or 3, more preferably 1 or 2 amino acid substitutions, deletions or additions.
  • the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise variable sequences as disclosed herein having up to 20, preferably up to 15, more preferably up to 10, amino acid substitutions, deletions or additions.
  • the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences as disclosed herein having 1 or more, preferably 1 , 2 or 3, more preferably 1 or 2, amino acid substitutions, preferably conservative amino acid substitutions.
  • the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise variable sequences as disclosed herein having up to 20, preferably up to 15, more preferably up to 10, amino acid substitutions, preferably conservative amino acid substitutions.
  • the present invention provides antibodies comprising CDRs and/or variable sequences as described herein, or variants thereof, as disclosed above. Methods are known to those skilled in the art to modify the sequence of an existing antibody (parent antibody) to derive variant antibodies with high sequence homology to the sequence of the existing antibody that retain the capacity to bind the original target epitope.
  • Variant DDRl binding antibodies (with similar or improved affinity, with modified selectivity, antigenicity, with modified pharmacokinetic characteristics) can be readily derived from the DDRl antibodies disclosed herein through variation of the sequence of the claimed antibodies, using methods that have been described in the literature.
  • Mutations can be introduced randomly into the variable regions of antibody genes by error-prone polymerase chain reaction (PCR) or E. coli mutator strains, site-directed mutagenesis, saturation mutagenesis, parsimonious mutagenesis, CDR walking or look-through mutagenesis targeting certain regions like the CDRs, hence generating limited collections of the specific variants of the parent antibody.
  • Shuffling approaches include DNA shuffling, chain shuffling, or CDR shuffling to obtain shuffled variants of the parent antibody.
  • Random mutation efforts can be combined with in vitro selection procedures (i.e. display methods) to identify binders.
  • Directed mutagenesis is preferentially performed after in silico modeling of the DDR1 protein - DDR1 antibody using the sequence and structure information of the (extracellular part of) the DDR1 protein and the DDR1 antibody.
  • Modeling can be done using the experimentally determined 3D crystal structure of the complex formed between the (extracellular domain of) DDR1 protein with the DDR1 antibodies of the invention as a starting point. Alternatively, modeling can also be done by using an in silico docking model of the (extracellular domain of) DDR1 protein and the antibodies disclosed herein based on published 3D structures of the individual protein. The extracellular part of DDR1 has been crystallized with an antibody and its 3D structure has been deposited (EBI PDBsum Database Entry 4ag4).
  • the 3D structure of the DDR1 antibody can be predicted with one of different algorithms available in the art that are rapidly increasing in accuracy like: Web Antibody Modeling (WAM) (Whitelegg and Rees, Protein Eng. 2000; 14(12):819-824), Prediction of ImmunGlobulin Structure (PIGS) (Marcatili et al, Bioinformatics. 2008;14(17): 1953-1954), or RosettaAntibody (Sivasubramanian et al, Proteins. 2009; 14(2):497-514. ),).
  • WAM Web Antibody Modeling
  • PIGS ImmunGlobulin Structure
  • RosettaAntibody RosettaAntibody
  • the algorithms cited above can be used to dock the antibodies to the (extracellular domain of the) target protein; and to analyze sequence tolerance to variation with respect to the antibody- target protein binding capacity, i.e. the algorithms can be used by a skilled user to design variant antibodies binding the same epitope (see e.g. Barderas et al. Proc Natl Acad Sci U S A. Jul 1, 2008; 105(26): 9029-9034) and this principle can be applied to the DDR-1 (extracellular domain) binding antibodies with the CDRs and/or variable sequences as disclosed herein.
  • variations in a limited number of amino acids will be evaluated during in silico modeling.
  • the effects of the variation may vary the affinity of the antibody to the DDR1 target epitope, typically it will be desirable that the affinity is similar or higher than that of the DDR-1 binding antibodies as disclosed herein.
  • Focused libraries containing candidate daughter sequences with the desired variations can then be synthesized or produced by directed mutagenesis into the DDR1 antibody sequences listed herein.
  • the retention of the DDR1 binding capacity can be verified after expressing the derived protein(s), and competition experiments can be used to demonstrate that the variant DDR-1 antibodies derived from the antibodies as disclosed herein bind to the same original epitope.
  • Stabilizing mutations can be identified by assessing melting curves using thermal scanning or light scattering [aggregation (agg)] of antibodies. Stabilizing mutations have been shown to stabilize antibodies independently of their target binding capacities.
  • Mutations stabilizing the antibodies of the invention can be identified either directly starting from these antibodies, or using antibodies derived from the antibodies disclosed herein that have lost the DDR1 binding capacity and then introduced into the antibodies of the invention or from the antibodies with DDR1 binding capacity derived from them as described above.
  • antibodies of the invention may also be produced in different hosts to modify glycosylation, but in all cases said antibodies will contain the DDR1 binding region from the antibody sequences as disclosed herein or they will be directly derived from them following established methods as disclosed above and will thus retain the binding capacity to the original epitope, as described above.
  • the antibody/binding molecule of the present invention may be a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody.
  • the antibodies/binding molecules of the invention may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross-cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc.
  • the antibody may also be a multispecific antibody, including a bispecific antibody.
  • the antibodies of the invention may be multifunctional, i.e. they may exert their effects via more than one mode of action, such as for example by promoting internalization of DDR1 and activating ADCC or CDC pathways.
  • the antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single- chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above.
  • synthetic antibodies monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single- chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope- binding
  • antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a DDR1 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-DDRl antibody).
  • a DDR1 antigen e.g., one or more complementarity determining regions (CDRs) of an anti-DDRl antibody.
  • the antibodies are humanized or human and/or deimmunized. More preferably, the antibodies are humanized and most preferably the antibodies are fully humanized/human.
  • Said “fully humanized antibody” are also characterized and described as "completely human” antibodies. All these antibodies can be generated by methods known in the art. For example, by phage display technology, recombinant antibody molecules may be generated due to the use of in vitro maturation which is the usage of a complete human immunoglobulin ⁇ , subclass- 1 framework (IgGl) as described by Knappik (2000) J Mol Biol. 296(1), 57-86, and Rauchenberger (2003) J Biol Chem. 278(40), 38194-205.
  • IgGl immunoglobulin ⁇ , subclass- 1 framework
  • CDR-grafted As used herein, the term "CDR-grafted”, “humanized” or “humanization” are used interchangeably to refer to a human antibody as defined herein (preferably a IgGl antibody) comprising in its binding domains at least one complementarity determining region (“CDR") from a non-human antibody or fragment thereof.
  • CDR complementarity determining region
  • the term encompasses the case in which a variable region of the binding domain comprises a single CDR region, for example the third CDR region (CDR-H3) of the VH, from another non-human animal, for example a rodent, as well as the case in which a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal.
  • CDR-H3 third CDR region
  • a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal.
  • humanized also encompasses cases in which, in addition to replacement of one or more CDR regions within a VH and/or VL of the binding domain further mutation/s (e.g. substitutions) of at least one single amino acid residue/s within the framework ("FR") regions between the CDRs has/have been effected such that the amino acids at that/those positions correspond/s to the amino acid/s at that/those position/s in the animal from which the CDR regions used for replacement is/are derived.
  • FR framework
  • humanized may further encompass (an) amino acid substitution(s) in the CDR regions from a non-human animal to the amino acid(s) of a corresponding CDR region from a human antibody, in addition to the amino acid substitutions in the framework regions as described above.
  • humanized antibodies or related terms encompass antibodies having the amino acid sequence of a human immunoglobulin with a variable region comprising non-human CDR- and/or framework region- sequences.
  • a humanized antibody or a human antibody (or fragment thereof) will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a "foreign", that is non-human protein. This means that no host, i.e.
  • an antibody as defined herein may also be regarded as humanized if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germline sequence(s) by no more than would be expected due to the imprint of somatic hypermutation.
  • the humanized antibodies as defined herein have a human constant region and one or more of the CDR sequences which may be of, but are not limited to, CDRs of non-human, preferably rodent, origin.
  • the present invention also provides for "fully-human” antibodies.
  • the term “chimeric antibody” encompasses antibodies having human constant regions on the light and heavy chains and non-human variable regions on the light and heavy chains.
  • the non-human regions are from a rodent sequence.
  • the variable regions of the heavy and light chain could be amplified by RT-PCR using R A extracted from a mouse hybridoma cell which produces the antibody of interest. The amplified sequence could be cloned in frame with the constant heavy-chain or the constant light chain respectively of a human IgG also included in a mammalian expression vector.
  • an expression vector encoding a chimeric IgG could be transfected into the right cell line, like for example CHO or HEK293, for chimeric antibody production.
  • the term “deimmunized” or “deimmunization” denotes modification of the binding domain vis-a-vis an original wild type construct by rendering said wild type construct non-immunogenic or less immunogenic in humans. Deimmunization approaches are shown e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/10755.
  • the term “deimmunized” also relates to constructs, which show reduced propensity to generate T cell epitopes.
  • the term "reduced propensity to generate T cell epitopes” relates to the removal of T-cell epitopes leading to specific T-cell activation. Furthermore, “reduced propensity to generate T cell epitopes” means substitution of amino acids contributing to the formation of T cell epitopes, i.e. substitution of amino acids, which are essential for formation of a T cell epitope. In other words, “reduced propensity to generate T cell epitopes” relates to reduced immunogenicity or reduced capacity to induce antigen independent T cell proliferation.
  • T cell epitope relates to short peptide sequences which can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatibility complex (MHC) in order to trigger the activation of T cells; see inter alia WO 02/066514.
  • MHC major histocompatibility complex
  • Reduced propensity to generate T-cell epitopes" and/or “deimmunization” may be measured by techniques known in the art. Preferably, de-immunization of proteins may be tested in vitro by T cell proliferation assay.
  • PBMCs from donors representing > 80 % of HLA-DR alleles in the world are screened for proliferation in response to either wild type or de- immunized peptides.
  • cell proliferation is only detected upon loading of the antigen- presenting cells with wild type peptides.
  • binding of e.g. fluorescence-labeled peptides on PBMCs can be measured.
  • deimmunization can be proven by determining whether antibodies against the deimmunized molecules have been formed after administration in patients.
  • antibody derived molecules are deimmunized in the framework regions and most of the CDR regions are not modified in order to generate reduced propensity to induce T cell epitope so that the binding affinity of the CDR regions is not affected. Even elimination of one T cell epitope results in reduced immunogenicity.
  • the above approaches help to reduce the immunogenicity of the antibodies provided herein when being administered to patients.
  • the invention also involves one or more of the disclosed CDR sequences above or a CDR sequence at least 75 % (at least 80%, at least 90%, at least 95%, at least 96 %, at least 97 %, at least 98 % or at least 99 %) identical in their amino acid sequence hereto wherein said CDR sequences is in the context of an antibody framework/framework region.
  • the antibody framework is a human antibody framework.
  • frameworks include an IgG framework, such as IgGl, IgG4, IgG2a and IgG2b, preferably a human IgG framework such as IgGl , IgG2, IgG3 and IgG4.
  • the antibodies of the invention may also comprise cross-cloned antibodies, i.e. antibodies comprising different antibody regions (e.g. CDR-regions) from one or more parental or affinity-optimized antibody(ies) as described herein.
  • These cross- cloned antibodies may be antibodies in several, different frameworks, e.g. an IgG-framework, e.g. a IgGl-, IgG4, IgG2a or an IgG2b-framework.
  • said antibody framework is a mammalian, e.g. a human framework such as IgGl , IgG2, IgG3 or IgG4.
  • antibodies described herein may be presented in a preferred (human) antibody framework, but also antibody molecules comprising CDRs from antibodies as described herein, may be introduced in an immunoglobulin framework.
  • frameworks include IgG frameworks such as IgGl , IgG4, IgG2a and IgG2b. Most preferred are human frameworks, and particularly human IgGl or IgG4 frameworks.
  • a "human antibody framework” relates to an antibody framework that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the antibody framework of a naturally occurring human immunoglobulin.
  • a "human framework region” relates to a framework region that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the framework region of a naturally occurring human immunoglobulin.
  • a framework region relates, accordingly, to a region in the V domain (VH or VL domain) of immunoglobulins and T-cell receptors that provides a protein scaffold for the hypervariable complementarity determining regions (CDRs) that make contact with the antigen.
  • VH or VL domain the V domain of immunoglobulins and T-cell receptors that provides a protein scaffold for the hypervariable complementarity determining regions (CDRs) that make contact with the antigen.
  • CDRs hypervariable complementarity determining regions
  • Framework 1 encompasses the region from the N-terminus of the V domain until the beginning of CDR1
  • framework 2 relates to the region between CDR1 and CDR2
  • framework 3 encompasses the region between CDR2 and CDR3
  • framework 4 means the region from the end of CDR3 until the C-terminus of the V domain; see, inter alia, Janeway, Immunobiology, Garland Publishing, 2001 , 5th ed.
  • the framework regions encompass all the regions outside the CDR regions in VH or VL domains.
  • the term "transition sequence between a framework and a CDR region” relates to a direct junction between the framework and CDR region.
  • transition sequence between a framework and a CDR region means the sequence directly located N- and C-terminally of the CDR regions or amino acids surrounding CDR regions.
  • frameworks may also comprise sequences between different CDR regions.
  • the person skilled in the art is readily in a position to deduce from a given sequence the framework regions, the CDRs as well as the corresponding transition sequences; see Kabat (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services, Chothia (1987). J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.
  • the antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody, preferably IgG.
  • an "antibody” may denote immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to DDR1. Such antibodies are constructed in the same way. They form paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. Within this general category, however, five different classes of immunoglobulins - IgM, IgD, IgG, IgA, and IgE - can be distinguished by their C regions.
  • IgG antibodies are large molecules, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chain.
  • Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites, and thus the ability to bind simultaneously to two identical structures.
  • lambda and kappa Two types of light chain, termed lambda and kappa, are found in antibodies.
  • a given immunoglobulin either has lambda chains or kappa chains, never one of each. No functional difference has been found between antibodies having lambda or kappa light chains, and either type of light chain may be found in antibodies of any of the five major classes.
  • the ratio of the two types of light chain varies from species to species. In mice, the average kappa to lambda ratio is 20: 1, whereas in humans it is 2: 1 and in cattle it is 1 :20. The reason for this variation is unknown.
  • the class, and thus the effector function of an antibody is defined by the structure of its heavy chain.
  • immunoglobulin M immunoglobulin M
  • IgD immunoglobulin D
  • IgG immunoglobulin G
  • IgA immunoglobulin A
  • IgE immunoglobulin E
  • IgG is by far the most abundant immunoglobulin and has several subclasses (IgGl, 2, 3, and 4 in humans, IgGl, IgG2a, IgG2b and IgG3 in mice). Their distinctive functional properties are conferred by the carboxy-terminal part of the heavy chain, where it is not associated with the light chain. The general structural features of all the isotypes are similar. The IgG antibody is the most abundant isotype in plasma.
  • the antibodies as defined herein are IgG antibodies.
  • an IgG comprises not only the variable antibody regions responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in endogenously produced antibodies and, in some cases, even decoration at one or more sites with carbohydrates.
  • Such glycosylation is generally a hallmark of the IgG format, and portions of these constant regions make up the so called Fc region of a full antibody which is known to elicit various effector functions in vivo, such as e.g. antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • the Fc region mediates binding of the IgG to an Fc receptor, hence prolonging half life in vivo as well as facilitating homing of the IgG to locations with increased Fc receptor presence.
  • the IgG antibody is an IgGl or IgG4 antibody specifically binding to DDR1.
  • exemplary methods for the generation of antibodies to DDR1 like polyclonal, monoclonal, humanized, human antibodies or antibody fragments are described.
  • Antibodies and fragments thereof to a DDR1 protein or a DDR1 epitope for therapeutic and/or diagnostic uses can be obtained in any number of ways known to those of ordinary skill in the art. These antibodies can be used in the methods of the invention and/or as the basis of engineering new antibodies. Phage display techniques can be used to generate or isolate an antibody and/or fragment thereof to a DDR1 protein or a DDR1 epitope. Standard hybridoma technologies can be used to generate antibodies and fragments thereof to a DDR1 protein or a DDR1 epitope. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a monoclonal antibody or a fragment thereof.
  • the antibody or fragment thereof to a DDRl or a DDRl epitope is a polyclonal antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a recombinant antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a humanized antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a fully human antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a chimeric antibody or fragment thereof. In one aspect, the antibody or fragment thereof (e.g., CDR(s)) to DDRl is derived from an animal source (e.g., mouse, rat, or rabbit).
  • an animal source e.g., mouse, rat, or rabbit.
  • the target protein antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the target protein polypeptide DDRl (or fragment or epitope thereof) or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the DDRl protein antibodies may, alternatively, be monoclonal antibodies and/or fragments thereof.
  • Monoclonal antibodies may be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495.
  • a hybridoma method a mouse, hamster, or other appropriate host animal (e.g., rabbit, goat etc.), is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the target protein polypeptide DDRl (or fragment thereof) or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non- human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT- deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor (1984) Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against target protein.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem. 107:220.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No.
  • non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies and fragments thereof may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Human and Humanized Antibodies
  • the DDRl protein antibodies of the invention may further comprise humanized antibodies or human antibodies (and/or fragments thereof).
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies (and/or fragments thereof) may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody (and/or fragments thereof) will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature, 321 :522-525; Riechmann et /.(1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596).
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature, 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies (and/or fragments thereof) can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter (1991) J. Mol. Biol. 227:381 ; Marks et al.
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al.
  • the antibodies (and/or fragments thereof) may also be affinity matured using known selection and/or mutagenesis methods as described above.
  • Preferred affinity matured antibodies have an affinity which is 5 times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.
  • Antibody fragments can also be produced directly by recombinant host cells and the antibody phage libraries discussed above. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al (1992) Bio/Technology 10: 163-167). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).
  • scFv single chain Fv fragment
  • the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641 ,870, for example. Such linear antibody fragments may be monospecific or bispecific.
  • Bispecific antibodies with binding specificities for at least two different epitopes may bind to two different epitopes of the DDRl protein.
  • An anti- DDR1 arm may be combined, for example, with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the DDRl -expressing cell.
  • a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the DDRl -expressing cell.
  • Bispecific antibodies may also be used to localize cytotoxic agents to cells which express DDRl (WO 96/16673; U.S. Pat. No. 5,837,234; WO98/02463; U.S. Pat. No. 5,821,337). Purification methods for bispecific antibodies have been disclosed (WO 93/08829; Traunecker et al (1991) EMBO J. 10:3655-3659; WO 94/04690; Suresh et al (1986) Methods in Enzymology 121 :210; U.S. Pat. No. 5,731,168). Bispecific antibodies can be produced using leucine zippers (Kostelny et al (1992) J. Immunol. 148(5): 1547- 1553), and single-chain Fv (sFv) dimers (Gruber et al (1994) J. Immunol. 152:5368).
  • bispecific antibodies from antibody fragments
  • Techniques for generating bispecific antibodies from antibody fragments have also been described, such as using chemical linkage wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (Brennan et al (1985) Science 229:81).
  • Fab'-SH fragments can be recovered from E. coli and chemically coupled to form bispecific antibodies (Shalaby et al (1992) J. Exp. Med. 175:217-225.
  • the "diabody” technology provides an alternative method for making bispecific antibody fragments (Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Antibodies with more than two valencies are contemplated.
  • Multivalent, "Octopus" antibodies with three or more antigen binding sites and two or more variable domains can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody (US 2002/0004586; WO 01/77342).
  • trispecific antibodies can be prepared (Tutt et al (1991) J. Immunol. 147:60.)
  • the antibody is conjugated to one or more therapeutic agents. This is particularly envisaged when the antibodies are to be used in medicine, for example, in the therapy/treatment of cancer (and/or a proliferative disorder, etc).
  • Antibody conjugates with antibodies to DDR1 can prepared for various types of antibodies (and/or fragments thereof) including chimeric antibodies, humanized antibodies, and fully human antibodies.
  • conjugated means that the antibody/binding molecule is bound to the therapeutic agent(s) via any type of bonding, and thus includes bonding via fusion proteins (in case the therapeutic agent is of peptidic nature) or any other type of coupling or linkage between the therapeutic agent and the antibody/binding molecule.
  • “Conjugated to a therapeutic agent” is thus to be understood as including fused to, linked to or coupled to a therapeutic agent.
  • “Therapeutic agent” as used herein refers to any molecule (including small molecules, macromolecules, peptides, polypeptides, proteins (including other therapeutic antibodies), radioactive isotopes, etc) exerting a beneficial effect in the treatment of diseases in humans or other mammals.
  • therapeutic agents are suitable for the therapy of cancer, tumorous disorders and/or proliferative disorders.
  • Further medical use comprise the medical intervention in proliferative disorders, like inflammations, inflammation disorders related to the undesired proliferation of immune cells, auto-immune disorders (such as atherosclerosis).
  • therapeutic agents also comprises toxins, in particular toxins used in cancer therapy or, inter alia, anti-inflammatory therapy, etc.
  • a molecule of antibody may conjugate with more than one molecule of the therapeutic agent (as used herein, "conjugation agent"), depending on the number of sites in the antibody available for conjugation and the experimental conditions employed for performing the conjugation.
  • conjugation agent a molecule of the therapeutic agent
  • a preparation of the antibody conjugate may analyze for a non-integer ratio of conjugation agent molecules per molecule of antibody, reflecting a statistical average.
  • therapeutic agents that can be conjugated to the antibodies/binding molecules of the invention targeting DDR1 include, but are not limited to, anticancer agents such as antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, capecitabine and decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, carboplatin, oxaliplatin nedaplatin, satraplatin, triplatin tetranitrate, procarbazine, altre
  • anti-mitotic agents e.g., vinca alkaloids such as vincristine, vinorelbine, vindesine and vinblastine, taxanes such as paclitaxel (or taxol) and docetaxel, and other tubulin polimeryzation inhibitors such as auristatins like monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) and maytansine derivatives (a.k.a maytansinoids) like mertansine (also known as DM1) and DM4).
  • MMAE monomethyl auristatin E
  • MMAF monomethyl auristatin F
  • maytansine derivatives a.k.a maytansinoids
  • DM1 and DM4 mertansine
  • inhibitory peptide means any peptide that inhibits cell proliferation or affects cell viability via any mechanism of action. Non-limiting examples are provided herein below.
  • anticancer agents that can be conjugated to the antibodies/binding molecule of the invention to DDRl include, but are not limited, to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, irinotecan, topotecan, camptothecin, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, calicheamicin, duocarmycin, actinomycin D, glucocorticoids, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine derivatives like mertansine (also known as DM1) and DM4, and puromycin and analogs or homologs thereof.
  • inhibitory peptides that can be conjugated to the antibodies/binding
  • YARAAARQARAGRGYVSTT (wherein Y represents a phosphotyrosine), which is a peptide inhibitor of the transcription factor STAT6 which binds only to the phosphorylated form of STAT6 to prevent its dimerization and activity
  • PYLKTK (wherein Y represents a phosphotyrosine), which is a phosphopeptide which inhibits the activity of the transcription factor STAT3 in vitro and in vivo
  • the therapeutic agent for conjugation is a toxin.
  • the toxin is an enzyme.
  • toxins that can be conjugated to the antibodies/binding molecules of the invention to DDR1 include, but are not limited to plant toxins such as saporin, Ricin or Gelonin, and bacterial toxins such as Pseudomona exotoxin or diphteria toxin, and derivatives thereof.
  • ribonucleases can be considered as toxins due to their ability to degrade RNA and cause cell death.
  • Rnases which are considered to have cytotoxic effects and can be used also as toxins are Binase (from Bacillus intermedius), a-sarcin (from Aspergillus giganteus), Ranpirnase (from amphinian), Onconase (from Rana pipiens), and human RNAses like inhibitor-resistant variant of human pancreatic RNase (HP-DDADD- RNase)
  • the antibodies/binding molecules of the invention may also be conjugated to nanoparticles comprising human serum albumin (HSA) to optimize preparation and uptake of antibodies in cancer cells, as described, for example, by Steinhauser et al., Biomaterials 2006 Oct;27(28):4975-83.
  • HSA human serum albumin
  • the therapeutic agent(s), such as toxin(s), are preferably suitable for the treatment of cancer and/or a proliferative disorders, etc.
  • Such antibody conjugates with antibodies/binding molecules to DDR1 can readily be prepared for various types of antibodies (and/or fragments thereof) as described above, including chimeric antibodies, deimmunized antibodies, humanized antibodies, fully humanized/human antibodies, single chain antibodies, diabodies and the like.
  • Techniques for conjugating agents, such as the therapeutic agents described above, to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
  • Conjugates can be prepared using a variety of cleavable linkers such as for example disulfide-based linkers, hydrazone linkers or peptide linkers (Alley et al. (2010) Curr Opin Chem Biol 14(4):529-37; Webb (201 1) Nat.Biotech, 29(4):297-8) or the TAP linker technology from ImmunoGen.
  • the conjugate may be prepared via fusion proteins, as disclosed below.
  • the antibodies of the invention may also be a fusion wherein the antibody portion (comprising one or more CDRs) is fused to another protein or polypeptide.
  • an antibody according to the invention can be fused to another protein or polypeptide wherein said protein or polypeptide is an agent which improve the properties of said antibody e.g., enhances therapeutic effect.
  • proteins or polypeptides which e.g., can enhance therapeutic effect through a number of mechanisms like attracting or enhancing an immune response or delivering a therapeutic agent such a cytotoxic peptide or inhibitory peptide as defined above.
  • proteins or polypeptides are cytokines like IL2 or a IL2 homolog or GM-CSF.
  • a nucleic acid encoding the antibody of the invention operably linked to the desired protein or polypeptide can be prepared and introduced into a suitable expression vector, which is then inserted into a host cell for production of the fusion protein.
  • the antibodies (and fragments thereof) of the invention can also be conjugated to or have a detectable label to molecules for diagnostic purposes.
  • an antibody to DDR1 can be conjugated to a detectable label (e.g., for imaging purposes) for diagnosing or detecting cancer (like e.g. epidermoid or endometrial cancer) and/or a proliferative disorder.
  • Suitable detectable markers include, but are not limited to, a radioisotope, a nanoparticle, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • kits for conjugating agents are commercially available.
  • the present invention relates to a nucleic acid molecule having a sequence encoding the antibody as defined and provided herein.
  • the nucleic acid molecules of the invention for example, those encoding anti-DDRl antibodies, and its subsequences/alternative transcripts, can be inserted into a vector, which will facilitate expression of the insert.
  • the nucleic acid molecules and the antibodies they encode can be used directly or indirectly as therapeutic (or diagnostic) agents (directly in the case of the antibody or indirectly in the case of a nucleic acid molecule). Accordingly, the present invention relates also to a vector comprising the nucleic acid molecule.
  • the vector may further comprise a nucleic acid molecule having a regulatory sequence which is operably linked to the nucleic acid molecule.
  • the vector may be an expression vector.
  • the present invention relates to a host, host cell or host cell line transformed or transfected with the vector as defined above.
  • the host, host cell or host cell line expresses the antibody as provided herein.
  • Said host, host cell or host cell line can be prokaryotic or eukaryotic.
  • the host is preferably a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.
  • the antibody of the invention can be made by any number of methods.
  • the antibody can be synthesized in a cell line harboring a nucleic acid encoding the antibody as described above and culturing said cell line under conditions sufficient to allow expression of said antibody.
  • the present invention relates in one embodiment to a process for the production of the antibody as defined herein, said process comprising culturing a host as defined herein under conditions allowing the expression of the antibody and recovering the produced antibody from the culture.
  • the antibody thus obtained can then be conjugated to a therapeutic agent or to a detectable label for diagnostic purposes, as described above.
  • a vector encoding the sequence for the fusion protein would be incorporated into the host cell line, which would then be cultured as described above.
  • Techniques for producing and purifying antibodies are well known (see e.g. Liu et al. (2010) MAbs. 2(5):480-99; Shukla et al. (2010) Trends Biotechnol. 28(5):253-61; and Backliwal et al. (2008) Nucleic Acids Res. 36(15):e96).
  • transformed (host) cell or "transfected (host) cell” (and the like) means a cell into which (or into predecessor or an ancestor of which) a nucleic acid molecule encoding an antibody (or a fragment thereof) of the invention has been introduced, by means of, for example, recombinant DNA techniques or viruses.
  • isolated DNA molecule is a fragment of DNA that has been separated from the chromosomal or genomic DNA of an organism. Isolation also is defined to connote a degree of separation from original source or surroundings.
  • cDNA Complementary DNA
  • copy DNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase.
  • cDNA double-stranded DNA molecule that comprises such a single-stranded DNA molecule and its complement DNA strand.
  • expression refers to the biosynthesis of a gene product, such as a protein or an mRNA molecule.
  • an "expression vector” is a nucleic acid construct, generated recombinant or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue -preferred regulatory elements, and enhancers.
  • the invention provides an expression vector comprising a nucleic acid encoding an antibody of the invention (or binding molecule or antibody fragment),
  • said vector is DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 201 1; or
  • said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1; or
  • said vector is DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 201 1.
  • the invention provides an expression vector comprising a nucleic acid encoding an antibody of the invention (or binding molecule or antibody fragment), wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1.
  • the invention also provides an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 2011 , using
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 67 and a 3 '-primer as shown in SEQ ID NO: 68 for amplification of a first binding domain;
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 69 and a 3 '-primer as shown in SEQ ID NO: 70 for amplification of a second binding domain.
  • the invention also provides in a preferred embodiment an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 using
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
  • the invention also provides an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011 , using
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 75 and a 3 '-primer as shown in SEQ ID NO: 76 for amplification of a first binding domain; and/ or
  • a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 77 and a 3 '-primer as shown in SEQ ID NO: 78 for amplification of a second binding domain.
  • first binding domain means, in relation to each DNA contained in each of the deposited vectors DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl, the VL domain
  • second binding domain means, in relation to the same DNA, the VH domain.
  • primers for molecular technologies like PCR for the amplification of relevant nucleic acid molecules, is also well known in the art.
  • the conditions for PCR may be stringent conditions.
  • Exemplified, but not binding conditions, for amplification of the relevant portions of the above described nucleic acid molecules comprise the following: A PCR reaction of a 20 ⁇ volume which comprises the following components: 2 ⁇ 1 of a 10X Taq Polymerase Buffer reaction, a final concentration of MgC of 1.5mM (to be supplemented in case the Buffer reaction does not include it already), 0.5pMol of each of the below indicated primers pairs, 2 ⁇ 1 of 25mM dNTPs, 2 units of Taq Polymerase and 10 to 20 ng of the deposited plasmid D A.
  • the following cycle conditions can be used: A denaturation first step of 10 minutes at 95°C; a denaturation step of 45 seconds at 95°C, an annealing step of 40 seconds at 58°C, an extension step of 40 seconds at 72°C, the last three steps will be repeated for 30-35 cycles. After the last cycle, a last step of 5 minutes at 72°C can be added. After that the PCR reaction will be complete and the PCR product can be loaded on a 1.5% agarose gel for checking.
  • the following cycle conditions can be used: A denaturation first step of 10 minutes at 95°C; a denaturation step of 45 seconds at 95°C, an annealing step of 40 seconds at 60°C, an extension step of 40 seconds at 72°C, the last three steps will be repeated for 30-35 cycles. After the last cycle, a last step of 5 minutes at 72°C can be added. After that the PCR reaction will be complete and the PCR product can be loaded on a 1.5% agarose gel for checking.
  • a “recombinant host” may be any prokaryotic or eukaryotic cell that contains a cloning vector, expression vector, or other heterologous nucleic acid sequences. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.
  • the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • the antibodies provided herein (as well as the nucleic acids encoding them, the herein provided vectors and hosts) are preferably “isolated” “purified” or “biologically pure” as defined herein.
  • isolated denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences.
  • a nucleic acid or antibody of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or antibody gives rise to essentially one band in an electrophoretic gel. For an antibody that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. Various levels of purity may be applied as needed according to this invention in the different methodologies set forth herein. The customary purity standards known in the art may be used if no standard is otherwise specified.
  • isolated nucleic acid molecule can refer to a nucleic acid molecule, depending upon the circumstance, which is separated from the 5' and 3' coding sequences of genes or gene fragments contiguous in the naturally occurring genome of an organism.
  • isolated nucleic acid molecule also includes nucleic acid molecules which are not naturally occurring, for example, nucleic acid molecules created by recombinant DNA techniques.
  • Nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • nucleic acids containing known nucleotide analogs or modified backbone residues or linkages which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide - nucleic acids (PNAs).
  • nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • a "host cell” is a naturally occurring cell or a transformed cell or a transfected cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells may be cultured cells, explants, cells in vivo, and the like.
  • Host cells may be prokaryotic cells, for example, E. coli, or eukaryotic cells, for example, yeast, insect, amphibian, or mammalian cells, for example, Vero, CHO, HEK293, HeLa, and others.
  • the present invention relates to a vector encoding an antibody/ binding molecule as defined herein deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively. Accordingly, the present invention relates to an antibody/binding molecule obtained or obtainable by the expression of a coding nucleic acid molecule comprised in a vector as deposited under these accession numbers.
  • the present invention relates to an antibody/binding molecule obtained or obtainable by the expression of a coding nucleic acid molecule comprised in a vector as deposited under DSM accession number 25530.
  • an antibody as defined herein above prepared by a process comprising (a) providing a cell line capable of producing the antibody; and
  • the cell line comprises a vector as deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 2011 under the designation DDRl-ablpBhl , DDRl-ab2pBhl, and DDRl-ab3pBhl , respectively.
  • the present invention relates to a vector encoding a binding molecule as defined herein deposited under accession number DSM 25529, preferably DSM 25530, and/or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 2011 under the designation DDRl-ablpBhl , DDRl-ab2pBhl and DDRl-ab3pBhl , respectively. Accordingly, the present invention relates to an antibody obtained or obtainable by the expression of a vector as deposited under these accession numbers.
  • the present invention also relates to anti-DDRl binding molecules/antibodies that comprise CDRs and/or variable regions that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) to the amino acid sequence of these (individual) CDRs or said variable regions of the sequences disclosed herein or as obtainable from the vectors as deposited under accession number DSM 25529, DSM 25530, or DSM 25531 with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively. Accordingly, the methods of preparation of these binding molecules/antibodies are also provided herein and as laid down herein above.
  • the present invention also relates to antibody/binding molecules that show in their amino acid sequences of their individual CDRs and/or their variable regions at least 75% identity (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) to the antibody molecules/binding molecules defined herein by their sequences as obtainable from the deposited vectors deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively.
  • the present invention also relates to antibodies/binding molecules that bind to and/or recognize the same epitope on the extracellular domain of DDR1 and/or that have the same functional properties as the antibodies/binding molecules obtainable from the deposited nucleic acid vectors DDRl-ablpBhl , preferably DDRl-ab2pBhl and DDR1- ab3pBhl .
  • the ability of an antibody or binding molecule to bind specifically to DDR1 can be determined using well known assays. Affinity or specificity can be determined experimentally by methods known in the art such as Flow Cytometry (FC), Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Other methods include the use of siRNA agents against DDR1.
  • FC Flow Cytometry
  • FC Flow Cytometry
  • Western blots ELISA-, RIA-, ECL-, IRMA-tests and peptide scans.
  • Other methods include
  • the invention therefore, also provides pharmaceutical compositions for use in the treatment of cancer (and/or proliferative disorders, etc) comprising an antibody/binding molecule as disclosed herein or having essentially the same biological activity of an antibody/binding molecule obtained or obtainable by expression of the nucleic acid molecule comprised in any of the vectors as deposited with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively.
  • the present invention also relates to pharmaceutical compostions and, as discussed herein below, to diagnostic compositions that comprise the herein disclosed antibodies/binding molecules directed against/binding to the extracellular domain of DDR1 , whereby said pharmaceutical composition and said diagnostic composition have their medical use in the medical amelioration of cancer (and/or proliferative disorders, etc) and in the diagnostic of e.g. cancer (and/or proliferative disorders, etc).
  • said cancer is , epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer (also known as squamous cell cancer) or endometrial cancer.
  • the present invention relates to a composition
  • a composition comprising the antibody/binding molecule directed against/specifically binding to DDR1 and as defined herein or as produced by the above described process, a nucleic acid molecule as described herein, a vector as described herein and/or a host as described herein.
  • the composition comprises the antibody/binding molecule as defined and provided herein.
  • the composition may further comprise (a) secondary antibody/antibodies that is/are specifically binding to the primary antibody as defined and provided in the present invention, whereby said secondary antibody/antibodies is/are conjugated to a therapeutic agent as defined above (in particular an anticancer/cyto toxic agent or a toxin such as Saporin) or a diagnostic agent as defined and explained herein above.
  • the primary antibody is preferably an IgG antibody.
  • the secondary antibody may be a goat anti-human IgG secondary antibody.
  • the secondary antibody may also be any of the antibody types as described herein above in context of the anti-DDRl antibodies provided herein
  • the herein above described composition is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s) like, inter alia, stabilizers or carriers.
  • pharmaceutically acceptable excipients are also provided herein below as non-limiting examples.
  • the antibody as provided herein, or the antibody as produced by the herein above described process, the nucleic acid molecule described herein, the vector described herein, the host as described herein and/or the composition (in particular the pharmaceutical composition) is for use in medicine.
  • the antibody as provided herein (optionally contained in the composition as defined above) is for use in medicine.
  • the antibody is conjugated to a therapeutic agent or to a diagnostic agent (like a label etc.).
  • the present invention relates to the use of the antibody as defined or provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for the preparation of a pharmaceutical composition for the treatment of e.g., cancer and/or a tumorous disease or proliferation disorder.
  • the present invention relates to the use of the antibody as defined or provided herein for the preparation of a pharmaceutical composition for the treatment of cancer and/or a tumorous disease or a proliferative disorder.
  • the present invention relates to the antibody as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for use in the treatment of cancer etc.
  • the invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, ovarian cancer.
  • epidermoid cancer also known as squamous cell cancer
  • endometrial cancer also known as squamous cell cancer
  • bladder cancer colorectal cancer
  • breast cancer breast cancer
  • lung cancer stomach cancer
  • prostate cancer pancreatic cancer
  • hepatic cancer hepatic cancer
  • urothelial cancer head
  • the present invention relates to the antibody as defined and provided herein for use in the treatment of cancer selected from the group consisting of epidermoid cancer (squamous cell cancer), endometrial cancer, bladder cancer, colon cancer, stomach cancer, lung cancer, breast cancer, urothelial cancer, prostate cancer, head and neck cancer and skin cancer. More preferably, the present invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer (also known as squamous cell cancer) or bladder cancer. In one embodiment, the present invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer or endometrial cancer. .
  • epidermoid cancer also known as squamous cell cancer
  • the present invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer or endometrial cancer. .
  • a method for the treatment of cancer, a proliferative disorder, etc. is subject of the present invention, said method comprising the administration of the antibody/binding molecule as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein to a subject in need of such a treatment.
  • a "patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the subject is a mammal, and in the most preferred embodiment the subject is a human.
  • a “cancer” in an animal refers to the presence of cells possessing one or more characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers.
  • the cancer to be treated is selected from epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer and ovarian cancer.
  • the cancer to be treated is epidermoid cancer or endometrial cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • precancerous refers to cells or tissues having characteristics relating to changes that may lead to malignancy or cancer.
  • a “proliferative disorder” comprises, but is not limited to cancer and tumorous, cancerous or pre-cancerous disorders.
  • a proliferative disorder may also comprise autoimmune or inflammatory (particularly chronic inflammatory) disorders.
  • One such inflammatory disorder is atherosclerosis.
  • treatment means obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a subject and includes: (a) preventing a disease related to an insufficient immune response from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • Treating does not necessarily require a complete cure. It means that the symptoms of the underlying disease are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that reduced, as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease.
  • the invention provides a method of treating an individual having cancer, tumorous disease and/or a proliferative disorder comprising administering to said individual a therapeutically effective amount of an antibody or fragment thereof to DDRl . In one aspect of this embodiment, the antibody reduces levels of activity of DDRl .
  • the antibody or fragment thereof induces internalization or aggregation of DDRl .
  • the antibody or fragment thereof to DDRl inhibits or reduces proliferation; causes cytotoxicity; inhibits or reduces metastasis; modulates, inhibits or reduces cell adhesion; modulates, inhibits or reduces migration; or modulates, inhibits or reduces invasion of cancer or cancer cells expressing DDRl .
  • the antibody or fragment thereof to DDRl inhibits or reduces proliferation of cancer or cancer cells expressing DDRl .
  • the antibody or fragment thereof to DDRl causes cytotoxicity to cancer or cancer cells expressing DDRl .
  • the antibody or fragment thereof to DDRl reduces or inhibits metastasis of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits cell adhesion of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits invasion of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits migration of cancer or cancer cells expressing DDRl .
  • said cancer is endometrial cancer, epidermoid cancer (also known as squamous cell cancer), bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer
  • Testing anti-DDRl Antibody for anti-cancer properties can be done using standard assays, well known in the art.
  • a cancer cell line e.g. an epidermoid cancer cell line or an endometrial cancer cell line
  • Various dosages of potentially therapeutic antibodies or fragments thereof or conjugates thereof according to the invention are applied to various cultures of the (cancer) cell line.
  • the treated cultures and control cultures are then followed over time and scored for reduction in proliferation; reduction in cellular growth; reduction in colony formation; appearance of cytotoxicity; reduction in cell-adhesion; reduction of cell invasion; reduction of degradation of the extracellular matrix; or reduction in cell migration or reduction in cell inavtion through differents extracellular matrix proteins.
  • the antibodies/binding molecules of the invention or conjugates thereof can be tested in animals having a tumor and/or an animal that have had a cancer cell line implanted subcutaneusly (see for example Talmadge et al. Am J Pathol 2007 Mar 170(3), Cespedes et al.
  • mice are treated with the antibodies to be tested.
  • Routes of antibody administration into mice include intravenous or intraperitoneal administration.
  • Various dosages of potentially therapeutic antibodies or fragments thereof according to the invention can be tested in in vivo models.
  • the treated animals and control animals (treated with a sham antibody or fragment or no antibody at all) are then followed over time and scored for reduction in tumor size, reduction in tumor weight, reduction in tumor cell proliferation, reduction in tumor cellular growth; appearance of cytotoxicity; reduction in tumor cell-adhesion; reduction in metastasis, reduction in tumor cell invasion, reduction in tumor cell migration or increase in survival.
  • the antibody to DDR1 induces, enhances, or mediates ADCC (antibody dependent cellular cytotoxicity) against cells to which it binds.
  • ADCC is one of the mechanism by which an antibody can have a therapeutic effect.
  • ADCC is a cell mechanism where an effector cell of the immune system, mainly Natural Killer cells (NK), lyses a target cell which has been previously bound by specific antibodies.
  • NK cells have specific receptors such as FcyRIIIa which recognize the Fc fragment of immunoglobulins and are responsible for the ADCC response.
  • an in vitro assay can be performed in which target cells will be incubated with different antibodies and natural killer cells from human or mouse origin. The effect of the antibodies on the cells can be measured by the occurred lyses.
  • the antibody to DDR1 induces, enhances, or mediates CDC (complement dependent cytotoxicity) against cells to which it binds.
  • CDC is another immune mechanism to exert cytotoxicity on tumor cells.
  • CDC is a cytolytic cascade mediated by complement proteins in the serum. CDC is initiated by the binding of CI q to the constant region of cell bound antibody molecule.
  • the antibody to DDR1 is conjugated to another molecule.
  • the antibody is conjugated to a therapeutic agent, such as a toxin, a radioactive agent, inhibitory peptide, or an anti-tumor drug.
  • a therapeutic agent such as a toxin, a radioactive agent, inhibitory peptide, or an anti-tumor drug.
  • the antibody (or fragment thereof) of this embodiment can be provided as a pharmaceutical composition comprising the antibody (or fragment thereof) conjugated to the therapeutic agent and a pharmaceutically acceptable excipient.
  • Pharmaceutical compositions of this invention also can be administered in combination therapy ("cotherapy"), i.e., combined with other agents.
  • the combination therapy can include an anti-DDRl antibody of the present invention combined with at least one other therapeutic agent (e.g.
  • the at least one other therapeutic agent is not conjugated (as defined above) to the herein provided anti-DDRl antibody (however, the antibody used in cotherapy with one or more other therapeutic agents may, in itself, be conjugated to one or more of the therapeutic agents as defined herein above).
  • the administration of the other therapeutic agent can be prior to, concurrent to or after the administration of the antibody of the invention.
  • the antibody of the invention and the one or more other therapeutic agents may also be combined into a single dosage unit.
  • the invention includes a pharmaceutical composition comprising two or more antibodies to DDR1. Without wishing to be bound by theory, it may be believed that treatment with two or more therapeutic antibodies to DDR1 can have synergistic effects in terms of therapy.
  • the method comprises identifying a patient having a risk factor for cancer and/or a proliferative disorder, obtaining a sample from said patient having a risk factor for such a disorder, and determining the level of DDR1 in said sample wherein a patient having an increased level of DDR1 is treated with an antibody that binds to or modulates DDR1.
  • the risk factor for cancer and/or a proliferative disorder is chosen from age, ethnicity, family history of cancer and/or a proliferative disorder, or genetic predisposing gene or variant thereof. Risk factors for cancer and/or a proliferative disorder are known to the skilled artisan.
  • the risk factor for cancer and/or a proliferative disorder is one or more SNPs that indicated a higher risk of having cancer and/or a proliferative disorder.
  • the subject or patient to be treated was previously treated or is currently being treated with radiation therapy.
  • the invention provides a method of treatment of cancer and/or a proliferative disorder in a patient wherein said patient was previously treated or is currently being treated with radiation therapy.
  • the treatment comprises identifying a patient previously treated or is currently being treated with radiation therapy and administering to said patient a DDR1 therapeutic antibody as defined herein.
  • Radiation therapy for cancer and/or a proliferative disorder is generally classified as external or internal. External radiation therapy usually involves the focusing of high energy beams of energy (e.g., x-rays) on the affected area.
  • Internal radiation therapy involves implanting a radioactive substance or composition comprising a radioactive substance near or inside the cancer (also referred to as brachytherapy, internal radiation therapy, and/or radiation brachytherapy).
  • the subject or patient will be treated or is currently being treated with a chemotherapy or a radiotherapy.
  • the invention provides a method for treating cancer and/or a proliferative disorder in a patient wherein said patient had discontinued a prior treatment due to disease progression.
  • disease progression occurred due to the cancer developed chemoresistance to the prior treatment.
  • said cancer chemoresistance was or is correlated to increased DDRl expression or activation.
  • the antibodies to DDRl confer chemosensitivity to chemoresistant cancer or cancer cells, or increase chemosensitivity of the cancer or cancer cells. The ability of an antibody of the invention to confer or increase chemosensitivity to chemoresistant cancer cells can be tested as follows.
  • Chemoresistant target cells e.g, expressing DDRl or overexpressing DDRl are plated on 96 well plates and incubated with the DDRl antibodies to be tested (e.g., of the invention) with and without a chemotherapeutic agent under conditions sufficient for cell growth and proliferation.
  • a cell line from epidermoid cancer, endometrial cancer, bladder cancer, colon cancer, lung cancer, breast cancer,or prostate cancer overexpressing DDRl and which is resistant to a chemotherapeutic agent is tested in the presence of (1) DDRl antibody (as described herein) and (2) the presence of DDRl antibody (as described herein) and chemotherapeutic agent and (3) chemotherapeutic agent, wherein an increase in sensitivity to the chemotherapeutic agent in the presence of DDRl antibody indicates said antibody increases chemosensitivity or overcomes chemoresistance.
  • the subject or patient to be treated has hormone dependent endometrial cancer.
  • the treatment comprises identifying a patient having hormone dependent endometrial cancer and administering to said patient a DDRl antibody.
  • the method comprises administering to said patient having hormone dependent endometrial cancer a DDRl antibody and another therapeutic agent which is hormone therapy.
  • the subject or patient to be treated has hormone -refractory or resistant endometrial cancer.
  • the therapy comprises identifying a patient having treatment-refractory endometrial cancer and administering to said patient a DDR1 antibody.
  • the treatment comprises administering to said patient having treatment-refractory endometrial cancer a DDR1 antibody and another therapeutic agent which is hormone therapy.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or binding molecule of the invention, as described herein, optionally further comprising one or more pharmaceutically acceptable excipient(s).
  • pharmaceutically acceptable excipient relates to any component of a pharmaceutical composition other than the active ingredient and includes any and all carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the excipient is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., antibody
  • the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the pharmaceutical compounds of this invention may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N.N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition of this disclosure also may include a pharmaceutically acceptable anti-oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA) 5 butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of this disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization micro filtration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient (i.e. the herein provided antibody, nucleic acid molecules etc.) which can be combined with a excipient to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a excipient to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with (a) pharmaceutically acceptable excipient(s).
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient.
  • the dosage unit forms of this disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • the dosage typically ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 10 mg/kg, of the host body weight.
  • the ADC will be administered at a dose of less than 1 mg/kg.
  • Antibody/binding molecules etc. can also be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient.
  • human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a “therapeutically effective dosage”, “therapeutically effective amount” or “effective amount” of an anti-DDRl antibody (or nucleic acid etc.) of this invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • a “therapeutically effective dosage” preferably inhibits cell growth or tumor growth by at least about 5%, more preferably by at least about 10%, even more preferably by at least about 20%, and still more preferably by at least about 60% relative to untreated subjects (or cells in cell based studies).
  • the ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth. Such inhibition can be measured in vitro by assays known to the skilled practitioner (cell proliferation, metastasis, cytotoxicty, invasion, migration, etc.).
  • a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • a composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • Preferred routes of administration for antibodies of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • an antibody of this disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • the active compounds can be prepared with excipients that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
  • compositions can be administered with medical devices known in the art.
  • a therapeutic composition of this disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824; or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824; or 4,596,556.
  • Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No.
  • therapeutic antibodies of this disclosure can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of this disclosure cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,81 1 ; 5,374,548; and 5,399,331.
  • the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.
  • targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); marmosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pi 20 (Schreier et al. (1994) J. Biol.
  • the antibodies of the invention can be coadministered (i.e. administered in combination) with one or more other therapeutic agents.
  • the antibody of the invention may be, as described above, a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody and may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross- cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc.
  • a full antibody immunoglobulin
  • an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment
  • a single-chain antibody may be a monoclonal antibody, a
  • the antibody of the invention may itself be linked to another agent like an anticancer (i.e. cytotoxic agent), i.e. be a conjugated antibody as described above.
  • chemotherapeutic agents that may be used in combination with the DDR1 antibodies of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, decarbazine, capecitabine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), cis
  • antimetabolites e
  • irinotecan topotecan and camptothecin
  • anti-mitotic agents e.g., vinca alkaloids such as vincristine and vinblastine, taxanes such as paclitaxel (also known as taxol), cabazitaxel and docetaxel, and other tubulin polimeryzation inhibitors such as monomethyl auristatin E (MMAE), maytansine derivatives like mertansine (also known as DM1) and DM4), and protein kinase inhibitors such as imatinib (gleevec), nilotinib and dasatinib.
  • MMAE monomethyl auristatin E
  • DM1 mertansine
  • DM4 protein kinase inhibitors
  • the following drugs/agents may be employed: steroids such as Glucocorticoids, Non-Steroidal anti-inflammatory drugs such as aspirin, ibuprofen, naproxen or Immune Selective Anti-Inflammatory Derivatives (ImSAIDs) such as the peptide phenylalanine-glutamine-glycine (FEG).
  • steroids such as Glucocorticoids
  • Non-Steroidal anti-inflammatory drugs such as aspirin, ibuprofen, naproxen or Immune Selective Anti-Inflammatory Derivatives (ImSAIDs)
  • FEG Immune Selective Anti-Inflammatory Derivatives
  • the antibodies of the invention can be combined with e.g. statins or niacin.
  • the invention relates to a DDR1 antibody that induces, enhances, or mediates antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC as described above is a type of immune reaction in which a target cell is coated with antibodies and killed by certain types of white blood cells, particularly NK cells. The white blood cells bind to the antibodies and release substances that kill the target cells or microbes. Not all antibodies produce ADCC.
  • the invention relates to an antibody to DDR1 that can induce, enhance or mediate ADCC.
  • antibodies of the invention to DDR1 can be engineered to have improved, increased or enhanced ADCC.
  • an antibody of the invention that does not induce, enhance, or mediate ADCC can be engineered, e.g., by making certain amino acid modifications to the antibody or by producing the antibody in certain strains of cells, to induce, enhance or mediate ADCC or have improved/enhanced ADCC properties.
  • an antibody to DDRl has antibody-dependent cellular cytotoxicity when used in a human subject.
  • One example of an antibody with increased or improved ADCC activity is a antibody to DDRl that is defucosylated.
  • an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that lacks or has decreased alpha- 1 ,6-fucosyltransferase activity.
  • an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks GDP-fucose transporter activity. In another aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell- line that has reduced or lacks GDP-mannose 4,6-dehydratase activity. In another aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks both alpha- 1 ,6-fucosyltransferase activity and GDP-mannose 4,6- dehydratase activity. See e.g., Yamane-Ohnuki et al. (2004) Biotechnol Bioeng. 87(5):614-22; Imai-Nishiya et al. (2007) BMC Biotechnology 7:84.
  • ADCC can be enhanced or improved by increasing the levels of interleukin-21 (IL-21) in a patient or by treating the patient with IL-21 in combination with the antibody of the invention. See e.g., Watanabe et al. Br J Cancer. 2010, 102(3), 520-9.
  • IL-21 interleukin-21
  • the invention relates to a anti-DDRl antibody/binding molecule that enhances, induces or mediates complement dependent cytotoxicty (CDC).
  • CDC complement dependent cytotoxicty
  • the invention relates to an antibody/binding molecule to DDRl that induces, enhances or mediates CDC.
  • antibodies of the invention to DDRl can be engineered to have improved, increased or enhanced CDC.
  • an antibody of the invention that does not induce or mediate CDC can be engineered, e.g., by making certain modifications to the antibody like amino acid mutations in Fc or the hinge region thereby improving or enhancing CDC.
  • Another method of producing CDC or enhancing an antibody's CDC is by shuffling IgGl and IgG3 sequences within the heavy chain constant region. See e.g., Natsume et al. (2008) Cancer Res. 68:3863-3872.
  • the composition provided herein is a diagnostic composition further comprising, optionally, means and methods for detection.
  • a method for diagnosing cancer and/or a proliferative disorder comprising detecting or assaying DDRl in a biological sample of an individual suspected of suffering from cancer and/or a proliferative disorder using the antibody as defined herein, in particular the antibody conjugated with a detectable label as described above.
  • Suitable detectable labels or markers include, but are not limited to, a radioisotope, a nanoparticle, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • a “label” or a “detectable moiety” is a compound that when linked with the antibody of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer.
  • the usefulness of the antibodies/binding molecules to DDRl or fragments thereof, such as those identified or created by any of the Examples disclosed herein, in the diagnosis of cancer (or a proliferative disorder) and/or increased risk for developing cancer (or a proliferative disorder) can be tested as follows:
  • a cohort of subjects is identified and a sample collected from each subject.
  • the sample is tested for levels of DDRl using the antibodies or fragments thereof to DDRl .
  • All subjects may be further tested for the presence of cancer and/or said proliferative disorder using techniques standard in the art.
  • All subjects may be followed and periodically tested using the inventive antibodies/binding molecules or fragments thereof to DDRl and further tested for the presence of cancer and/or said proliferative disorder using techniques standard in the art. After each round of testing, the levels of DDRl are correlated with the presence of cancer and/or said proliferative disorder and/or increased risk for developing cancer.
  • the present invention relates to the use of the antibody as defined and provided herein, the antibody as produced by the herein above described process, the nucleic acid molecule as described above, the vector as described herein and/or the host as described herein for the preparation of a diagnostic composition for the diagnosis of cancer (and/or a proliferative disorder).
  • the present invention relates to the use of the antibody as defined and provided herein for the preparation of a diagnostic composition for the diagnosis of cancer (and/or a proliferative disorder).
  • said cancer is epidermoid (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer.
  • the present invention relates to the antibody/binding molecule as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for use in the diagnosis of cancer (and/or a proliferative disorder).
  • the present invention relates to the antibody as defined and provided herein for use in the diagnosis of cancer (and/or a proliferative disorder).
  • said cancer is epidermoid (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer.
  • the present invention relates to the use of the antibody/binding molecule as defined and provided herein, the antibody/binding molecule as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for the preparation of a diagnostic kit for the diagnosis of cancer (and/or a proliferative disorder).
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer.
  • DDR1 refers to a “DDR1 nucleic acid” or a “DDR1 protein” that can be specifically detected for diagnostic purposes.
  • DDR1 especially of DDR1 proteins and nucleic acids encoding DDR1 have been provided herein above. These explanations apply, mutatis mutandis, in this context.
  • a DDR1 nucleic acid can be a R A molecule, DNA molecule, or other nucleic acid that corresponds to the DDR1 gene or a fragment thereof.
  • a DDR1 gene may correspond to a human DDR1 gene.
  • a DDR1 nucleic acid can be a cDNA, or fragment thereof, corresponding to a DDRl mRNA molecule.
  • a DDRl protein refers to a protein (or fragment thereof) encoded or expressed by the DDRl gene.
  • a "DDRl gene” can be a region on the genome that is capable of being transcribed to an RNA that encodes a DDRl protein as well as the regulatory sequences associated with or operably linked to the coding region.
  • the skilled artisan will appreciate that the present invention encompasses all encoding transcripts that may be found, including splice variants, allelic variants and transcripts that occur because of alternative promoter sites or alternative polyadenylation sites of DDRl .
  • a "full-length" gene or RNA therefore encompasses any naturally occurring splice variants, allelic variants, other alternative transcripts, splice variants generated by recombinant technologies which bear the same function as the naturally occurring variants, and the resulting RNA molecules.
  • a "fragment" of a gene can be any portion from the gene, which may or may not represent a functional domain, for example, a catalytic domain, a DNA binding domain, etc.
  • a fragment may preferably include nucleotide sequences that encode for at least 25 contiguous amino acids, and preferably at least about 30, 40, 50, 60, 65, 70, 75 or more contiguous amino acids or any integer thereabout or therebetween.
  • detecting cancer (and/or a proliferative disorder) or “diagnosing cancer (and/or a proliferative disorder)” refers to determining the presence or absence of cancer or a precancerous condition in an subject, preferably in a human.
  • Detecting cancer (and/or a proliferative disorder) or “diagnosing cancer (and/or a proliferative disorder)” also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the subject or assessing the predisposition of a subject to the development of cancer (and/or a proliferative disorder).
  • Detecting cancer (and/or a proliferative disorder) can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the subject.
  • the "diagnosis of cancer (and/or a proliferative disorder)" may, in particular, comprise (i) the diagnosis for curative purposes stricto sensu representing the deductive medical or veterinary decision phase as a purely intellectual exercise, (ii) the preceding steps which are constitutive for making that diagnosis, and (iii) the specific interactions with the human or animal body (or biological sample, like a blood sample or a tissue sample) which occur when carrying those out among these precedings steps which are of a technical nature.
  • the invention provides a method for diagnosis of cancer (and/or a proliferative disorder) by determining the level or activity of a DDRl biomarker in a biological sample from an individual wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder), whereby the DDRl biomarker (preferably the DDRl protein as defined herein) is detected with an anti-DDRl antibody (i.e. the antibody/binding molecule specifically binding to DDRl) of the invention as provided and described herein.
  • an anti-DDRl antibody i.e. the antibody/binding molecule specifically binding to DDRl
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer
  • said cancer is epidermoid cancer or endometrial cancer.
  • the invention therefore relates to a method for the diagnosis of cancer (and/or a proliferative disorder) by contacting an antibody/binding molecule of the invention that specifically binds to DDRl and that is capable of detecting a DDRl biomarker with a biological sample from an individual and determining the level of the DDRl biomarker, wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder).
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer
  • said cancer is epidermoid cancer or endometrial cancer.
  • the invention provides a method for the diagnosis of cancer (and/or a proliferative disorder) by contacting the herein provided antibody/binding molecule to DDRl that is capable of detecting a DDRl biomarker with a tissue, tumor, blood, serum, plasma, body fluid or urine sample from an individual and determining the level of the DDRl biomarker, wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder).
  • the biological sample is a blood, serum, plasma, body fluid or urine.
  • the biological sample is a blood, serum, or plasma sample. In a more preferred aspect, the biological sample is a plasma sample. In a preferred aspect, an increased or elevated level of DDRl biomarker as compared to a control or normal value is indicative of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder).
  • the invention includes a method of diagnosing cancer (and/or a proliferative disorder) disease associated with DDRl expression comprising:
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer or endometrial cancer.
  • the sample is a serum, blood, body fluid, endometrial fluid aspirate, plasma, or urine sample.
  • the sample is a plasma sample.
  • the sample in another specific aspect, in a method of diagnosing endometrial cancer, is an endometrial fluid aspirate.
  • the DDRl biomarker is a nucleic acid biomarker. In one aspect, the DDRl biomarker is a protein biomarker.
  • the present invention provides a method for characterizing a sample obtained from a patient for prognostic, diagnostic and/or pharmaco genomic uses in respect of cancer (and/or a proliferative disorder).
  • Characterization of a sample obtained from a patient by determining the level of a DDRl biomarker can be used to provide information regarding diagnosis of cancer (and/or a proliferative disorder), prognosis of cancer (and/or a proliferative disorder), disease progression, diagnosis of cancer type (and/or subtype), and selection of an appropriate therapeutic treatment for the cancer (and/or a proliferative disorder).
  • a biological sample is obtained from an individual.
  • the individual can be a healthy person, an individual diagnosed with cancer (and/or a proliferative disorder), an individual suspected of having endometrial cancer, an individual displaying one or more symptoms of cancer (and/or a proliferative disorder) and/or an individual desiring screening for cancer (and/or a proliferative disorder).
  • the method comprises the step of determining the level of a DDRl biomarker in a sample obtained for a patient wherein the DDRl biomarker is a protein or nucleic acid biomarker.
  • the DDRl biomarker is a DDRl protein biomarker.
  • characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding prognosis of the cancer (and/or a proliferative disorder). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding disease progression of the cancer (and/or a proliferative disorder). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information diagnosis of cancer (and/or a proliferative disorder) cell type (and/or subtype).
  • characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding selection of an appropriate therapeutic, for example, chemotherapy and/or an antibody of the invention (for example antibodies to DDRl that are coupled or conjugated with a therapeutic agent).
  • an appropriate therapeutic for example, chemotherapy and/or an antibody of the invention (for example antibodies to DDRl that are coupled or conjugated with a therapeutic agent).
  • Subject as used herein in context of diagnosis of cancer (and/or a proliferative disorder) refers in particular to a biological subject that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDRl .
  • the subject may be a mammalian subject, preferably a human.
  • Biological sample refers to a sample obtained from a subject, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDRl .
  • a biological sample also includes samples from a region of a biological subject containing or suspected of containing precancerous or cancer (and/or a proliferative disorder) cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from mammals including humans.
  • Biological samples also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes.
  • a biological sample can be obtained using commonly employed clinical practices (e.g. fine needle biopsy, fluid aspirate, blood from a blood draw, serum or plasma derived from blood, tumor sections, circulating tumor cells and the like).
  • a biological sample can be a "control” or a "control sample” or a "test sample”.
  • a "control” refers to a representative of healthy, cancer-free biological subject or information obtained from a different individual or a normalized value, which can be based on baseline data obtained from a population or other acceptable sources.
  • a control also can refer to a given level of DDR1, representative of the cancer- free population, that has been previously established based on measurements from normal, cancer- free animals.
  • a control also can be a reference data point in a database based on data obtained from control samples representative of a cancer-free population. Further, a control can be established by a specific age, sex, ethnicity or other demographic parameters. In some situations, the control is implicit in the particular measurement.
  • control sample refers especially to a sample of biological material representative of healthy, cancer- free animals or a normal biological subject obtained from a cancer- free population.
  • the level of DDR1, in a control sample is desirably typical of the general population of normal, cancer-free animals of the same species.
  • This sample either can be collected from an animal for the purpose of being used in the methods described in the present invention or it can be any biological material representative of normal, cancer-free animals suitable for use in the methods of this invention.
  • a control sample also can be obtained from normal tissue from the animal that has cancer or is suspected of having cancer.
  • test sample refers especially to a biological sample, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDR1.
  • a test sample also includes biological samples containing or suspected of containing precancerous or cancer cells or tissues.
  • a test sample also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes.
  • Providing a sample, a biological sample, or a test sample means to obtain from a subject a sample, in vivo, ex-vivo, or in situ, including tissue or cell sample for use in the methods described in the present invention. Most often, this will be done by removing a sample of cells from an animal, but also can be accomplished in vivo, ex-vivo, or in situ, or by using previously isolated cells (for example, isolated from another subject, at another time, and/or for another purpose).
  • Data includes, but is not limited to, information obtained that relates to "biological sample,” “test sample.” “control sample,” and/or “control,” as described above, wherein the information is applied in generating a test level for diagnostics, prevention, monitoring or therapeutic use.
  • the present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper formats.
  • Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk; tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above.
  • “Overexpression” of a gene or an "increased,” or “elevated,” level of a ribonucleotide or protein refers to a level of the gene, ribonucleotide or polypeptide that, in comparison with a control level of gene, ribonucleotides or polypeptide, is detectably (preferably statistically significantly) higher. Comparison may be carried out by statistical analyses on numeric measurements of the expression; or, it may be done through visual examination of experimental results by qualified researchers.
  • a level of ribonucleotide or polypeptide, that is "expected" in a control sample refers to a level that represents a typical, cancer-free sample, and from which an elevated, or diagnostic, presence of the polypeptide or polynucleotide, can be distinguished.
  • an "expected” level will be controlled for such factors as the age, sex, medical history, etc., of the mammal, as well as for the particular biological subject being tested.
  • phrases "functional effects" in the context of an assay or assays for testing compounds that modulate a particular gene's activity includes the determination of any parameter that is indirectly or directly under the influence of the gene, for example, a functional, physical, or chemical effect, for example, of the genes activity, activity of a polypeptide encoded by the gene, the ability to induce gene amplification or overexpression in cancer cells, and to aggravate cancer cell proliferation.
  • “Functional effects” include in vitro, in vivo, and ex vivo activities.
  • Determining the functional effect refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of the gene or the polypeptide encoded by the gene, for example, functional, physical, and chemical effects.
  • Such functional effects can be measured by any means known to those skilled in the art, for example, changes in spectroscopic characteristics (for example, fluorescence, absorbance, refractive index), hydrodynamic (for example, shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the gene, measuring binding activity or binding assays (for example, substrate binding, and measuring cellular proliferation), measuring signal transduction, or measuring cellular transformation.
  • the present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper format.
  • Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk, tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above.
  • the data of this embodiment is information regarding the results of the analysis of DDR1.
  • the compounds, targets, assays, tests, inquiries and methodologies described herein can be employed in a variety of contexts, including diagnostic and therapeutic discovery, diagnostic and therapeutic development, safety and efficacy monitoring, compound and treatment regimen potency determination and validation, treatment assessment, comparative studies, marketing and the like.
  • the information provided by the invention can be communicated to regulators, physicians and other healthcare providers, manufacturers, owners, investors, patients, and/or the general public. This information and the like can be used in exploratory research, pre-clinical and clinical settings, labeling, production, advertising, and sales, for example.
  • the present invention relates to a kit comprising the antibody as provided and described herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, the host and/or the composition as described herein.
  • the kit comprises the antibody as provided and described herein.
  • the kit is used for the diagnosis of cancer (and/or a proliferative disorder).
  • said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer.
  • said cancer is epidermoid cancer orendometrial cancer.
  • the kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s).
  • said instruction manual(s) may guide the skilled person (how) to diagnose cancer (and/or a proliferative disorder) in accordance with the present invention.
  • said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.
  • the kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention.
  • substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the expression level of DDR1 as defined herein.
  • Figure 1 shows the results of a immunofluorescent staining of DDRl-ab2 with a fluorescent Cy2 labeled secondary anti -human antibody on DDR1 overexpressed cells from clone 44 which have been previously incubated with DDRl-ab2 and fixed at different times.
  • the pictures show the different results when the staining is done under permeable (bottom row), or non permeable conditions (top row) (see Example 7).
  • Top row the staining under non-permeable conditions visualizes the DDRl-ab2 on the outer cell surface.
  • Bottow row the staining under permeable conditions visualizes the efficient internalization of DDRl-ab2 on outer cell surface towards the inside of the cell.
  • Figure 2 shows the results of cell proliferation assays for the indicated cells treated with DDR1- ab2 and a secondary antibody to human IgG carrying saporin.
  • the two cells types are CHO (o) and a CHO line overexpressing DDR1(B) (Clone 44) (see Example 8).
  • Figure 3 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-abl (white diamond) or DDRl-abl plus a secondary antibody to human IgG carrying saporin ( ⁇ ).
  • Figure 4 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-ab3 ( ⁇ ) or DDRl-ab3 plus a secondary antibody to human IgG carrying saporin ( ⁇ ).
  • Figure 5 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-ab2 (white triangle) or DDRl-ab2 plus a secondary antibody to human IgG carrying saporin ( ⁇ ). (see Example 10).
  • Figure 6 shows the results obtained in an in vivo tumor model in mice with DDRl-ab2-tox, see Example 12.
  • Figure 7 shows the results obtained with DDRl-ab2-tox an in vivo model of AN3CA human endometrial tumor in mice (see Example 13) Examples
  • DDR1 is overexpressed in several types of cancers.
  • DDR1 has been shown to be upregulated in endometrial cancer primary tissue as compared to normal tissue by a microarray experiment and further studies using RT-PCR demonstrated that DDR1 was overexpressed in endometrial cancer tissue as compared to normal tissue. Studies also demonstrated DDR1 was overexpressed in endometrial cancer tissue at the protein level as compared to normal tissue. See WO201 1/009637.
  • DDR1 is significantly over-expressed in human malignant glioma, breast, colon, ovarian, lung, esophageal, and brain cancers (see e.g., Turashvili et al. (2007) BMC Cancer.
  • DDR1 is also reported to be overexpressed in injured arteries and has been implicated in additional diseases such as inflammation (Hachehouche et al. (2010) Mol Immunol.;47(9): 1866- 9); cirrhotic liver (Song et al. (2011) Am J Pathol. 178(3): 1134-44.), pulmonary fibrosis , pulmonary fibrosis (C Avivi-Green et al (2006) Am J Respir Crit Care Med, 174(4), 420-27), pituitary adenoma (Yoshida et al. (2007) J. Neuro-Oncol. 82:29-40), congestive heart failure (Andersson et al. (2006) Acta Physiol.
  • Monoclonal antibodies to DDR1 can be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495.
  • a host animal e.g., mammal
  • an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent (or the lymphocytes may be immunized in vitro).
  • the immunizing agent will typically include the target protein polypeptide DDRl (or fragment thereof) or a fusion protein thereof or epitope thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
  • HGPRT medium hypoxanthine, aminopterin, and thymidine
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against DDRl target protein or epitope.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem. 107:220.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No.
  • non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • This example sets out a procedure for humanization of a murine anti-DDRl antibody. Design of Genes for Humanized DDR1 Antibody Light and Heavy Chains
  • VL and VH amino acid sequences for murine antibodies are known or can be determined using standard molecular biology techniques (e.g., from a monoclonal antibody as prepared according to Example 2 or commercially available hybridomas encoding such an antibody).
  • the sequence of a human antibody identified using the National Biomedical Foundation Protein Identification Resource or similar database can be used to provide the framework of the humanized antibody.
  • the murine heavy chain sequence is aligned with the sequence of the human antibody heavy chain.
  • the human antibody amino acid is selected for the humanized sequence, unless that position falls in any one of four categories defined below, in which case the murine amino acid is selected: (1) The position falls within a complementarity determining region (CDR), as defined by Kabat (1980) J. Immunol. 125:961-969;
  • CDR complementarity determining region
  • the human antibody amino acid is rare for human heavy chains at that position, whereas the murine amino acid is common for human heavy chains at that position;
  • the murine light chain sequence is aligned with the sequence of the human antibody light chain.
  • the human antibody amino acid is selected at each position for the humanized sequence, unless the position again falls into one of the categories described above and repeated below:
  • the actual nucleotide sequence of the heavy and light chain genes is selected as follows:
  • nucleotide sequences code for the amino acid sequences chosen as described above;
  • nucleotide sequences code for a leader (signal) sequence.
  • leader sequences are chosen as typical of antibodies;
  • nucleotide sequences are the sequences that follow the mouse light chain J5 segment and the mouse heavy chain J2 segment, which are part of the murine sequence. These sequences are included because they contain splice donor signals;
  • a specific restriction site e.g., Xba I site
  • Xba I site e.g., Xba I sites
  • cloning into the restriction site e.g., Xba I site
  • the genes encoding the humanized light and heavy chain genes can be prepared by any method.
  • One method involves annealing fragments of the gene together to synthesize the full length genes.
  • oligonucleotides are synthesized using a DNA synthesizer (e.g., Applied Biosystems 380B DNA synthesizer). Two of the oligonucleotides are part of each strand of the heavy chain, and each oligonucleotide overlaps the next one by about 20 nucleotides to allow annealing. Together, the oligonucleotides cover the entire humanized heavy chain variable region with a few extra nucleotides at each end to allow cutting at the restriction site (e.g., Xba I sites). The oligonucleotides are purified from polyacrylamide gels.
  • a DNA synthesizer e.g., Applied Biosystems 380B DNA synthesizer.
  • oligonucleotide is phosphorylated using ATP and T4 polynucleotide kinase by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).
  • TA e.g., 33 mM Tris acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate
  • the complete gene can be synthesized from the oligonucleotides by synthesizing the opposite strand of each oligonucleotide, the following components are added in a final volume of 100 ⁇ :
  • Construction of plasmids to express humanized light and heavy chains can be accomplished, - e.g., by isolating the light and heavy chain Xba I fragments from the pUC19 plasmid in which it had been inserted and then inserting it into the Xba I site of an appropriate expression vector which will express high levels of a complete heavy chain when transfected into an appropriate host cell.
  • the expression vectors are transfected into mouse Sp2/0 cells, and cells that integrate the plasmids are selected on the basis of the selectable marker(s) conferred by the expression vectors by standard methods.
  • supernatant from the cells are incubated with cells that are known to express DDR1. After washing, the cells they can be, e.g., incubated with fluorescein-conjugated goat anti-human antibody, washed, and analyzed for fluorescence on a FACSCAN cytofluorometer.
  • the cells producing the humanized antibody are cultured in vitro.
  • Humanized antibody is purified to substantial homogeneity from the cell supernatants by passage through an affinity column of Protein A (Pro-Chem. Inc., Littleton, Mass. or equivalent) according to standard techniques.
  • the affinity of the humanized antibody relative to the original murine antibody is determined according to techniques known in the art.
  • Example 4 Selection of Anti-DDRl protein Fabs and IgGs via phage display
  • Phage display libraries can be used to identify antibodies against a target of interest.
  • a phage display library is a library of genetically engineered phage (virus that can infect bacteria).
  • the phage library is engineered in such a way that it encodes a diversity of antibodies, single chain antibodies or fragments thereof which are expressed in a bacteriophage library as the form of fusions with the bacteriophage coat protein where the antibodies, single chain antibodies or fragments thereof are displayed on the surface of the phage.
  • Each individual phage corresponds to and displays an antibody, single chain antibody or fragment thereof that corresponds to the genetic sequence within the phage.
  • Phage with specific binding properties can be isolated and the gene for the antibody can be sequenced, cloned or otherwise isolated.
  • the phage-display library (e.g., Fab or scFv) is added to a microtiter plate containing the DDR1 target protein or DDR1 epitope and incubated for a length of time. After incubation for a time sufficient to allow phage to bind to the target protein or epitope, the dish is washed. Phage- displayed antibodies that bind with the target protein or epitope remain attached to the dish, while the other phage are washed away. Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant (i.e. non-binding phage) than were present in the initial mixture.
  • the DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.
  • the Fab region can be cloned onto vectors expressing IgGs, such IgGl , IgG4 or IgG2a, to express a complete IgG in eukaryotic cells, such as pBhl , pBh4 or pBm2a (Dyax).
  • IgGs such as IgGl , IgG4 or IgG2a
  • eukaryotic cells such as pBhl , pBh4 or pBm2a (Dyax).
  • a DDR1 overexpressed cell line to be used on the selection of anti DDR1 Fab fragments on cells was constructed by routinary techniques.
  • the plasmid IOH5763 (Invitrogen) encoding the cDNA sequence of human DDR1 (sequence shown below) was cloned on the eukaryotic plasmid pEF5_FRT_V5-DEST from Invitrogen.
  • a transient transfection using liposomes was done in order to introduce the plasmid onto the Flpin Cho cell line (Invitrogen).
  • a plasmid able to express the enzyme recombinase once inside a eucariotic cell was co-transfected with the vector pEF5_FRT_V5-DEST incorporating the DDR1 cDNA.
  • the antibiotic Hygromicin was added to the cell culture media 48 hours after transfection.
  • the advantage of Flpin system is that the sequence of interest is not incorporated into the cell genome at random but does so at a particular place, avoiding changes in expression patterns from different clones due to position effects.
  • Transfected cells were kept on antibiotic media (Hygromycin) for several weeks in order to select stable overexpressing clones.
  • Antibiotic resistant clones were tested by different techniques such as immunofluorescence and western blot with commercial antibodies from Santa Cruz : anti-DDRl C-20 in order to choose the ones which showed higher expression of DDR1.
  • the selected clone was named Clone 44.
  • DDRl-abl Using the above described method, the following 3 antibodies, designated DDRl-abl, DDR1- ab2 and DDRl-ab3, were isolated. Their CDRs and variable sequences are listed below. Vectors encoding these 3 antibodies designated DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively, were deposited on December 20, 201 1 with the depositary institute DSMZ (Braunschweig, Germany) under deposit numbers DSM 25529, DSM 25530 and DSM 25531.
  • This antibody has the following CRD sequences:
  • SEQ ID No. 2 Amino acid sequence of VL_CDR1_ DDRl -ab l
  • SEQ ID No. 8 Amino acid sequence of VH CDR1 DDR1 -ab 1
  • SEQ ID No. 10 Amino acid sequence of VH_CDR2_DDRl -ab l : VIGPSGGHTYYADSV
  • SEQ ID No. 12 Amino acid sequence of VH_CDR3_DDRl -abl
  • EGITGISAAFDI This antibody has the following variable L and H region sequences:
  • SEQ ID No. 38 Amino acid sequence of VL DDRl -ab l
  • SEQ ID No. 40 Amino acid sequence of VH DDRl -abl
  • This antibody has the following CRD sequences: SEQ ID No. 14: Amino acid sequence of VL_CDR1_ DDRl -ab2
  • SEQ ID No. 16 Amino acid sequence of VL_CDR2_DDRl -ab2
  • SEQ ID No. 18 Amino acid sequence of VL_CDR3_DDRl -ab2
  • SEQ ID No. 20 Amino acid sequence of VH_CDRl_DDRl -ab2
  • SEQ ID No. 22 Amino acid sequence of VH_CDR2_DDRl -ab2
  • SIGPSGGHTSYADSV SEQ ID No. 24 Amino acid sequence of VH_CDR3_DDRl -ab2
  • This antibody has the following variable L and H region sequences:
  • SEQ ID No. 42 Amino acid sequence of VL_DDRl -ab2 LTQPPSASGPPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIDSNNQRPSGVP DRFSGSKSGTSASLAIGGLQSEDEADYYCAVWDDSLSVPVFGGGTKLT
  • SEQ ID No. 44 Amino acid sequence of VH_DDRl -ab2
  • This antibody has the following CRD sequences:
  • SEQ ID No. 26 Amino acid sequence of VL_CDR1_ DDRl -ab3
  • RASQSISSYLN SEQ ID No. 28: Amino acid sequence of VL_CDR2_DDRl -ab3
  • SEQ ID No. 30 Amino acid sequence of VL_CDR3_DDRl -ab3
  • SEQ ID No. 32 Amino acid sequence of VH_CDRl_DDRl -ab3
  • SEQ ID No. 34 Amino acid sequence of VH_CDR2_DDRl -ab3
  • SEQ ID No. 36 Amino acid sequence of VH_CDR3_DDRl -ab3
  • This antibody has the following variable L and H region sequences: SEQ ID No. 46: Amino acid sequence of VL_DDRl -ab3
  • SEQ ID No. 48 Amino acid sequence of VH_DDRl -ab3
  • siRNAs small interfering R A technique
  • the level of DDRl protein detected by flow cytometry in the original CHO cell was compared to the level of protein detected in the same cell line overexpressing DDRl protein (Clone 44).
  • Clone 44 cells were transiently transfected with a siRNA control or a siRNA which blocked the expression of DDRl (s2298, Ambion) and labelled with the antibodies described herein. Briefly, cells were plated on 6 well plate dishes and transiently transfected with lipofectamine 2000 with the corresponding siRNA. Cell were transfected at 70-80% confluence with 50 pmoles of siRNA per well for two consecutive days. 48 hours after the second transfection, the expression of cell surface DDRl was evaluated by flow cytometry.
  • DDRl_ab3 0.10% 92.56% 17.96% 77.66% 14.00%
  • the signal detected with the antibodies of the invention is drastically reduced when Clone 44 cells were analyzed 48 hours after transfection with siRNA against DDR1.
  • Clone 44 cells were plated on 6 well plates. Once the cells were seed, cells were serum starved for a day prior incubation with the antibodies. The antibody of interest was incubated on live cells at 37C for an hour before stimulating the cells with lOug/ml rat tail collagen type I at different times in order to promote DDR1 internalization. After collagen treatment, cells were washed with PBS to remove all traces of collagen and incubated directly with a goat anti-human secondary antibody conjugated to cyanine dye Cy2 at 4C in order to detect the amount of anti- DDR1 antibodies that remained on the cell surface. To stop internalization, the incubation with the secondary antibody was done at 4C which will slow down any natural cell process. Cells were then harvested in PBS /1%FBS and the remaining DDR1 on plasma membrane was measured by flow cytometry in order to test any differences due to collagen treatment. A IgG which does not specifically bind to DDRl was used as a control.
  • the signal detected on the cell surface of unstimulated cells using the antiDDRl antibodies is compared with the signal detected upon collagen stimulation, we see that the longer the stimulation with collagen, the lower is the detected signal on the cell surface. This indicates that once the antibody binds to DDRl, the complex DDRl -antibody internalizes inside the cells upon collagen stimulation. The internalization process seems to be faster for DDRl_ab2 than for the DDRl abl or DDRl ab3.
  • Example 7 Antibodies of invention are able to internalize without collagen stimulation
  • Cells from Clone 44 were plated onto coverslips with a complete growth media suplemented with 10% serum and left at 37C and 5% C02. After 24 hours DDRl_ab2 was added onto live cells and was left for different periods of time from 5 minutes to 30 minutes to allow for antibody binding and internalization. After that, cells were fixed with 4%PFA (paraformaldehide) .
  • an immunofluorescence assay was performed.
  • the coverslips with the fixed cells were incubated with a goat anti-human antibody coupled with the fluorochrome Cy2 under permeable and non- permeable conditions.
  • this Cy2-coupled secondary antibody is not able to go through the plasma membrane, so only the anti-DDRl antibodies localized outside the plasma membrane will be detected.
  • the secondary antibody will be able to detect not only the primary antibodies outside cells but also the ones that could have been internalized.
  • the dots inside the cells correspond to endosomes.
  • the antibodies of the invention internalized inside cells which express DDR1 on the plasma membrane, as shown in Examples 6 and 7.
  • the following assay shows these antibodies can be used as carriers to introduce inside cells conjugates or bound agents such as toxins and thus are able to work as immunotoxins.
  • Alamar Blue was used to measure the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability— and cytotoxicity. Measure of Alamar Blue is proportional to the number of living cells and corresponds to the cells metabolic activity.
  • Cells were incubated with serial dilutions of the isolated antibodies of the invention with a goat anti-human IgG secondary antibody conjugated to saporin.
  • Saporin is a ribosome-inactivating protein which comes from the seeds of the plant Saponaria officinali. Once inside the cells Saporin causes inhibition of protein synthesis which leads to cell death. Recognition and internalization of the primary antibody thus results in delivery of the saporin-antibody complex to the cell interior followed by cell killing.
  • Cells from Clone 44 or control cell line CHO were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, serial dilutions of the antibodies to be tested were added to the corresponding well, from 10 ⁇ 6 to 10 ⁇ 13 M.
  • the antibodies were added in combination with a human secondary antibody coupled to the toxin Saporin, the Hum-ZAP secondary antibody from Advanced Targeting Systems.
  • This Hum-ZAP is a chemical conjugate of a purified goat anti-human IgG and the ribosome-inactivating protein, saporin.
  • the secondary antibody was used at a constant concentration of 100 ng per well. 72 hours after the addition of the antibodies, an Alamar Blue® assay was performed.
  • AlamarBlue® (Invitrogen) is a proven cell viability indicator that uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin.
  • the active ingredient of AlamarBlue® (resazurin) is a nontoxic, cell permeable compound that is blue in color and virtually nonfluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces very bright red fluorescence. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability— and cytotoxicity.
  • AlamarBlue® was added to the cells and after 4 hours incubation fluorescence signal was read with an spectrophotometer.
  • the amount of fluorescence is proportional to the number of living cells and corresponds to the cells metabolic activity. Damaged and nonviable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells.
  • Antibodies DDRl-abl and DDRl-ab3 were also tested in this assay and exhibited an EC50 value of 6.35 nM and 26 nM, respectively.
  • the antibodies of the invention have excellent internalization properties, which makes them particularly suitable for use in antibody-drug conjugate (ADC) therapy to introduce drugs such as toxins into cells, like cancer cells.
  • ADC antibody-drug conjugate
  • Cells from Clone 44 or control cell line CHO were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, different concentrations of the antibodies to be tested (as indicated in table 3 below) were added to the corresponding well. The antibodies were added in combination with an appropriate secondary antibody coupled to Saporin, as indicated below. No secondary antibody is required for DDRl-abl-tox since the antibody is conjugated to saporin.
  • the secondary antibody was used at a constant concentration of 100 ng per well. 72 hours after the addition of the antibodies, an Alamar Blue® assay was performed and the proliferation rate was determined. The results obtained using clone 44 are shown in table 3, below. No effects on proliferation were observed with any of the tested antibodies in the DDRl -negative control cell line, CHO.
  • the antibodies of the invention can be used to identify cell lines which express DDR1. This assay is performed as follows:
  • DDRl-abl DDRl-ab2 or DDRl-ab3
  • PBS/1 %FBS PBS/1 %FBS
  • a goat anti-human IgG Cy2 lallebed antibody Signal was detected using a flow cytometer. The experiment was done in no-permeabilized cells so the levels of DDR1 detected correspond to the plasma membrane protein.
  • Tables 4 and 5 show different human cancer cell lines that were found to express DDR1.
  • Table 4 Expression level of DDR1 detected in different human endometrial cancer cell lines
  • (+++) strong signal detection which means high expression levels of DDRl at the membrane surface.
  • the cell line express different isoforms of DDRl which show different binding affinities for the described antibodies 3) Because DDR1 could be present on cells as a monomer or as a dimer, the described antibodies could have different affinities for the monomer or the dimer of DDR1 or even be able to recognise only one the two aspects of DDR1. So the different signals detected upon cell for the three antibodies could indicate distinct amounts of monomer and dimer present on the surface of specific cells.
  • Example 10 The isolated antibody is used to target different cell lines which express DDR1.
  • the isolated antibody is used to introduce a toxin inside DDR 1 -expressing cells and inhibit cell proliferation.
  • a further proliferation assay was done in different human cancer cell lines.
  • Table 6 shows the expression level of DDR1 detected on the indicated cell line with each antibody (column 4, corresponding to the same data shown in Example 9) and the corresponding proliferation rate obtained after treating the cells with the antibody plus the Hum-ZAP for 72 hours (shown in column 5). The lower the proliferation, the higher is the effect of the antibody upon cell proliferation inhibition.
  • the cell line U87MG was used as a negative control to prove that the effect upon cell proliferation is due to the use of DDR1 antibodies. Since U87MG does not express DDR1, no effect on cell proliferation is observed when cells are incubated with the antibodies.
  • a further proliferation assay was done in human endometrial adenocarcinoma AN3 CA cell line. Cells were incubated with serial dilutions of the three isolated antibodies DDRl -abl , DDRl -ab2 and DDRl -ab3 with or without a goat anti-human IgG secondary antibody conjugated to saporin.
  • Cells from the endometrial cell line AN3 CA were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, serial dilutions of the tested antibodies were added to the corresponding well. The antibodies were added alone or in combination with a human secondary antibody coupled to the toxin Saporin (hum-ZAP, ATSBio). The secondary antibody was used on a constant concentration of l OOng per well of a 96 well plate. 72 hours after the addition of the antibodies an Alamar Blue assay was performed on the plates to check cell viability, following the procedure disclosed in Example 8. The results obtained with DDRl -abl , DDRl -ab2 and DDR1 - ab3 are shown in Figures 3 to 5.
  • EC50 values of 1.46, 2.47 and 1.34 nM were obtained for DDRl-abl, DDRl-ab3 and DDRl-ab2, respectively, indicating potent cell proliferation inhibitory activity.
  • the above data further show that the antibodies of the invention can be used as carriers of a therapeutic agent such as a toxin into DDR1 -expressing cells and inhibit proliferation of said cells.
  • Antibodies of the invention can be conjugated to toxins for use in therapy using any of the conjugation procedures available in the art.
  • Antibodies DDRl-abl , DDRl-ab2 and DDRl-ab3 were conjugated to saporin using Advanced Targeting Systems technology for conjugation to saporin.
  • the corresponding Ab-saporin conjugates are designated DDRl-abl-tox, DDRl-ab2-tox and DDRl-ab3-tox.
  • Example 12 Anti-tumor effects in a human epidermoid cancer model in mice
  • mice An A431 Human Epidermoid tumor model in mice was used to evaluate the anticancer activity of DDRl-ab2, administered as a conjugate with the toxin saporin (DDRl-ab2-tox, see example 11).
  • mice were s.c. injected in Balb lc nude mice (6-8-week-old, females) on the lower back on day 0. On day 7, mice were randomized and divided into control group (group 1 , 10 animals) and treatment group (group 2, DDRl-ab2-tox, 9 animals). Six 100 ⁇ i.v injections were given to each group on days 7, 8, 9, 12, 15, 18 at the doses specified: group 1 : PBS alone, group 2: 250 ⁇ g/kg ab2_DDRl-tox.
  • Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was
  • V 0.5 a x b , where a and b are the long and short diameters of the tumor, respectively.
  • a and b are the long and short diameters of the tumor, respectively.
  • Example 13 Anti-tumor effects in a human endometrial cancer model in mice
  • mice An AN3CA human endometrial tumor model in mice was used to evaluate the anticancer effects of DDRl-ab2, administered as a conjugate with the toxin saporin (ab2-DDRl-tox, see example 11). Briefly, 5xl0 6 cells were s.c injected in Balb/c nude mice (6-8-week-old females) on the lower back on day 0. On day 12, mice were randomized and divided into control group (15 animals) and treatment group (DDRl-ab2-tox, 12 animals). Eight 100 ⁇ i.v injections were given to each group on days 13, 14, 15, 17, 19, 21 ,23,25 at the doses specified: group 1 : PBS alone, group 2: 250 ⁇ g/kg ab2-DDRl-tox. Primary tumors were measured twice weekly. On day 26 primary tumors were removed from all mice. The results of the change in tumour weight are shown in Figure 7.
  • Erythrocytes and mononuclear cells were isolated from 20 tubes with 10 ml of human whole blood. Histopaque 1077 (Sigma 10771) was used for a density gradient separation. 3 ml of Histopaque 1077 and 3 ml blood were mixed. After 30 minutes of centrifugation at 300g separate pools were obtained for mononuclear cells and erythrocytes. Subsequently, successive washings were performed with PBS. The erythrocytes were resuspended en 4ml of PBS and divided in 4 tubes (concentrated RBC). Each tube was diluted with 19 mL of PBS to obtain the RBC working solution.
  • % Hemolysis 100 * (OD sample-OD negative control) / (OD Total - OD negative control)
  • OD Total is the maximum OD of positive control hemolysis.
  • group 1 PBS alone
  • group 2 250 ⁇ g/kg DDRl-ab2-tox
  • group 3 50 ⁇ g/kg DDRl-ab2-tox
  • group 4 50 ⁇ g/kg DDRl-ab3-tox
  • group 5 250 ⁇ g/kg DDR1- ab3-tox
  • group 6 30 mg/kg DDRl-ab2
  • group 7 30 mg/kg DDRl-ab3.
  • Mouse weight and overall health were monitored every day. Animals were sacrificed at 14 days post- administration. Blood samples and major organs (liver, spleen, kidney, mesenteric ganglia) were collected.
  • Hematological study (hemoglobin content, RBC, WBC count) and biochemical analysis (AST, ALT, Creatinin, Total Protein) were examined as well as histopathological analysis of the major organs. No significant differences were found between the control and treated animals.
  • Flow cytometry is a technique that allows measuring certain physical and chemical characteristics of cells or microscopic particles as they pass in a fluid stream by a beam of laser light. Millions of cells can be analyzed by staining their proteins with specific fluorescent antibodies. Antibody staining of cell membrane proteins and its analysis by flow cytometry is the best way to characterize cell populations. A major application is to separate cells according to subtype or epitope expression for further biological studies. This process is called cell sorting or FACS. Other interesting applications are detection of protein expression levels, comparation of membrane protein levels, monitorization of internalization of receptors or even studies of antibody affinities and specificities. See e.g., Handbook of Flow Cytometry Methods by J. Paul Robinson, et al. ISBN 0471596345.
  • Example 5 The results of studies with various cell lines and various antibodies of the invention disclosed in Example 5, 6 and 9 show that flow cytometry can be used to detect the binding of anti-DDRl antibodies of the invention to DDR1 to measure DDR1 protein expression levels.
  • Enzyme-linked immunosorbent assay is a plate -based technique designed for detecting and quantifying substances such as peptides, proteins, antibodies or hormones using specific labeled antibodies.
  • the antigen must be immobilized to a solid surface, usually a polystyrene microtiter plate, either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich” ELISA). If the antigen is in a live cell, this can be growth in the same place where they are going to be detected with a specific antibody.
  • the detection requires using a specific fluorescent or covalently linked to an enzyme antibody that is applied over the antigen coated surface of the plates.
  • the most commonly used enzyme labels horseradish peroxidase (HRP) and alkaline phosphatase (AP).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • the detection antibody is biotin labeled a secondary antibody linked to a protein such as streptavidin is required. Therefore the assay combines the specificity of antibodies with the sensitivity of simple enzyme assays or fluorescence intensity signal.
  • the ELISA is very useful as a diagnostic tool in basic research, medicine.
  • the present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.
  • QQANSFPLT SEQ ID No. 7 Nucleotide sequence encoding VH_CDRl_DDRl -abl ttcactttctcttattactggatgtgg
  • VH_CDR2_DDRl -abl VIGPSGGHTYYADSV
  • SEQ ID No. 49 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 1 , mRNA with the NCBI Reference Sequence: NM_001954.4
  • DDR1 discoidin domain receptor tyrosine kinase 1
  • variant 1 amino acid sequence with the NCBI Reference Sequence NP 001945.3
  • SEQ ID No. 51 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDR1), transcript variant 2, mRNA with the NCBI Reference Sequence: NM_013993.2
  • DDR1 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDR1), variant 2, amino acid sequence with the NCBI Reference Sequence:NP_054699.2
  • SEQ ID No. 53 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 3, mRNA with the NCBI Reference Sequence: NM_013994.2
  • SEQ ID No. 54 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 3, amino acid sequence with the NCBI Reference Sequence NP 054700.2
  • SEQ ID No. 55 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 4, mRNA with the NCBI Reference Sequence: NM_001202521.1 AGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCATCTTTAC
  • SEQ ID No. 56 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 4, amino acid sequence with the NCBI Reference Sequence: NP 001 189450.1
  • SEQ ID No. 57 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 5, mRNA with the NCBI Reference Sequence: NM_001202522.1
  • SEQ ID No. 58 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 5, amino acid sequence with the NCBI Reference Sequence NP 001 189451 .1
  • SEQ ID No. 59 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 6, mRNA with the NCBI Reference Sequence: NM_001202523.1
  • SEQ ID No. 60 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 6, amino acid sequence with the NCBI Reference Sequence NP 001 189452.1 MSLPRCCPHPLRPEGSGAMGPEALSSLLLLLLVASGDADMKGHF
  • SEQ ID No 61 corresponding to plasmid IOH5763 from Invitrogen corresponding to the cDNA sequence of human DDR1
  • SEQ ID No. 62 aminoacid sequence corresponding to plasmid IOH5763
  • SEQ ID No. 63 Epitope corresponding to the extracellular domain of human DDR1 :
  • Amino acid sequence of an inhibitory peptide (peptide inhibitor of the transcription factor STAT6) to be conjugated to the anti-DDRl antibodies
  • Amino acid sequence of an inhibitory peptide (phosphopeptide which inhibits the activity of the transcription factor STAT3 in vitro and in vivo) to be conjugated to the anti-DDRl antibodies
  • PYLKTK (Y represents a phosphotyrosine) SEQ ID No. 66:
  • Amino acid sequence of an inhibitory peptide (part of the N-terminal sequence of pl4ARF that is able to induce apoptosis) to be conjugated to the anti-DDRl antibodies MVRRFLVTLRIRRACGPPRVRV
  • SEQ ID No. 77 Nucleotide sequence of 5 '-primer for amplification of a second binding domain of vector DDRl -ab3pBhl

Abstract

The present invention relates to antibodies/binding molecules that specifically bind to discoidin domain receptor 1 (DDR1). The use of these antibodies/binding molecules in human and veterinary medicine, for example in the treatment and diagnosis of cancer, tumorous disease and/or proliferative disorder is also subject of the present invention. Further, compositions and kits comprising the antibodies/binding molecules are provided herein.

Description

ANTI-DDR1 INTERNALIZING ANTIBODIES AND THEIR MEDICAL USE
Field of the invention
The present invention relates to antibodies/binding molecules that specifically bind to discoidin domain receptor 1 (DDR1). The use of these antibodies/binding molecules in human and veterinary medicine, for example in the treatment and diagnosis of cancer, tumorous diseases and/or proliferative disorders is also subject of the present invention. Further, compositions and kits comprising the antibodies are provided herein. Background art
Discoidin domain receptor 1 (DDR1) is a Receptor Tyrosine Kinase (RTK) encoded by the DDR1 gene. In general, RTKs play a key role in the communication of cells with their microenvironment typically through a variety of signal transduction mechanisms where extracelluar signals are transduced into an intercellular response. A number of RTKs are involved in the regulation of cell growth, differentiation and metabolism.
The DDR1 protein belongs to a subfamily of tyrosine kinase receptors with a homology region to the Dictyostelium discoideum protein discoidin I in the extracellular domain (ECD). DDR1 is activated by various types of collagen and its autophosphorylation is stimulated by all collagens so far tested (type I to type VI). DDR1 is one of two non-integrin tyrosine kinase receptors activated by collagen. The interaction of DDR1 with collagen induces receptor activation and downstream signaling which regulates cellular processes such as cell adhesion, migration, differentiation, cytokine and chemokine production (see e.g., Mihai et al. (2009) J. Mol. Biol. 385(2):432-445 and Xu et al. (2011) Matrix Biology, 30(1), 16-26). The known functions of DDR1 include promotion of cell adhesion and migration on collagen matrices at least partially due to upregulation of matrix metallopro teases (MMPsl , 2 and 9), and modulation of cell proliferation (see Kamohara et al, 2001 FASEB J 15(14): 2724-6; Vogel et al, 2001 Mol. Cell Biol. 21(8):2906-17; Franco et al, 2010 Circ Res 106(11): 1775-83; Dejmek et al, 2003 Int J Cancer 103(3):344-51). In primary lung fibroblasts collagen stimulated DDR1 activation was reported to signal through a JAK-2 ERK1/2 mediated pathway (Ruiz et al. (2011) J Biol Chem.;286(15): 12912-23.)· DDRl is important for axon growth of cerebrellar granule neurons (Bhatt et al., 2000 Genes Dev. 14(17):2216-28) and for normal development of the mammary gland (Vogel et al, 2001 Mol. Cell Biol. 21(8):2906-17). DDRl expression and activation are triggered by apoptotic stimuli, and promote cell survival by a mechanism dependent on activation of MAPK and induction of p53 (Ongusaha et al., 2003 EMBO J. 22(6): 1289-301). DDRl is widely expressed in normal and transformed epithelial cells. In situ studies and Northern-blot analysis showed that expression of DDRl protein is restricted to epithelial cells, particularly in the kidney, lung, gastrointestinal tract, and brain. In addition, DDRl is significantly over-expressed in several human tumors including endometrial cancer (WO2011/009637), as well as malignant glioma, breast, colon, ovarian, lung, esophageal, and brain cancers (see e.g., Turashvili et al. (2007) BMC Cancer. 7:55; Yamanaka et al. (2006) Oncogene 25:5994-6002); Yang et al. (2010) 24(2):311-9; Nemoto et al. (1997) Pathobiology 65(4): 195-203; Johansson et al. (2005) Oncogene 24:3896-3905; Heinzelmann- Schwartz et al. (2004) Clin Cane. Res. 10:4427-4436). DDRl is also reported to be overexpressed in injured arteries and has been implicated in additional diseases such as inflammation (Hachehouche et al. (2010) Mol Immunol.;47(9): 1866-9), cirrhotic liver (Song et al. (2011) Am J Pathol. 178(3): 1134-44.), pulmonary fibrosis (C Avivi-Green et al (2006) Am J Respir Crit Care Med, 174(4), 420-27) pituitary adenoma (Yoshida et al. (2007) J. Neuro-Oncol. 82:29-40), congestive heart failure (Andersson et al. (2006) Acta Physiol. 186: 17-27), atherosclerosis (Ahmad et al. (2009) Am J Pathol.l75(6):2686-96; Franco et al.(2008) Circ Res.102(10): 1202-1 1), Alport syndrome (a hereditary type IV collagen disease which symptoms include renal inflammation and fibrosis; Gross et al. (2010) Matrix Biol. 29(5):346-56), obstructive nephropathy (Guerrot et al. (201 1) Am J Pathol. 179(1):83-91) and lymphangioleiomyomatosis (Ferri et al. (2004) Am. J. Pathol. 5: 1575-1585). Anti-DDRl antibodies have been proposed in the art in the treatment of various types of cancer in WO 2010/019702 and WO2013/034933; these prior art antibodies are not reported to have the capacity to internalize into cells. This capacity is highly advantageous, as it allows the internalization of antibodies that are coupled with a therapeutic agent (like, inter alia, a toxin) into cancer cells, thereby effectively targeting and killing tumor cells.
Known means and methods for treating or diagnosing cancer are in need of improvement. Thus, the technical problem underlying the present invention lies in the provision of means and methods for effective medical intervention in proliferative disorders, in particular in cancer. In particular, the invention seeks to solve the problem of providing antibodies against human DDRl that are suitable for use in Antibody-Drug Conjugate (ADC) therapy for the targeted delivery of cytotoxic agents into cancer cells. Anti-DDRl antibodies for use in ADC should exhibit high affinity and specificity for human DDRl , should be efficiently internalized into DDRl -expressing cells and exhibit in vivo antitumor activity at low doses when administered as an Antibody-Drug Conjugate (ADC).
The technical problem is solved by provision of the embodiments characterized in the claims.
Summary of the invention The present invention relates to antibodies or functional fragments or functional derivatives thereof specifically binding to discoidin domain receptor 1 (DDRl). The term "binding molecule" in accordance with this invention relates to functional fragments or functional derivatives of the herein disclosed and the herein defined antibodies. Accordingly, the present invention relates to antibodies/binding molecules specifically binding to discoidin domain receptor 1 (DDRl). In one embodiment, said antibodies specifically binding to DDRl are capable of internalizing into cells. In one embodiment, these antibodies, antibody fragments or antibody derivatives comprise the variable regions and/or CDRs as provided herein. The invention also provides for binding molecules/antibodies that comprise variable regions and/or CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the variable regions and/or CDRs as provided herein and that specifically bind to discoidin domain receptor 1. In one embodiment, the inventive antibodies/binding molecules comprise variable regions and/or CDRs as comprised in a (binding) molecule obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ablpBhl under accession number DSM 25529 with the depositary institute DSMZ; or obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ab2pBhl under accession number DSM 25530 with the depositary institute DSMZ; or obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDRl-ab3pBhl under accession number DSM 25531 with the depositary institute DSMZ. Said three vectors were deposited, as mentioned, under the designation DDRl-ablpBhl , DDRl-ab2pBhl and DDRl-ab3pBhl, respectively, by Oryzon Genomics S.A., with address at Sant Ferran 74, 08940 Cornelia de Llobregat, Spain, on December 20th, 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures ), InhoffenstraBe 7B, D-38124 Braunschweig GERMANY. The invention also provides for binding molecules/antibodies that comprise CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the CDRs as comprised in these molecules encoded by the nucleic acid molecule comprised in said deposited vectors and that specifically bind to discoidin domain receptor 1 (DDRl). The invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the variable regions as comprised in these molecules encoded by the nucleic acid molecule comprised in said deposited vectors and that specifically bind to discoidin domain receptor 1 (DDRl). Furthermore, the present invention also provides for antibodies that bind to/recognize the same epitope as any of the binding molecules obtainable upon expression of the nucleic acid molecule comprised in any of the vectors as described under the designation DDR1- ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively deposited by Oryzon Genomics S.A., Spain on December 20th, 201 1 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
It was shown herein that DDRl is overexpressed in a variety of cancer cells. As explained in more detail below, the present invention provides new anti-DDRl antibodies/binding molecules that have the unexpected property to internalize into cells and that allow therefore for an effective targeting of DDRl -expressing cells, such as DDRl -expressing epidermoid (also called squamous cell), endometrial, bladder, colon, breast, stomach, lung, pancreas, hepatic, and prostate cancer cells. This capacity to internalize is highly advantageous, as it allows the internalization of antibodies that are coupled with a therapeutic agent (like, inter alia, a toxin) into cancer cells, thereby effectively targeting and killing tumor cells. DDRl is known to internalize upon collagen binding/stimulation, i.e. collagen binding is believed to be essential for internalization of DDRl . Suprisingly, the anti-DDRl antibodies provided herein are capable of inducing internalization of DDRl in the absence of collagen stimulation (see, e.g., example 7). Moreover, not only DDRl is internalized upon antibody stimulation: the entire DDRl -antibody complex (optionally including therapeutic or diagnostic agents coupled to the antibody) is internalized. Thus, the antibodies provided herein are capable of specifically targeting DDRl -expressing cells and to effectively introduce therapeutic or diagnostic agents into such cells. Due to said specific targeting and destruction of DDR1- expressing cancer cells, a more effective cancer therapy as compared to conventional therapy is expected. A reduction of side-effects is also expected. In sum, the antibodies/binding molecules of the present invention are expected to have therapeutic utility in diseases where DDRl expression, internalization and/or activation is implicated. One of such disease is cancer and/or tumorous diseases, for example epidermoid (also called squamous cell), endometrial, bladder, colon, breast, stomach, lung, pancreas, hepatic, or prostate cancer. The antibodies/binding molecules of the present invention may also be useful in certain further proliferative disorders such as inflammatory disorders or atherosclerosis. Moreover, the anti-DDRl antibodies as provided herein may also be useful in the medical intervention of other disorders where DDR1 has been reported to play a role, like cirrhotic liver, pulmonary fibrosis, pituitary adenoma, congestive heart failure, Alport syndrome, obstructive nephropathy and lymphangioleiomyomatosis.
The present invention relates to the following items:
1. An antibody that specifically binds to discoidin domain receptor 1 (DDR1), wherein the variable region of the heavy chain of said antibody comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24, or a CDR sequence having 75% or more amino acid identity to said CDR.
2. The antibody of item 1 , wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, or a CDR sequence having 75% or more amino acid identity to said CDR.
3. The antibody of item 1 or 2, wherein the variable region of the heavy chain of said antibody comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, or a CDR sequence having 75% or more amino acid identity to said CDR.
4. The antibody of any one of items 1 to 3, wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24, or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
5. The antibody of item 4, wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24. The antibody of any one of items 1 to 5, wherein the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14, or a CDR sequence having 75% or more amino acid identity to said CDR. The antibody of any one of items 1 to 6, wherein the variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16, or a CDR sequence having 75% or more amino acid identity to said CDR. The antibody of any one of items 1 to 7, wherein the variable region of the light chain of said antibody comprises a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO: 18, or a CDR sequence having 75% or more amino acid identity to said CDR. The antibody of any one of items 1 to 8, wherein the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18, or a CDR sequence having 75% or more amino acid identity to one of said CDRs. The antibody of item 9, wherein the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18. An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. The antibody of item 1 1 ,
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-Hl region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24. An antibody that specifically binds to discoidin domain receptor 1 (DDRl),
wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43, or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VH-region; or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 , or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region. The antibody of item 13,
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 , or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 42, or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region. An antibody that specifically binds to discoidin domain receptor 1 (DDRl ), wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region or a variable VR- region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region; and
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region. The antibody of item 15,
wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44; and wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or
a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), wherein said antibody is DDRl-ab2 as encoded by a nucleic acid molecule comprised in vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1. The antibody of any one of items 1 to 17, wherein said antibody specifically binds to discoidin domain receptor 1 (DDRl), wherein the antibody is capable of internalizing into cells. The antibody of item 18, wherein the antibody is capable of internalizing into cells in the absence of collagen. The antibody of any one of items 18 or 19, wherein said cell is a DDRl expressing cell. The antibody of item 20, wherein said cell is a cancer cell. 22. The antibody of any one of items 1 to 21, wherein said antibody is a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment or a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a bispecific single-chain antibody, a synthetic antibody or a cross-cloned antibody.
23. The antibody of item 22, wherein said antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody. 24. The antibody of any one of items 1 to 23, wherein said antibody is conjugated to one or more therapeutic agents.
25. The antibody of item 24, wherein the therapeutic agent is a toxin. 26. The antibody of item 24, wherein the therapeutic agent is an anticancer agent.
27. A nucleic acid molecule having a sequence encoding the antibody as defined in any one of items 1 to 26. 28. A nucleic acid molecule comprised in a vector as defined in item 17.
29. A vector comprising a nucleic acid molecule according to item 27 or 28.
30. The vector of item 29, which further comprises a nucleic acid molecule having a regulatory sequence which is operably linked to said nucleic acid molecule according to item 27 or
28.
31. The vector of item 29 or 30, wherein the vector is an expression vector. 32. The vector of any one of items 29 to 31 , wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011.
33. A host transformed or transfected with a vector according to any of items 29 to 32. The host of item 33, wherein said host is a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell. A process for the production of the antibody as defined in any one of items 1 to 26, said process comprising culturing a host of item 33 or 34 under conditions allowing the expression of the antibody and recovering the produced antibody from the culture. A composition comprising the antibody as defined in any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 . The composition of item 36, further comprising a secondary antibody that is specifically binding to the primary antibody as defined in any one of items 1 to 26, whereby said secondary antibody is conjugated to a therapeutic agent. The composition of item 37, wherein said therapeutic agent is a toxin. The composition of item 37 or 38, wherein said primary antibody is an IgG antibody and said secondary antibody is a goat anti-human IgG secondary antibody. The antibody of item 25 or the composition of item 38 or 39, wherein said toxin is Saporin. The composition of any one of items 36 to 40 which is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s). The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for use in medicine. Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 for the preparation of a pharmaceutical composition for the treatment of cancer. The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for use in the treatment of cancer. 45. A method for the treatment of cancer comprising the administration of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 to a subject in need of such a treatment. 46. The method of item 45, wherein said subject is a human.
47. The use of item 43, the antibody of item 21 or 44, the nucleic acid molecule of item 44, the vector of item 44, the host of item 44, the composition of item 44, or the method of item 45 or 46, wherein said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer.
48. An antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in item 32.
49. An antibody obtainable by a process comprising culturing a host transfected or transformed with the vector as defined in item 28 under conditions that provide for the production of the antibody by the host and allow for the recovering of the antibody from the culture.
50. The composition of item 36, which is a diagnostic composition further comprising, optionally, means and methods for detection.
51. A method for diagnosing cancer, comprising detecting or assaying DDR1 in a biological sample of an individual suspected of suffering from cancer using the antibody according to any one of items 1 to 26.
52. Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 for the preparation of a diagnostic composition for the diagnosis of cancer. 53. The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 40 for use in the diagnosis of cancer.
54. Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 40 for the preparation of a diagnostic kit for the diagnosis of cancer.
55. Kit comprising the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41 and 50.
56. The use of item 52 or 54, the antibody of item 53, the nucleic acid molecule of item 53, the vector of item 53, the host of item 53 and/or the composition of item 53, wherein said cancer is epidermoid cancer (also known as squamous cell cancer) endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer.
57. An antibody having essentially the same biological activity of an antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in item 32.
58. An antibody that binds to the same epitope as an antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in item 32. 59. An antibody that specifically binds to discoidin domain receptor 1 (DDR1), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 using a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
Furthermore, the present invention relates to the following items:
1. An antibody that specifically binds to discoidin domain receptor 1 (DDR1), whereby the antibody is capable of internalizing into cells.
2. The antibody of item 1 , wherein the antibody is capable of internalizing into cells in the absence of collagen.
3. An antibody that specifically binds to discoidin domain receptor 1 (DDR1), wherein the variable region of the heavy chain of said antibody comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 , SEQ ID NO.: 24 or SEQ ID NO.: 36, or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
4. The antibody of item 3, wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , SEQ ID NO: 20 , or SEQ ID NO: 32 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
5. The antibody of any one of items 3 or 4, wherein the variable region of the heavy chain of said antibody comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 , SEQ ID NO: 22 , or SEQ ID NO: 34 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
6. The antibody of any one of items 3 to 5, wherein the variable region of the heavy chain of said antibody comprises
(a) a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
(b) a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 or a CDR sequence having 75% or more amino acid identity to one of said CDRs; or
(c) a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
The antibody of any one of items 3 to 6, wherein the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 , SEQ ID NO: 14 , or SEQ ID NO: 26 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
The antibody of any one of items 3 to 7, wherein the variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , SEQ ID NO: 16 , or SEQ ID NO: 28 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
The antibody of any one of items 3 to 8, wherein the variable region of the light chain of said antibody comprises a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO: 6 , SEQ ID NO: 18 , or SEQ ID NO: 30 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs.
The antibody of any one of items 3 to 9, wherein the variable region of the light chain of said antibody comprises
(a) a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , and a CDR- L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs;
(b) a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 or a CDR sequence having 75% or more amino acid identity to one of said CDRs; or
(c) a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 2 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 26 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 , or a CDR sequence having 75% or more amino acid identity to one of said CDRs. 14. An antibody that specifically binds to discoidin domain receptor 1 (DDRl),
wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 , SEQ ID NO:43 , or SEQ ID NO:47 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable Vn-regions; or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 , SEQ ID
NO:44 , or SEQ ID NO:48 , or a variable Vn-region having an amino acid sequence which has 75% or more identity to one of said variable Vn-regions.
The antibody of item 14,
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 , SEQ ID NO:41 , or SEQ ID NO:45 , or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to one of said variable VL-regions or
a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 , SEQ ID NO:42 , or SEQ ID NO:46 or a variable VL-region having an amino acid sequence which has 75% or more identity to one of said variable VL-regions.
An antibody that specifically binds to discoidin domain receptor 1 (DDRl ), wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region; and
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region. An antibody that specifically binds to discoidin domain receptor 1 (DDRl ), wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region; and
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.
An antibody that specifically binds to discoidin domain receptor 1 (DDRl ), wherein said antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:47 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:48 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region; and
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:46 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable VL-region.
An antibody that specifically binds to discoidin domain receptor 1 (DDRl), wherein said antibody is DDRl-abl as encoded by a nucleic acid molecule comprised in vector DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 201 1; or wherein said antibody is DDRl-ab2 as encoded by a nucleic acid molecule comprised in vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1; or
wherein said antibody is DDRl-ab3 as encoded by a nucleic acid molecule comprised in vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 201 1. The antibody of any one of items 1 or 2, wherein said cell is a DDR1 expressing cell. The antibody of item 20, wherein said cell is a cancer cell. The antibody of any one of items 1 to 21, wherein said antibody is a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment or a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a bispecific single-chain antibody, a synthetic antibody or a cross-cloned antibody. The antibody of item 22, wherein said antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody. The antibody of any one of items 1 to 23, wherein said antibody is conjugated to one or more therapeutic agents. The antibody of item 24, wherein the therapeutic agent is a toxin. The antibody of item 24, wherein the therapeutic agent is an anticancer agent. A nucleic acid molecule having a sequence encoding the antibody as defined in any one of items 1 to 26. A nucleic acid molecule comprised in any of the vectors as defined in item 19. A vector comprising a nucleic acid molecule according to item 27 or 28. 30. The vector of item 29, which further comprises a nucleic acid molecule having a regulatory sequence which is operably linked to said nucleic acid molecule according to item 27 or 28. 31. The vector of item 29 or 30, wherein the vector is an expression vector.
32. The vector of any one of items 29 to 31 ,
wherein said vector is DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 2011 ; or
wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 ; or
wherein said vector is DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011. 33. A host transformed or transfected with a vector according to any of items 29 to 32.
34. The host of item 33, wherein said host is a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell. 35. A process for the production of the antibody as defined in any one of items 1 to 26, said process comprising culturing a host of item or 34 under conditions allowing the expression of the antibody and recovering the produced antibody from the culture.
36. A composition comprising the antibody as defined in any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34.
37. The composition of item 36, further comprising a secondary antibody that is specifically binding to the primary antibody as defined in any one of items 1 to 26, whereby said secondary antibody is conjugated to a therapeutic agent.
38. The composition of item 37, wherein said therapeutic agent is a toxin.
39. The composition of item 37 or 38, wherein said primary antibody is an IgG antibody and said secondary antibody is a goat anti-human IgG secondary antibody. The antibody of item 25 or the composition of item 38 or 39, wherein said toxin is Saporin. The composition of any one of items 36 to 40 which is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s). The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for use in medicine. Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 for the preparation of a pharmaceutical composition for the treatment of cancer. The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for use in the treatment of cancer. A method for the treatment of cancer comprising the administration of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 to a subject in need of such a treatment. The method of item 45, wherein said subject is a human. The use of item 43, the antibody of item 44, the nucleic acid molecule of item 44, the vector of item 44, the host of item 44, the composition of item 44, or the method of item 45 or 46, wherein said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer. 48. An antibody obtained or obtainable by expression of the nucleic acid molecule contained in any of the vectors as defined in item 32.
49. An antibody obtainable by a process comprising culturing a host transfected or transformed with any one of the vectors as defined in item 32 under conditions that provide for the production of the antibody by the host and allow for the recovering of the antibody from the culture.
50. The composition of item 36, which is a diagnostic composition further comprising, optionally, means and methods for detection.
51. A method for diagnosing cancer, comprising detecting or assaying DDR1 in a biological sample of an individual suspected of suffering from cancer using the antibody according to any one of items 1 to 26.
52. Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 for the preparation of a diagnostic composition for the diagnosis of cancer. 53. The antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for use in the diagnosis of cancer.
Use of the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32, a host of item 33 or 34 and/or the composition of any one of items 36 to 41 for the preparation of a diagnostic kit for the diagnosis of cancer.
Kit comprising the antibody of any one of items 1 to 26 or as produced by the process of item 35, a nucleic acid molecule of item 27 or 28, a vector of any one of items 29 to 32 and/or a host of item 33 or 34 and/or the composition of any one of items 36 to 41.
56. The use of item 52 or 54, the antibody of item 53, the nucleic acid molecule of item 53, the vector of item 53, and/or the host of item 53, wherein said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer. An antibody having essentially the same biological activity of an antibody obtained or obtainable by expression of the nucleic acid molecule contained in any of the vectors as defined in item 32. An antibody that binds to the same epitope as an antibody obtained or obtainable by expression of the nucleic acid molecule contained in any of the vectors as defined in item 32. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 2011 using a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 67 and a 3 '-primer as shown in SEQ ID NO: 68 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 69 and a 3 '-primer as shown in SEQ ID NO: 70 for amplification of a second binding domain. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 using a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011 , using
a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 75 and a 3 '-primer as shown in SEQ ID NO: 76 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 77 and a 3 '-primer as shown in SEQ ID NO: 78 for amplification of a second binding domain.
Detailed description of the invention As used herein, the term "anti-DDRl antibody", "antiDDRl antibody", "antibody to DDR1", "DDR1 antibody" and "DDR1 binding molecule" are used interchangeably to refer to an antibody or a functional fragment or a functional derivative thereof specifically binding to DDR1 according to the invention. The "anti-DDRl antibodies or functional fragments or functional derivatives thereof bind specifically to the extracellular domain of DDR1. Within the scope of this invention are antibodies/binding molecules/antibody fragments and/or antibody derivatives that are functional in this binding capacity, whereby said molecules bind to the same epitope(s) as any of the binding molecules obtainable by the expression of the nucleic acid molecule comprised in a vector as deposited under the designation DDR-ablpBhl, DDR1- ab2pBhl and DDRl-ab3pBhl, respectively by Oryzon Genomics S.A., Spain on December 20th, 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures). As will be detailed herein, the term "antibody" is not limited to immunoglobulins, but also comprises, inter alia, antibody fragments, artificial antibodies, antibody derivatives, single chain antibodies (also bispeciflc single chain antibodies), diabodies, triabodies, etc. The term also relates to binding molecules that comprise CDRs or binding portions of the antibodies described herein.
As documented in the appended examples, antibodies of this invention are, inter alia, capable of internalizing into cells. The term "antibody is capable of internalizing into cells" means, in context of this invention, that the antibody has the capacity to induce internalization of the DDR1 protein. Antibodies, antibody fragments, antibody derivatives and/or corresponding binding molecules of this invention preferably have the functional feature being capable of "internalization into cells" as defined herein. As the internalization is believed to be induced upon binding of the antibody/antibody fragment/antibody derivative to DDR1 , the antibodies provided herein are "capable of internalizing upon binding to DDR1". This term means that the antibodies first bind to DDR1 and induce in a second step internalization. In other words, the antibodies activate the internalizing capacity of DDRl . Accordingly, the present invention provides antibodies/antibody fragments/antibody derivatives specifically binding to DDRl , whereby the antibodies are capable of being internalized. "Internalization" as used herein may refer to (i) the internalization of only DDRl (for example, if the DDRl -antibody-binding is dissolved after induction of internalization; this is thought to occur only in rare cases) or (ii) the internalization of the antibody-DDRl complex (for example, if the anti-DDRl -antibody remains bound to DDRl after induction of internalization). Accordingly, the antibodies of the present invention comprise antibodies that are capable of inducing the internalization of the antibody- DDRl complex , i.e. the antibodies have the capacity to penetrate inside a DDRl -expressing cell upon binding to DDRl . Therefore, the present invention relates to an antibody specifically binding to DDRl, whereby the antibody-DDRl complex is internalized. Such an antibody- DDRl complex may include further compounds or agents, such as therapeutic or diagnostic agents coupled to the antibody. In one embodiment, the antibody of the invention is a DDRl internalizing antibody. A DDRl internalizing antibody can bind cell surface DDRl and be internalized into the cell.
As mentioned, the antibodies of the present invention are advantageous as they do not necessitate the use of a ligand (like collagen) for the stimulation or induction of internalization. Nonetheless, internalization may be enhanced by the use of such ligands. Accordingly, the herein provided antibodies may internalize by or upon ligand stimulation (such as collagen) or the internalization may additionally be stimulated by a ligand (such as collagen). The term "ligand" as used herein refers in particular to compounds that differ from the antibodies of the present invention and are capable of inducing internalization as defined above. Preferably, such ligands induce or enhance internalization by binding or upon binding to DDRl . Exemplary ligands known in the art are type I, type II, type III, type IV, type V and/or type VI collagen.
The ability of an antibody to internalize can be determined by a variety of standard assays. For example, upon a signal that induces protein internalization, comparison between the protein level detected on the cell surface of unstimulated cells versus stimulated cells can be done in fixed cells or in vivo cells. The localization of the protein can be detected with a primary antibody which specifically binds the protein of interest and a secondary antibody coupled to a chromophore that recognises the primary antibody. The chromophore can then be detected for example by fluorescent microscopy or flow cytometry techniques.
The ability of an antibody to internalize can also be determined for example using the assays disclosed in Examples 6, 7 and 8 below. The herein provided antibodies specifically bind to discoidin domain receptor 1 (DDRl), preferably to DDRl of a mammal, most preferably to DDRl of a human. As known to the person skilled in the art and as also illustrated herein below, several discoidin domain receptor 1 (DDRl) isoforms are known. Currently, there are six human isoforms of DDRl described, each of which have the same extracellular domain. The antibodies of this invention bind to said extracellular domain of the DDRl receptor/polypeptide/protein as defined herein.
The term "specifically binding" means in accordance with this invention that the antibody/binding molecule is capable of specifically interacting with and/or binding to DDRl as defined herein below. Therefore, said term relates to the specificity of the antibody, i.e. to its ability to discriminate between DDRl and another, non-DDRl protein. A "non-DDRl protein" is to be understood as a protein that does not present any domain that is included in the DDRl extracellular region (for example the discoidin domain). Specificity can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Other methods include the use of siR A agents against DDRl . Such methods also comprise the determination of KD- values. As used herein, the term "antibody specifically binding to DDRl" (or short "antibody to DDRl ,") therefore refers to an antibody or a functional fragment thereof that specifically binds to a DDRl polypeptide (or a fragment or epitope of a DDRl polypeptide) and that does not specifically bind to other non-DDRl polypeptides. Preferably, antibodies (or functional fragments thereof) binding specifically to a DDRl polypeptide or fragment thereof do not non- specifically cross-react with other antigens (e.g., binding cannot be competed away with a non- DDRl polypeptide/protein, e.g., BSA in an appropriate immunoassay). Antibodies or functional fragments that specifically (or immunospecifically) bind to a DDRl polypeptide/protein can be identified, for example, by immunoassays or other techniques known to those of skill in the art.
As mentioned, the term "DDRl" refers preferably to a DDRl protein/polypeptide or a fragment or an epitope of a DDRl polypeptide. The terms "DDRl protein" or "DDRl polypeptide" (or short "DDRl" which is used interchangeably herein) can refer to and include polymorphic variants, alleles, mutants, and interspecies homologs that (i) are encoded by a nucleotide sequence having substantial nucleotide sequence identity (for example, at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, still more preferably at least 90% identity and even more preferably at least 95% identity) with the nucleotide sequence indicated in the respective database for the indicated ID number (an exemplary sequence can be retrieved from NCBI under accession number NM 001954.4 and is shown in SEQ ID NO: 49); or (ii) have substantial amino acid sequence identity (for example, at least 60% identity, preferably at least 70% identity, more preferably at least 80% identity, still more preferably at least 90% identity and even more preferably at least 95% identity) with the amino acid sequence as set forth in the respective database for the indicated ID number (an exemplary sequence can be retrieved from NCBI under accession number NP 001945.3 and is shown in SEQ ID NO: 50). In example 1 the NCBI accession number for the various known human isoforms for DDR1 are disclosed. Preferably, DDR1 refers to a mammal DDR1, most preferred to a human DDR1 polypeptide/protein or a fragment or an epitope of a human DDR1 polypeptide. The antibodies/binding molecules to be employed in context of this invention and as disclosed herein bind to the extracellular domain (or an epitope or a fragment thereof) of DDR1.
As used in herein, and unless otherwise specified, these terms refer to the entire gene sequence, mRNA sequence, and/or protein sequence as well as fragments of these sequences. In a more specific definition, these terms refer to the minimal amount of nucleic acid or amino acid sequence that can be used to identify such sequences in a specific manner. The skilled artisan recognizes that the DDR1 genes can have numerous splice forms and variants. When referring to a specific DDR1 gene or locus by a reference number (e.g., NCBI accession number), all splices forms and variants are included in the various embodiments of the invention. The gene can also comprise a regulatory element.
The exemplary sequences provided herein may only be representative of one particular individual in a population (in particular a human population). Individuals (in particular humans) vary from one to another in their gene sequences. These variations are very minimal, sometimes occurring at a frequency of about 1 to 10 nucleotides per gene. Nonetheless, different forms of any particular DDR1 gene exist within a population, such as the human population. These different forms are called allelic variants. Allelic variants often do not change the amino acid sequence of the encoded protein; such variants are termed synonymous. Even if they do change the encoded amino acid (non-synonymous), the function of the protein is typically not affected. Such changes are evolutionarily or functionally neutral. When a gene ID (e.g., NCBI database) is referred to in the present application all allelic variants are intended to be encompassed by the term. The gene ID sequences given for a DDR1 gene are provided merely as representative examples of a wild-type sequence, in particular a wild-type human sequence. The invention is therefore not limited to a single allelic form of the amplified genes or regions (and proteins they encode). In a preferred aspect, the antibody, antibody fragment thereof or antibody derivatives of this invention bind selectively or specifically to a DDR1 epitope. The peptide scan (pepspot assay) is routinely employed to map linear epitopes in a polypeptide antigen. The primary sequence of the polypeptide is synthesized successively on activated cellulose with peptides overlapping one another. The recognition of certain peptides by the antibody to be tested for its ability to detect or recognize a specific antigen/epitope is scored by routine colour development (secondary antibody with horseradish peroxidase and 4-chloronaphthol and hydrogenperoxide), by a chemoluminescence reaction or similar means known in the art. In the case of, inter alia, chemoluminescence reactions, the reaction can be quantified. If the antibody reacts with a certain set of overlapping peptides one can deduce the minimum sequence of amino acids that are necessary for reaction. The same assay can reveal two distant clusters of reactive peptides, which indicate the recognition of a discontinuous, i. e. conformational epitope in the antigenic polypeptide (Geysen (1986), Mol. Immunol. 23, 709-715). In addition to the pepspot assay, standard ELISA assay can be carried out. Small hexapeptides may be coupled to a protein and coated to an immunoplate and reacted with antibodies to be tested. The scoring may be carried out by standard colour development (e.g. secondary antibody with horseradish peroxidase and tetramethyl benzidine with hydrogenperoxide). The reaction in certain wells is scored by the optical density, for example at 450 nm. Typical background (=negative reaction) may be 0.1 OD, typical positive reaction may be 1 OD. This means the difference (ratio) positive/negative can be more than 10 fold.
The antibody of the present invention is directed against and binds specifically to a DDR1 polypeptide/protein/receptor, a fragment thereof or an epitope of a DDR1 polypeptide/protein/receptor, preferably to the extracellular region of said DDR1 polypeptide/protein/receptor (or an extracellular DDR1 epitope or a extracellular DDR1 fragment) The DDR1 receptor is known to exist in vivo in dimeric and monomeric form, see, inter alia, Noordeen et al. J Biol Chem (2006) 281(32):22744-51; Mihai et al. (2009) J Mol Biol. 385(2):432-45; Leitinger et al. (2003) J. Biol. Chem. 278: 16761-69; Agarwal et al. J. Mol. Biol, 367: 443-455;or Abdulhussein et al. (2008) 283(18): 12026-33). In one embodiment, the antibody of this invention binds to or can be generated against a polypeptide having the full length sequence of a DDR1 protein. In another aspect of the embodiment the antibody only recognizes or binds the DDR1 protein when the protein is present in the plasma membrane as a dimer. In another aspect of the embodiment the antibody only recognises or binds the DDR1 protein when it is present in the plasma membrane as a monomer. The accession numbers for the nucleic acid and amino acid sequences for the various known human isoforms of DDRl are listed below (see example 1) :
Figure imgf000029_0001
Preferably, the binding site or epitope for the herein provided antibodies is located on an extracellular domain of the DDRl protein. The amino acid sequence of an exemplary extracellular domain is shown in SEQ ID NO:63 (extracellular domain of human DDRl protein, which is shared by all human isoforms of DDRl known to date).
SEQ ID NO: 63
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPDSDISASSSWSDSTAA RHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGTQGRHAGGLGKEFSRS YRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGWLKDLGPPMVARLVRFYPRADRV MS VCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGHTVGGLQYGGLG QLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDRLRAFQAMQV HCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAVSVPLGGR VARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPPTNFSSLELEP RGQQPVAKAEGSPT
The antibodies provided herein specifically bind to discoidin domain receptor 1 (DDRl) and are capable of internalizing into cells.
Generally, the antibodies provided and to be used in accordance with the present invention may comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of the specific CDR sequences provided and disclosed herein. It is understood that the identity is assessed/determined over the full length of the CDR sequence.
In one embodiment of the present invention, the variable region of the heavy chain of the antibody of this invention comprises a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 [CDR-H3 of DDRl -abl ], preferably SEQ ID NO.: 24 [CDR- H3 of DDRl -ab2], or SEQ ID NO.: 36 [CDR-H3 of DDRl -ab3] . The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
The term "CDR" as employed herein relates to "complementary determining region", which is well known in the art. The CDRs are parts of immunoglobulins and T cell receptors that determine the specificity of said molecules and make contact with specific ligand. 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 V domain. CDR-H depicts a CDR region of a variable heavy chain and CDR-L relates to a CDR region of a variable light chain. H means the variable heavy chain and L means the variable light chain. The CDR regions of an Ig- derived region may be determined as described in Kabat (1991), Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia (1987), J. Mol. Biol. 196, 901-917; and Chothia (1989) Nature, 342, 877-883.
Each CDR region of a variable heavy chain is herein interchangeably designated as CDR-H 1 or VH-CDRl , CDR-H2 or VH-CDR2, and CDR-H3 or VH-CDR3, respectively. Likewise, each CDR region of a variable light chain is designated herein CDR-L1 or VL-CDR1 , CDR-L2 or VL-CDR2, and CDR-L3 or VL-CDR3, respectively.
In a further embodiment of the present invention, the variable region of the heavy chain of the antibody of this invention comprises a CDR-H 1 region having an amino acid sequence as depicted in SEQ ID NO: 8 [CDR-H1 of DDRl-abl], preferably SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], or SEQ ID NO: 32 [CDR-H1 of DDRl-ab3]. The antibodies may also comprise a CDR-H1 sequence having 75% or more (e.g. more preferably 80 %, 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs. In a further embodiment, the variable region of the heavy chain of the antibody of this invention comprises a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 [CDR-H2 of DDRl-abl], preferably SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], or SEQ ID NO: 34 [CDR-H2 of DDRl-ab3]. The antibodies may also comprise a CDR-H2 sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In a preferred embodiment, the variable region of the heavy chain of said antibody comprises
(a) a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 [CDR-H1 of DDRl-abl], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10
[CDR-H2 of DDRl-abl], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 [CDR-H3 of DDRl-abl];
(b) preferably a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2]; or
(c) a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 [CDR-H1 of DDRl-ab3], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 [CDR-H2 of DDRl-ab3], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 [CDR-H3 of DDRl-ab3]. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In a more preferred embodiment, the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2]. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In one embodiment, the variable region of the light chain of said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 [CDR-L1 of DDRl-abl], preferably SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], or SEQ ID NO: 26 [CDR-L1 of DDR1- ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs. In a further embodiment, the variable region of the light chain of said antibody comprises a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 [CDR-L2 of DDRl-abl], preferably SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], or SEQ ID NO: 28 [CDR-L2 of DDR1- ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs. In a yet further embodiment, the variable region of the light chain of said antibody comprises a CDR- L3 region having an amino acid sequence as depicted in SEQ ID NO: 6 [CDR-L3 of DDRl- abl], preferably SEQ ID NO: 18 [CDR-L3 of DDRl-ab2], or SEQ ID NO: 30 [CDR-L3 of DDRl-ab3], or a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In a preferred embodiment of the present invention, the variable region of the light chain of the antibody of this invention comprises
(a) a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 [CDR-L1 of DDRl-abl], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 [CDR-L2 of DDRl-abl], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 [CDR-L3 of DDRl-abl];
(b) preferably a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2]; or
(c) a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 [CDR-L1 of DDRl-ab3], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 [CDR-L2 of DDRl-ab3], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 [CDR-L3 of DDRl-ab3]. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In a more preferred embodiment of the present invention, the variable region of the light chain of the antibody of this invention comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2]. The antibodies may also comprise a CDR sequence having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs. The antibodies/binding molecules etc. of the present invention may be characterized by at least one CDR sequence as described above. Preferably, the antibody comprises 2 or more CDRs. More preferably, the antibody comprises 3, 4, 5 or more CDRs. Still even more preferably, said antibody comprises 6 CDRs. Yet, even more preferably the antibody comprises a set of 6 CDRs: 3 CDRs in the variable region of the light chain of the antibody and 3 CDRs in the variable region of the heavy chain of the antibody.
Accordingly, in a very preferred embodiment of the invention the variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 [CDR-L1 of DDRl-abl], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 [CDR-L2 of DDRl-abl], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6 [CDR-L3 of DDRl-abl] and the variable region of the heavy chain of the antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 [CDR-H1 of DDRl-abl], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10 [CDR-H2 of DDRl-abl], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12 [CDR-H3 of DDRl-abl]. The invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs. In another very preferred embodiment of the invention, the variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 [CDR-L1 of DDRl-ab3], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 [CDR-L2 of DDRl-ab3], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30 [CDR-L3 of DDRl-ab3] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 [CDR-H1 of DDRl-ab3], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 [CDR-H2 of DDRl-ab3], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 [CDR-H3 of DDRl-ab3]. The invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
In an even more preferred embodiment of the invention, the variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2]. The invention also relates to antibodies that specifically bind to DDRl wherein the antibody comprises CDR sequences having 75% or more (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) amino acid identity to one of said CDRs.
The antibodies of the present invention may also comprise entire variable regions of the light chain of the antibody and/or entire variable regions of the heavy chain of the antibody as disclosed herein. Accordingly, the antibody comprises in one embodiment a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 [Vn-region of DDRl-abl], preferably SEQ ID NO:43 [VH-region of DDRl-ab2], or SEQ ID NO:47 [VH-region of DDRl-ab3], or it comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 40 [Vn-region of DDRl-abl], preferably SEQ ID NO:44 [VH-region of DDRl-ab2], or SEQ ID NO:48 [Vn-region of DDRl -ab3].
In a further embodiment, the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 [VL-region of DDRl-abl], preferably SEQ ID NO:41 [VL-region of DDRl-ab2], or SEQ ID NO:45 [VL-region of DDRl-ab3], or a variable VL- region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl], preferably SEQ ID NO:42 [VL-region of DDRl-ab2], or SEQ ID NO:46 [VL-region of DDRl - ab3].
In a very preferred embodiment, the antibody comprises a variable VH-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 [VH-region of DDRl-abl], or a variable VH- region having an amino acid sequence as shown in SEQ ID NO:40 [VH-region of DDRl-abl]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 [VL-region of DDRl-abl] or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl]. Preferably, the antibody comprises a variable VH-region having an amino acid sequence as shown in SEQ ID NO:40 [VH-region of DDRl-abl] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:38 [VL-region of DDRl-abl].
In another very preferred embodiment of the invention, the antibody comprises a variable VH- region as encoded by a nucleic acid molecule as shown in SEQ ID NO:47 [VH-region of DDR1- ab3], or a variable VH-region having an amino acid sequence as shown in SEQ ID NO:48 [VH- region of DDRl-ab3]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45 [VL-region of DDRl-ab3], or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:46 [VL-region of DDRl-ab3]. Preferably, , the antibody comprises a variable VH-region having an amino acid sequence as shown in SEQ ID NO:48 [VH-region of DDRl-ab3] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:46 [VL-region of DDRl-ab3].
In an even more preferred embodiment of the invention, the antibody comprises a variable VR- region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 [Vn-region of DDR1- ab2] or a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44 [Vn- region of DDRl-ab2]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 [VL-region of DDRl-ab2] or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 [VL-region of DDRl-ab2]. Preferably, the antibody comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 [Vn-region of DDRl-ab2] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 [VL-region of DDRl-ab2].
The herein provided antibodies can comprise one or more of the heavy or light chain variable sequences above or a sequence at least 75%, 80%, more preferably at least 85 %, 90 %, even more preferably at least 95 %, 96 %, 97 %, 98 %, or most preferably 99 % identical thereto.
In one aspect, the variation in the sequences occurs in the framework regions, i.e. outside of the CDR sequences. In one embodiment, the antibody comprises:
a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:39 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:40 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn- region;
and
a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:37 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or
a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 38 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL- region;
and
wherein said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 2 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 4 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 6;
and/or
wherein said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 8 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 10, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 12.
In another embodiment, the antibody comprises:
a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:47 , or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:48 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable VH- region;
and
a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:45, or a variable VH-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 46 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable VL- region;
and
wherein said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 26 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 28 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 30; and/or
wherein said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 32 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 34 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 36 .
In another, more preferred, embodiment, the antibody comprises
a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region, or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn- region;
and
a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region, or
a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 42 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL- region;
and
wherein said antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 , a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 , and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18; and/or
wherein said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 , a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 , and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24. In the above embodiments, the variation in the sequences occurs in the framework regions, i.e. outside of the CDR sequences. In other words, the antibodies of these embodiments contain specific CDR regions as defined above that are not subject to variation. Yet, the framework region of these antibodies can show a variation/identity of 75 % or more (or 80%, more preferably at least 85 %, 90 %, even more preferably at least 95 %, 96 %, 97 %, 98 %, or most preferably 99 %) to the framework region of the specific variable VL-region(s) and variable Vn-region(s) as defined above. The framework region(s) can be identified by methods known in the art. As used herein and in the above emdodiments the term "framework region" refers to the sequence of the variable VL-region(s) and/or the variable VR- region(s) that is outside of the CDR sequences.
A particularly preferred antibody according to the invention is the antibody designated herein as DDRl-ab2. This antibody has been found to exhibit high affinity and specificity for human DDR1 , is able to internalize into cells quickly and kill cancer cells when conjugated to a drug such as a toxin and has shown antitumor activity in in vivo xenograft models of epidermoid (squamous cell) cancer and endometrial cancer when conjugated to a toxin when administered at doses below 500 μg/kg of ADC. Moreover, DDRl-ab2 has been shown to be devoid of toxicity in a battery of in vitro and in vivo toxicity studies, as described in more detail in the appended Examples. Antibody DDRl-ab2 is thus an excellent candidate for drug development, in particular for use in antibody-drug conjugate (ADC) therapy for the treatment of cancer.
The rate/speed of internalization of an antibody can be determined by time course experiments in which the disappearance of the antibody from the cell surface or the intracellular appearance of the antibody is analyzed as a function of time. Suitable methods to determine the rate of internalization include in particular in vitro methods, inter alia, FACS analysis, real time quantitative confocal fluorescence analysis of fluorescently labeled anti-DDRl antibody, time series assessment of internalized antibody detected directly or with a secondary antibody, or time series assessment of the incorporation of radioactively labeled anti-DDRl antibody inside the cell, among others. Examples of cells suitable for testing anti-DDRl antibodies are, for example, Clone 44 cells (see Example 4), which over-express DDR1 , or other cells capable of expressing and internalizing DDR1. The internalization rate can be assessed in the presence or absence of collagen (for example collagen I). Different antibodies can be tested in parallel to assess their relative internalization rate and to rank said antibodies according to their delivery capacity. As used herein, an antibody is regarded as internalizing "quickly" if, under the experimental conditions described in example 6, the % of internalized antibody at 15 minutes (t=15) as compared to the antibody on the cell surface at t=0 is > 90%. Accordingly, in a very preferred embodiment, the invention provides an antibody that specifically binds to DDR1 , wherein the variable region of the light chain of the antibody comprises a CDR-L1 region having an amino acid sequence as depicted in SEQ ID NO: 14 [CDR-L1 of DDRl-ab2], a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16 [CDR-L2 of DDRl-ab2], and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18 [CDR-L3 of DDRl-ab2] and the variable region of the heavy chain of said antibody comprises a CDR-H1 region having an amino acid sequence as depicted in SEQ ID NO: 20 [CDR-H1 of DDRl-ab2], a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22 [CDR-H2 of DDRl-ab2], and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24 [CDR-H3 of DDRl-ab2]. The invention also provides for binding molecules/antibodies that comprise CDR sequences that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to the CDRs as provided herein and that specifically bind to discoidin domain receptor 1. In another very preferred embodiment, the invention provides an antibody that specifically binds to DDR1, wherein the antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 [Vn-region of DDRl-ab2] or a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 [Vn-region of DDRl-ab2]; and the antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 [VL-region of DDRl-ab2] or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 [VL-region of DDRl-ab2]. Preferably, the antibody comprises a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44 [Vn-region of DDRl-ab2] and a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 [VL-region of DDRl-ab2]. The invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to the variable regions as provided herein and that specifically bind to discoidin domain receptor 1. In another very preferred embodiment, the invention provides antibodies/binding molecules that specifically bind to DDR1, wherein said antibodies/binding molecules comprise variable regions and/or CDRs as comprised in a (binding) molecule obtainable from the expression of a vector (nucleic acid molecule) as deposited on December 20th, 2011 under the designation DDR1- ab2pBhl under accession number DSM 25530 with the depositary institute DSMZ by Oryzon Genomics S.A., with address at Sant Ferran 74, 08940 Cornelia de Llobregat, Spain. The invention also provides for binding molecules/antibodies that comprise CDRs that are at least 75% identical in their protein sequence (amino acid identity) to one or more of the CDRs as comprised in the molecule encoded by the nucleic acid molecule comprised in said deposited vector and that specifically bind to discoidin domain receptor 1 (DDR1). The invention also provides for binding molecules/antibodies that comprise variable regions that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) in their protein sequence (amino acid identity) to one or more of the variable regions as comprised in the molecule encoded by the nucleic acid molecule comprised in said deposited vector and that specifically bind to discoidin domain receptor 1 (DDR1).
In another very preferred embodiment, the invention provides antibodies/binding molecules that specifically bind to DDR1 , wherein said antibodies/binding molecules bind to or recognize the same epitope as any of the binding molecules obtainable upon expression of the nucleic acid molecule comprised in the vector as described under the designation DDRl-ab2pBhl deposited by Oryzon Genomics S.A., Spain on December 20th, 2011 with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, German Collection of Microorganisms and Cell Cultures).
As disclosed herein, the antibodies/binding molecules of the invention include the antibodies having the CDRs and/or variable sequences as disclosed herein as well as variants thereof having 75% or more (for example 80%, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, or 99 %) sequence identity.
As used herein, the terms "identity", "sequence identity", "homology" or "sequence homology" (the terms are used interchangeably herein) are used to describe the sequence relationships between two or more nucleic acids, polynucleotides, proteins, or polypeptides, and is understood in the context of and in conjunction with the terms including: (a) reference sequence, (b) comparison window, (c) sequence identity, (d) percentage of sequence identity, and (e) substantial identity or "homologous". A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence.
A "comparison window" includes reference to a contiguous and specified segment of a polynucleotide or polypeptide sequence, wherein the polynucleotide or polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. Generally, the comparison window may be at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a misleadingly high similarity to a reference sequence due to inclusion of gaps in the polynucleotide or polypeptide sequence a gap penalty is typically introduced and is subtracted from the number of matches. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2: 482, 1981; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48: 443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 7 Science Dr., Madison, Wise, USA; the CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73: 237-244; Corpet et al. (1988) Nucleic Acids Research 16:881-90; Huang, et al. (1992) Computer Applications in the Biosciences, 8: 1-6; and Pearson, et al. (1994) Methods in Molecular Biology, 24:7-331. The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York, 1995. New versions of the above programs or new programs altogether will undoubtedly become available in the future, and can be used with the present invention. Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs, or their successors, using default parameters. Altschul et al. (1997) Nucleic Acids Res, 2:3389-3402. It is to be understood that default settings of these parameters can be readily changed as needed in the future.
As those ordinary skilled in the art will understand, BLAST searches assume that proteins or nucleic acids can be modeled as random sequences. However, many real proteins and nucleic acids comprise regions of nonrandom sequences which may be homopolymeric tracts, short- period repeats, or regions enriched in one or more amino acids or nucleic acids. Such low- complexity regions may be aligned between unrelated proteins even though other regions of the protein or nucleic acid are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten et al. (1993) Comput. Chem. 17: 149-163) and XNU (Claverie et al. (1993) Comput. Chem. 17: 191-1) low-complexity filters can be employed alone or in combination.
"Sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window, and can take into consideration additions, deletions and substitutions. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) and therefore do not deleteriously change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have sequence similarity. Approaches for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, for example, according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 1 1-17, 1988, for example, as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif, USA). "Percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or nucleic acid sequence in the comparison window may comprise additions, substitutions, or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions, substitutions, or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
The term "substantial identity" or "homologous" in their various grammatical forms in the context of polynucleotides means that a polynucleotide comprises a sequence that has a desired identity, for example, at least 75% sequence identity, preferably at least 80%, more preferably at least 85 %, still more preferably at least 90% and even more preferably at least 95%, 96 %, 97 %, 98 % or 99 %, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Accordingly, the present invention provides for binding molecules/antibodies etc specifically binding to DDR1 which comprise CDRs and/or variable regions that have at least 75% sequence identity, more preferably at least 80%, even more preferably at least 85 %, still more preferably at least 90% and most preferably at least 95%, 96 %, 97 %, 98 % or 99 % sequence identity with either the encoding nucleic acid molecule or the expressed amino acid molecule of an antibody molecule (or variable regions or CDRs thereof) as obtainable from the vector deposits deposited under the designation DDRl-ablpBhl, preferably DDRl-ab2pBhl, or DDRl-ab3pBhl on December 20, 201 1 with the DSMZ Braunschweig/ Germany.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as, for example, the highly stringent hybridization conditions of 0.1 x SSC, 0.1% SDS at 65°C or 2 x SSC, 60°C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 55°C or 60°C. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical.
The term "substantial identity" or "homologous" in their various grammatical forms in the context of peptides indicates that a peptide comprises a sequence that has a desired identity, for example, at least 75% sequence identity to a reference sequence, preferably at least 80% sequence identity to a reference sequence, more preferably 85%, even more preferably at least 90% or 95% or even 96 %, 97 %, 98 % or 99 % sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol., 48:443. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide, although such cross-reactivity is not required for two polypeptides to be deemed substantially identical. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
Conservative amino acid substitutions are known to those skilled in the art and typically include, but are not limited to, substitutions listed in the following table:
Exemplary
Typical α-Aminoacid Symbol Nature Conservative
substitution substitutions
Alanine Ala (A) Aliphatic, hydrophobic, Val, He, Leu, Gly, Val neutral Ser
Arginine Arg (R) Polar, hydrophilic, charge (+) Lys, His, Gin, Asn Lys
Asparagine Asn (N) Polar, hydrophilic, neutral Lys, His, Gin, Arg Gin
Cysteine Cys (C) Polar, hydrophobic, neutral Ser, Ala Ser
Glutamine Gin (Q) Polar, hydrophobic, neutral Asn Asn
Glycine Gly (G) Aliphatic, neutral Pro, Ala Ala
Histidine His (H) Aromatic, polar, hydrophilic, Asn, Gin, Lys, Arg Arg charge (+)
Isoleucine He (I) Aliphatic, hydrophobic, Leu, Val, Met, Ala, Leu neutral Phe
Leucine Leu (L) Aliphatic, hydrophobic, He, Val, Met, Phe, He neutral Ala
Lysine Lys (K) polar, hydrophilic, charge (+) Arg, Gin, Asn, His Arg
Methionine Met (M) hydrophobic, neutral Leu, He, Phe Leu
Phenylalanine Phe (F) Aromatic, hydrophobic, Leu, He, Val, Ala, Leu neutral Tyr
Proline Pro (P) hydrophobic, neutral Ala, Gly Gly
Serine Ser (S) Polar, hydrophilic, neutral Thr, Ala, Cys Thr
Threonine Thr (T) Polar, hydrophilic, neutral Ser Ser
Tryptophan Trp (W) Aromatic, hydrophobic, Tyr, Phe Tyr neutral
Tyrosine Tyr (Y) Aromatic, polar, hydrophobic Trp, Phe, Thr, Ser Phe
Valine Val (V) Aliphatic, hydrophobic, He, Met, Leu, Phe, Leu neutral Ala,
Glutamic Acid Glu (E) Polar, hydrophilic, charge (-) Asp, Gin Asp
Aspartic Acid Asp (D) Polar, hydrophilic, charge (-) Glu, Asn Glu
In another embodiment, the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences and/or variable sequences as disclosed herein having 1 or more amino acid substitutions, deletions or additions. In a preferred embodiment, the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences as disclosed herein having 1 or more, preferably 1 , 2 or 3, more preferably 1 or 2 amino acid substitutions, deletions or additions. In another preferred embodiment, the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise variable sequences as disclosed herein having up to 20, preferably up to 15, more preferably up to 10, amino acid substitutions, deletions or additions. In another preferred embodiment, the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise CDR sequences as disclosed herein having 1 or more, preferably 1 , 2 or 3, more preferably 1 or 2, amino acid substitutions, preferably conservative amino acid substitutions. In another preferred embodiment, the invention relates to antibodies/binding molecules that specifically bind to DDRl wherein said antibodies or binding molecules comprise variable sequences as disclosed herein having up to 20, preferably up to 15, more preferably up to 10, amino acid substitutions, preferably conservative amino acid substitutions.
The present invention provides antibodies comprising CDRs and/or variable sequences as described herein, or variants thereof, as disclosed above. Methods are known to those skilled in the art to modify the sequence of an existing antibody (parent antibody) to derive variant antibodies with high sequence homology to the sequence of the existing antibody that retain the capacity to bind the original target epitope.
Variant DDRl binding antibodies (with similar or improved affinity, with modified selectivity, antigenicity, with modified pharmacokinetic characteristics) can be readily derived from the DDRl antibodies disclosed herein through variation of the sequence of the claimed antibodies, using methods that have been described in the literature.
Mutations can be introduced randomly into the variable regions of antibody genes by error-prone polymerase chain reaction (PCR) or E. coli mutator strains, site-directed mutagenesis, saturation mutagenesis, parsimonious mutagenesis, CDR walking or look-through mutagenesis targeting certain regions like the CDRs, hence generating limited collections of the specific variants of the parent antibody. Shuffling approaches include DNA shuffling, chain shuffling, or CDR shuffling to obtain shuffled variants of the parent antibody.
Introduction of variations can be random (radiation, chemical mutagens, error prone PCR, chain shuffling) or directed (site directed mutagenesis, (partial) gene synthesis using regular phosphoramidite chemistry or triplet synthesis). Random mutation efforts can be combined with in vitro selection procedures (i.e. display methods) to identify binders.
Directed mutagenesis is preferentially performed after in silico modeling of the DDR1 protein - DDR1 antibody using the sequence and structure information of the (extracellular part of) the DDR1 protein and the DDR1 antibody.
Modeling can be done using the experimentally determined 3D crystal structure of the complex formed between the (extracellular domain of) DDR1 protein with the DDR1 antibodies of the invention as a starting point. Alternatively, modeling can also be done by using an in silico docking model of the (extracellular domain of) DDR1 protein and the antibodies disclosed herein based on published 3D structures of the individual protein. The extracellular part of DDR1 has been crystallized with an antibody and its 3D structure has been deposited (EBI PDBsum Database Entry 4ag4).
The 3D structure of the DDR1 antibody can be predicted with one of different algorithms available in the art that are rapidly increasing in accuracy like: Web Antibody Modeling (WAM) (Whitelegg and Rees, Protein Eng. 2000; 14(12):819-824), Prediction of ImmunGlobulin Structure (PIGS) (Marcatili et al, Bioinformatics. 2008;14(17): 1953-1954), or RosettaAntibody (Sivasubramanian et al, Proteins. 2009; 14(2):497-514. ),).
The algorithms cited above can be used to dock the antibodies to the (extracellular domain of the) target protein; and to analyze sequence tolerance to variation with respect to the antibody- target protein binding capacity, i.e. the algorithms can be used by a skilled user to design variant antibodies binding the same epitope (see e.g. Barderas et al. Proc Natl Acad Sci U S A. Jul 1, 2008; 105(26): 9029-9034) and this principle can be applied to the DDR-1 (extracellular domain) binding antibodies with the CDRs and/or variable sequences as disclosed herein.
Typically, variations in a limited number of amino acids will be evaluated during in silico modeling. The effects of the variation may vary the affinity of the antibody to the DDR1 target epitope, typically it will be desirable that the affinity is similar or higher than that of the DDR-1 binding antibodies as disclosed herein. Focused libraries containing candidate daughter sequences with the desired variations can then be synthesized or produced by directed mutagenesis into the DDR1 antibody sequences listed herein. The retention of the DDR1 binding capacity can be verified after expressing the derived protein(s), and competition experiments can be used to demonstrate that the variant DDR-1 antibodies derived from the antibodies as disclosed herein bind to the same original epitope.
This process can be reiterated by submitting successful daughter sequence(s) to a new cycle of modifications, or to introduce stabilizing peripheral mutations. It has been described that the introduction of amino acid changes that increase affinity may reduce overall antibody protein stability, and that this may also lead to reduced expression/production of antibody (fragments) in mammalian cells (Wang et al, Proc Natl Acad Sci U S A. Mar 12, 2013; 1 10(11): 4261^266). Stabilizing mutations can be identified by assessing melting curves using thermal scanning or light scattering [aggregation (agg)] of antibodies. Stabilizing mutations have been shown to stabilize antibodies independently of their target binding capacities. Mutations stabilizing the antibodies of the invention can be identified either directly starting from these antibodies, or using antibodies derived from the antibodies disclosed herein that have lost the DDR1 binding capacity and then introduced into the antibodies of the invention or from the antibodies with DDR1 binding capacity derived from them as described above.
Additional changes may be introduced into the antibodies of the invention to modify potential antigenicity, glycosylation, and antibodies may also be produced in different hosts to modify glycosylation, but in all cases said antibodies will contain the DDR1 binding region from the antibody sequences as disclosed herein or they will be directly derived from them following established methods as disclosed above and will thus retain the binding capacity to the original epitope, as described above.
In all the embodiments described herein, the antibody/binding molecule of the present invention may be a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody. The antibodies/binding molecules of the invention may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross-cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc. The antibody may also be a multispecific antibody, including a bispecific antibody. The antibodies of the invention may be multifunctional, i.e. they may exert their effects via more than one mode of action, such as for example by promoting internalization of DDR1 and activating ADCC or CDC pathways. Thus, the antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single- chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a DDR1 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-DDRl antibody). In a further preferred embodiment of the invention, the antibodies are humanized or human and/or deimmunized. More preferably, the antibodies are humanized and most preferably the antibodies are fully humanized/human.
Said "fully humanized antibody" are also characterized and described as "completely human" antibodies. All these antibodies can be generated by methods known in the art. For example, by phage display technology, recombinant antibody molecules may be generated due to the use of in vitro maturation which is the usage of a complete human immunoglobulin γ, subclass- 1 framework (IgGl) as described by Knappik (2000) J Mol Biol. 296(1), 57-86, and Rauchenberger (2003) J Biol Chem. 278(40), 38194-205.
As used herein, the term "CDR-grafted", "humanized" or "humanization" are used interchangeably to refer to a human antibody as defined herein (preferably a IgGl antibody) comprising in its binding domains at least one complementarity determining region ("CDR") from a non-human antibody or fragment thereof. Humanization approaches are described for example in WO 91/09968 and US 6,407,213. As non-limiting examples, the term encompasses the case in which a variable region of the binding domain comprises a single CDR region, for example the third CDR region (CDR-H3) of the VH, from another non-human animal, for example a rodent, as well as the case in which a or both variable region/s comprise at each of their respective first, second and third CDRs the CDRs from said non-human animal. In the event that all CDRs of a binding domain of the antibody have been replaced by their corresponding equivalents from, for example, a rodent, one typically speaks of "CDR-grafting", and this term is to be understood as being encompassed by the term "humanized" as used herein. The term "humanized" also encompasses cases in which, in addition to replacement of one or more CDR regions within a VH and/or VL of the binding domain further mutation/s (e.g. substitutions) of at least one single amino acid residue/s within the framework ("FR") regions between the CDRs has/have been effected such that the amino acids at that/those positions correspond/s to the amino acid/s at that/those position/s in the animal from which the CDR regions used for replacement is/are derived. As is known in the art, such individual mutations are often made in the framework regions following CDR-grafting in order to restore the original binding affinity of the non-human antibody used as a CDR-donor for its target molecule. The term "humanized" may further encompass (an) amino acid substitution(s) in the CDR regions from a non-human animal to the amino acid(s) of a corresponding CDR region from a human antibody, in addition to the amino acid substitutions in the framework regions as described above.
More specifically, as used herein, "humanized antibodies" or related terms encompass antibodies having the amino acid sequence of a human immunoglobulin with a variable region comprising non-human CDR- and/or framework region- sequences. In contemplating an antibody intended for therapeutic administration to humans, it is highly advantageous that the major part of this antibody is of human origin. Following administration to a human patient, a humanized antibody or a human antibody (or fragment thereof) will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a "foreign", that is non-human protein. This means that no host, i.e. patient antibodies will be generated against the therapeutic antibody which would otherwise block the therapeutic antibody's activity and/or accelerate the therapeutic antibody's elimination from the body of the patient, thus preventing it from exerting its desired therapeutic effect. An antibody as defined herein may also be regarded as humanized if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germline sequence(s) by no more than would be expected due to the imprint of somatic hypermutation. Preferably, the humanized antibodies as defined herein have a human constant region and one or more of the CDR sequences which may be of, but are not limited to, CDRs of non-human, preferably rodent, origin. However, in context of this invention, also antibodies are provided that comprise not only human constant regions but also CDRs that are of human origin. Accordingly, the present invention also provides for "fully-human" antibodies. As used herein, the term "chimeric antibody" encompasses antibodies having human constant regions on the light and heavy chains and non-human variable regions on the light and heavy chains. Preferably the non-human regions are from a rodent sequence. For example, the variable regions of the heavy and light chain could be amplified by RT-PCR using R A extracted from a mouse hybridoma cell which produces the antibody of interest. The amplified sequence could be cloned in frame with the constant heavy-chain or the constant light chain respectively of a human IgG also included in a mammalian expression vector. An expression vector encoding a chimeric IgG could be transfected into the right cell line, like for example CHO or HEK293, for chimeric antibody production. As used herein, the term "deimmunized" or "deimmunization" denotes modification of the binding domain vis-a-vis an original wild type construct by rendering said wild type construct non-immunogenic or less immunogenic in humans. Deimmunization approaches are shown e.g. in WO 00/34317, WO 98/52976, WO 02/079415 or WO 92/10755. The term "deimmunized" also relates to constructs, which show reduced propensity to generate T cell epitopes. In accordance with this invention, the term "reduced propensity to generate T cell epitopes" relates to the removal of T-cell epitopes leading to specific T-cell activation. Furthermore, "reduced propensity to generate T cell epitopes" means substitution of amino acids contributing to the formation of T cell epitopes, i.e. substitution of amino acids, which are essential for formation of a T cell epitope. In other words, "reduced propensity to generate T cell epitopes" relates to reduced immunogenicity or reduced capacity to induce antigen independent T cell proliferation. The term "T cell epitope" relates to short peptide sequences which can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatibility complex (MHC) in order to trigger the activation of T cells; see inter alia WO 02/066514. For peptides presented by MHC class II such activation of T cells can then give rise to an antibody response by direct stimulation of T cells to produce said antibodies. "Reduced propensity to generate T-cell epitopes" and/or "deimmunization" may be measured by techniques known in the art. Preferably, de-immunization of proteins may be tested in vitro by T cell proliferation assay. In this assay PBMCs from donors representing > 80 % of HLA-DR alleles in the world are screened for proliferation in response to either wild type or de- immunized peptides. Ideally cell proliferation is only detected upon loading of the antigen- presenting cells with wild type peptides. Alternatively, one may test deimmunization by expressing HLA-DR tetramers representing all haplotypes. These tetramers may be tested for peptide binding or loaded with peptides substitute for antigen-presenting cells in proliferation assays. In order to test whether deimmunized peptides are presented on HLA-DR haplotypes, binding of e.g. fluorescence-labeled peptides on PBMCs can be measured. Furthermore, deimmunization can be proven by determining whether antibodies against the deimmunized molecules have been formed after administration in patients. Preferably, antibody derived molecules are deimmunized in the framework regions and most of the CDR regions are not modified in order to generate reduced propensity to induce T cell epitope so that the binding affinity of the CDR regions is not affected. Even elimination of one T cell epitope results in reduced immunogenicity. In summary, the above approaches help to reduce the immunogenicity of the antibodies provided herein when being administered to patients.
The invention also involves one or more of the disclosed CDR sequences above or a CDR sequence at least 75 % (at least 80%, at least 90%, at least 95%, at least 96 %, at least 97 %, at least 98 % or at least 99 %) identical in their amino acid sequence hereto wherein said CDR sequences is in the context of an antibody framework/framework region. Preferably, the antibody framework is a human antibody framework. Examples for frameworks include an IgG framework, such as IgGl, IgG4, IgG2a and IgG2b, preferably a human IgG framework such as IgGl , IgG2, IgG3 and IgG4. Accordingly, the antibodies of the invention may also comprise cross-cloned antibodies, i.e. antibodies comprising different antibody regions (e.g. CDR-regions) from one or more parental or affinity-optimized antibody(ies) as described herein. These cross- cloned antibodies may be antibodies in several, different frameworks, e.g. an IgG-framework, e.g. a IgGl-, IgG4, IgG2a or an IgG2b-framework. For example, said antibody framework is a mammalian, e.g. a human framework such as IgGl , IgG2, IgG3 or IgG4. It is of note that not only cross-cloned antibodies described herein may be presented in a preferred (human) antibody framework, but also antibody molecules comprising CDRs from antibodies as described herein, may be introduced in an immunoglobulin framework. Examples for frameworks include IgG frameworks such as IgGl , IgG4, IgG2a and IgG2b. Most preferred are human frameworks, and particularly human IgGl or IgG4 frameworks.
As used herein, a "human antibody framework " relates to an antibody framework that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the antibody framework of a naturally occurring human immunoglobulin.
As used herein, a "human framework region" relates to a framework region that is substantially identical (about 85% or more, usually 90 %, more preferably 95%, 96 %, 97 %, 98 %, 99 % or more) to the framework region of a naturally occurring human immunoglobulin.
In accordance with this invention, a framework region relates, accordingly, to a region in the V domain (VH or VL domain) of immunoglobulins and T-cell receptors that provides a protein scaffold for the hypervariable complementarity determining regions (CDRs) that make contact with the antigen. In each V domain, there are four framework regions designated FR1, FR2, FR3 and FR4. Framework 1 encompasses the region from the N-terminus of the V domain until the beginning of CDR1 , framework 2 relates to the region between CDR1 and CDR2, framework 3 encompasses the region between CDR2 and CDR3 and framework 4 means the region from the end of CDR3 until the C-terminus of the V domain; see, inter alia, Janeway, Immunobiology, Garland Publishing, 2001 , 5th ed. Thus, the framework regions encompass all the regions outside the CDR regions in VH or VL domains. Furthermore, the term "transition sequence between a framework and a CDR region" relates to a direct junction between the framework and CDR region. In particular, the term "transition sequence between a framework and a CDR region" means the sequence directly located N- and C-terminally of the CDR regions or amino acids surrounding CDR regions. Accordingly, frameworks may also comprise sequences between different CDR regions. The person skilled in the art is readily in a position to deduce from a given sequence the framework regions, the CDRs as well as the corresponding transition sequences; see Kabat (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services, Chothia (1987). J. Mol. Biol. 196, 901-917 and Chothia (1989) Nature, 342, 877-883.
In a further embodiment of the present invention, the antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody, preferably IgG. As used herein, an "antibody" may denote immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to DDR1. Such antibodies are constructed in the same way. They form paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. Within this general category, however, five different classes of immunoglobulins - IgM, IgD, IgG, IgA, and IgE - can be distinguished by their C regions. IgG antibodies are large molecules, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chain. One, of approximately 50 kDa, is termed the heavy or H chain, and the other, of 25 kDa, is termed the light or L chain. Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. In any given immunoglobulin molecule, the two heavy chains and the two light chains are identical, giving an antibody molecule two identical antigen-binding sites, and thus the ability to bind simultaneously to two identical structures. Two types of light chain, termed lambda and kappa, are found in antibodies. A given immunoglobulin either has lambda chains or kappa chains, never one of each. No functional difference has been found between antibodies having lambda or kappa light chains, and either type of light chain may be found in antibodies of any of the five major classes. The ratio of the two types of light chain varies from species to species. In mice, the average kappa to lambda ratio is 20: 1, whereas in humans it is 2: 1 and in cattle it is 1 :20. The reason for this variation is unknown. By contrast, the class, and thus the effector function of an antibody, is defined by the structure of its heavy chain. There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule such as, for example, complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The five major classes of immunoglobulin are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). Their heavy chains are denoted by the corresponding lower-case Greek letter (mu, delta, gamma, alpha, and epsilon, respectively). IgG is by far the most abundant immunoglobulin and has several subclasses (IgGl, 2, 3, and 4 in humans, IgGl, IgG2a, IgG2b and IgG3 in mice). Their distinctive functional properties are conferred by the carboxy-terminal part of the heavy chain, where it is not associated with the light chain. The general structural features of all the isotypes are similar. The IgG antibody is the most abundant isotype in plasma.
Preferably, the antibodies as defined herein are IgG antibodies. As is well known in the art, an IgG comprises not only the variable antibody regions responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in endogenously produced antibodies and, in some cases, even decoration at one or more sites with carbohydrates. Such glycosylation is generally a hallmark of the IgG format, and portions of these constant regions make up the so called Fc region of a full antibody which is known to elicit various effector functions in vivo, such as e.g. antibody-dependent cellular cytotoxicity (ADCC). In addition, the Fc region mediates binding of the IgG to an Fc receptor, hence prolonging half life in vivo as well as facilitating homing of the IgG to locations with increased Fc receptor presence. Advantageously, the IgG antibody is an IgGl or IgG4 antibody specifically binding to DDR1. In the following, exemplary methods for the generation of antibodies to DDR1 (like polyclonal, monoclonal, humanized, human antibodies or antibody fragments) are described.
Generation of Antibodies to DDR1
Antibodies and fragments thereof to a DDR1 protein or a DDR1 epitope (also referred to as a target protein) for therapeutic and/or diagnostic uses can be obtained in any number of ways known to those of ordinary skill in the art. These antibodies can be used in the methods of the invention and/or as the basis of engineering new antibodies. Phage display techniques can be used to generate or isolate an antibody and/or fragment thereof to a DDR1 protein or a DDR1 epitope. Standard hybridoma technologies can be used to generate antibodies and fragments thereof to a DDR1 protein or a DDR1 epitope. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a monoclonal antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a polyclonal antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a recombinant antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a humanized antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a fully human antibody or a fragment thereof. In one aspect, the antibody or fragment thereof to a DDRl or a DDRl epitope is a chimeric antibody or fragment thereof. In one aspect, the antibody or fragment thereof (e.g., CDR(s)) to DDRl is derived from an animal source (e.g., mouse, rat, or rabbit).
Polyclonal Antibodies
The target protein antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the target protein polypeptide DDRl (or fragment or epitope thereof) or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. Monoclonal Antibodies
The DDRl protein antibodies may, alternatively, be monoclonal antibodies and/or fragments thereof. Monoclonal antibodies may be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal (e.g., rabbit, goat etc.), is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the target protein polypeptide DDRl (or fragment thereof) or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non- human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Manassas, Va. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor (1984) Immunol. 133:3001; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against target protein. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem. 107:220.
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No. 4,816,567; Morrison et ah, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies and fragments thereof may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Human and Humanized Antibodies
The DDRl protein antibodies of the invention may further comprise humanized antibodies or human antibodies (and/or fragments thereof). Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies (and/or fragments thereof) include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies (and/or fragments thereof) may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody (and/or fragments thereof) will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature, 321 :522-525; Riechmann et /.(1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596).
Methods for humanizing non-human antibodies (and/or fragments thereof) are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. (1986) Nature, 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239: 1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies (and/or fragments thereof) can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter (1991) J. Mol. Biol. 227:381 ; Marks et al. (1991) J. Mol. Biol. 222:581). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al.
(1991) J. Immunol. 147(l):86-95). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al. (1992) Bio/Technology 10:779-783; Lonberg et al. (1994) Nature 368:856-859; Morrison (1994) Nature 368:812-13; Fishwild et al. (1996) Nature Biotechnology 14:845-51; Neuberger (1996) Nature Biotechnology 14:826; Lonberg and Huszar (1995) Intern. Rev. Immunol. 13 65-93.
The antibodies (and/or fragments thereof) may also be affinity matured using known selection and/or mutagenesis methods as described above. Preferred affinity matured antibodies have an affinity which is 5 times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.
Antibody Fragments
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see Morimoto et al
(1992) Journal of Biochemical and Biophysical Methods 24: 107-117; and Brennan et al (1985) Science 229:81). Antibody fragments can also be produced directly by recombinant host cells and the antibody phage libraries discussed above. Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al (1992) Bio/Technology 10: 163-167). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641 ,870, for example. Such linear antibody fragments may be monospecific or bispecific.
Multispecific and bispecific antibodies
Bispecific antibodies with binding specificities for at least two different epitopes (Millstein et al (1983), Nature 305:537-539) may bind to two different epitopes of the DDRl protein. An anti- DDR1 arm may be combined, for example, with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the DDRl -expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express DDRl (WO 96/16673; U.S. Pat. No. 5,837,234; WO98/02463; U.S. Pat. No. 5,821,337). Purification methods for bispecific antibodies have been disclosed (WO 93/08829; Traunecker et al (1991) EMBO J. 10:3655-3659; WO 94/04690; Suresh et al (1986) Methods in Enzymology 121 :210; U.S. Pat. No. 5,731,168). Bispecific antibodies can be produced using leucine zippers (Kostelny et al (1992) J. Immunol. 148(5): 1547- 1553), and single-chain Fv (sFv) dimers (Gruber et al (1994) J. Immunol. 152:5368).
Techniques for generating bispecific antibodies from antibody fragments have also been described, such as using chemical linkage wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments (Brennan et al (1985) Science 229:81). Fab'-SH fragments can be recovered from E. coli and chemically coupled to form bispecific antibodies (Shalaby et al (1992) J. Exp. Med. 175:217-225. The "diabody" technology provides an alternative method for making bispecific antibody fragments (Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448).
Antibodies with more than two valencies are contemplated. Multivalent, "Octopus" antibodies with three or more antigen binding sites and two or more variable domains can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody (US 2002/0004586; WO 01/77342). For example, trispecific antibodies can be prepared (Tutt et al (1991) J. Immunol. 147:60.)
Conjugated antibodies
In a preferred embodiment of the present invention, the antibody is conjugated to one or more therapeutic agents. This is particularly envisaged when the antibodies are to be used in medicine, for example, in the therapy/treatment of cancer (and/or a proliferative disorder, etc). Antibody conjugates with antibodies to DDR1 can prepared for various types of antibodies (and/or fragments thereof) including chimeric antibodies, humanized antibodies, and fully human antibodies. As used herein, "conjugated" means that the antibody/binding molecule is bound to the therapeutic agent(s) via any type of bonding, and thus includes bonding via fusion proteins (in case the therapeutic agent is of peptidic nature) or any other type of coupling or linkage between the therapeutic agent and the antibody/binding molecule. "Conjugated to a therapeutic agent" is thus to be understood as including fused to, linked to or coupled to a therapeutic agent. "Therapeutic agent" as used herein refers to any molecule (including small molecules, macromolecules, peptides, polypeptides, proteins (including other therapeutic antibodies), radioactive isotopes, etc) exerting a beneficial effect in the treatment of diseases in humans or other mammals. In a preferred embodiment, such therapeutic agents are suitable for the therapy of cancer, tumorous disorders and/or proliferative disorders. Further medical use comprise the medical intervention in proliferative disorders, like inflammations, inflammation disorders related to the undesired proliferation of immune cells, auto-immune disorders (such as atherosclerosis). The term "therapeutic agents" also comprises toxins, in particular toxins used in cancer therapy or, inter alia, anti-inflammatory therapy, etc. A molecule of antibody may conjugate with more than one molecule of the therapeutic agent (as used herein, "conjugation agent"), depending on the number of sites in the antibody available for conjugation and the experimental conditions employed for performing the conjugation. As it will be apparent to those skilled in the art, while each molecule of antibody is conjugated to an integer number of molecules of the conjugation agent, a preparation of the antibody conjugate may analyze for a non-integer ratio of conjugation agent molecules per molecule of antibody, reflecting a statistical average.
Examples of therapeutic agents that can be conjugated to the antibodies/binding molecules of the invention targeting DDR1 include, but are not limited to, anticancer agents such as antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, capecitabine and decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin, carboplatin, oxaliplatin nedaplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine, and tetrazines), anthracyclines (e.g., daunorubicin, doxorubicin, valrubicin, idarubicin, epirubicin and mitoxantrone), antibiotics (e.g., actinomycins like dactinomycin, bleomycins, mithramycins, calicheamicins, mitomycins, duocarmycins and anthramycins (AMC)), topoisomerase inhibitors (e.g. irinotecan, topotecan, camptothecin, etoposide and teniposide), and anti-mitotic agents (e.g., vinca alkaloids such as vincristine, vinorelbine, vindesine and vinblastine, taxanes such as paclitaxel (or taxol) and docetaxel, and other tubulin polimeryzation inhibitors such as auristatins like monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) and maytansine derivatives (a.k.a maytansinoids) like mertansine (also known as DM1) and DM4). The term "anticancer agent" as used herein refers to and includes cytotoxic agents.
Other therapeutic agents that can be conjugated to the antibodies of the invention include toxins and inhibitory peptides. As used herein, "inhibitory peptide" means any peptide that inhibits cell proliferation or affects cell viability via any mechanism of action. Non-limiting examples are provided herein below.
Specific examples of anticancer agents that can be conjugated to the antibodies/binding molecule of the invention to DDRl include, but are not limited, to taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, irinotecan, topotecan, camptothecin, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, calicheamicin, duocarmycin, actinomycin D, glucocorticoids, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine derivatives like mertansine (also known as DM1) and DM4, and puromycin and analogs or homologs thereof. Specific examples of inhibitory peptides that can be conjugated to the antibodies/binding molecule of the invention to DDRl include but are not limited to the following peptide sequences:
• YARAAARQARAGRGYVSTT ( wherein Y represents a phosphotyrosine), which is a peptide inhibitor of the transcription factor STAT6 which binds only to the phosphorylated form of STAT6 to prevent its dimerization and activity
• PYLKTK (wherein Y represents a phosphotyrosine), which is a phosphopeptide which inhibits the activity of the transcription factor STAT3 in vitro and in vivo
• MVRRFLVTLRIRRACGPPRVRV, which is part of the n-terminal sequence of p 14ARF and it is able to induce apoptosis. In one embodiment, the therapeutic agent for conjugation is a toxin. In a preferred embodiment, the toxin is an enzyme. Specific examples of toxins that can be conjugated to the antibodies/binding molecules of the invention to DDR1 include, but are not limited to plant toxins such as saporin, Ricin or Gelonin, and bacterial toxins such as Pseudomona exotoxin or diphteria toxin, and derivatives thereof. Also, ribonucleases can be considered as toxins due to their ability to degrade RNA and cause cell death. Some Rnases which are considered to have cytotoxic effects and can be used also as toxins are Binase (from Bacillus intermedius), a-sarcin (from Aspergillus giganteus), Ranpirnase (from amphinian), Onconase (from Rana pipiens), and human RNAses like inhibitor-resistant variant of human pancreatic RNase (HP-DDADD- RNase)
The antibodies/binding molecules of the invention may also be conjugated to nanoparticles comprising human serum albumin (HSA) to optimize preparation and uptake of antibodies in cancer cells, as described, for example, by Steinhauser et al., Biomaterials 2006 Oct;27(28):4975-83.
The therapeutic agent(s), such as toxin(s), are preferably suitable for the treatment of cancer and/or a proliferative disorders, etc. Such antibody conjugates with antibodies/binding molecules to DDR1 can readily be prepared for various types of antibodies (and/or fragments thereof) as described above, including chimeric antibodies, deimmunized antibodies, humanized antibodies, fully humanized/human antibodies, single chain antibodies, diabodies and the like. Techniques for conjugating agents, such as the therapeutic agents described above, to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62: 119- 58 (1982)). Conjugates can be prepared using a variety of cleavable linkers such as for example disulfide-based linkers, hydrazone linkers or peptide linkers (Alley et al. (2010) Curr Opin Chem Biol 14(4):529-37; Webb (201 1) Nat.Biotech, 29(4):297-8) or the TAP linker technology from ImmunoGen. Alternatively, the conjugate may be prepared via fusion proteins, as disclosed below.
The antibodies of the invention may also be a fusion wherein the antibody portion (comprising one or more CDRs) is fused to another protein or polypeptide. For example, an antibody according to the invention can be fused to another protein or polypeptide wherein said protein or polypeptide is an agent which improve the properties of said antibody e.g., enhances therapeutic effect. Such proteins or polypeptides which e.g., can enhance therapeutic effect through a number of mechanisms like attracting or enhancing an immune response or delivering a therapeutic agent such a cytotoxic peptide or inhibitory peptide as defined above. Examples of such proteins or polypeptides are cytokines like IL2 or a IL2 homolog or GM-CSF. A nucleic acid encoding the antibody of the invention operably linked to the desired protein or polypeptide can be prepared and introduced into a suitable expression vector, which is then inserted into a host cell for production of the fusion protein.
The antibodies (and fragments thereof) of the invention can also be conjugated to or have a detectable label to molecules for diagnostic purposes. For example, an antibody to DDR1 can be conjugated to a detectable label (e.g., for imaging purposes) for diagnosing or detecting cancer (like e.g. epidermoid or endometrial cancer) and/or a proliferative disorder. Suitable detectable markers include, but are not limited to, a radioisotope, a nanoparticle, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Techniques for conjugating diagnostic agents to antibodies are well known (Holmes et al. (2001) Curr Protoc Cytom. May; Chapter 4:Unit 4.2; Kumar et al (2008) ACS Nano. Mar;2(3):449-56; Rosenthal et al. (2006) Laryngoscope Sep; 116(9): 1636-41). Additionally kits for conjugating agents (such as a detectable label) to diagnostic antibodies are commercially available.
In one embodiment, the present invention relates to a nucleic acid molecule having a sequence encoding the antibody as defined and provided herein. The nucleic acid molecules of the invention, for example, those encoding anti-DDRl antibodies, and its subsequences/alternative transcripts, can be inserted into a vector, which will facilitate expression of the insert. The nucleic acid molecules and the antibodies they encode can be used directly or indirectly as therapeutic (or diagnostic) agents (directly in the case of the antibody or indirectly in the case of a nucleic acid molecule). Accordingly, the present invention relates also to a vector comprising the nucleic acid molecule. The vector may further comprise a nucleic acid molecule having a regulatory sequence which is operably linked to the nucleic acid molecule. The vector may be an expression vector. Further, the present invention relates to a host, host cell or host cell line transformed or transfected with the vector as defined above. In other words, the host, host cell or host cell line expresses the antibody as provided herein. Said host, host cell or host cell line can be prokaryotic or eukaryotic. The host is preferably a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.
The antibody of the invention can be made by any number of methods. For example, the antibody can be synthesized in a cell line harboring a nucleic acid encoding the antibody as described above and culturing said cell line under conditions sufficient to allow expression of said antibody. Accordingly, the present invention relates in one embodiment to a process for the production of the antibody as defined herein, said process comprising culturing a host as defined herein under conditions allowing the expression of the antibody and recovering the produced antibody from the culture. The antibody thus obtained can then be conjugated to a therapeutic agent or to a detectable label for diagnostic purposes, as described above. In the event the antibody is conjugated to a protein (for example a marker or label protein or a therapeutic or a toxic protein) via a fusion protein, a vector encoding the sequence for the fusion protein would be incorporated into the host cell line, which would then be cultured as described above. Techniques for producing and purifying antibodies are well known (see e.g. Liu et al. (2010) MAbs. 2(5):480-99; Shukla et al. (2010) Trends Biotechnol. 28(5):253-61; and Backliwal et al. (2008) Nucleic Acids Res. 36(15):e96).
As used herein, the term "transformed (host) cell" or "transfected (host) cell" (and the like) means a cell into which (or into predecessor or an ancestor of which) a nucleic acid molecule encoding an antibody (or a fragment thereof) of the invention has been introduced, by means of, for example, recombinant DNA techniques or viruses.
An "isolated DNA molecule" is a fragment of DNA that has been separated from the chromosomal or genomic DNA of an organism. Isolation also is defined to connote a degree of separation from original source or surroundings.
"Complementary DNA" (cDNA), often referred to as "copy DNA," is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Those skilled in the art also use the term "cDNA" to refer to a double-stranded DNA molecule that comprises such a single-stranded DNA molecule and its complement DNA strand. The term "expression" refers to the biosynthesis of a gene product, such as a protein or an mRNA molecule.
An "expression vector" is a nucleic acid construct, generated recombinant or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue -preferred regulatory elements, and enhancers. In one embodiment, the invention provides an expression vector comprising a nucleic acid encoding an antibody of the invention (or binding molecule or antibody fragment),
wherein said vector is DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 201 1; or
wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1; or
wherein said vector is DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 201 1.
In a preferred embodiment, the invention provides an expression vector comprising a nucleic acid encoding an antibody of the invention (or binding molecule or antibody fragment), wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1.
The invention also provides an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ablpBhl deposited under accession number DSM 25529 with the depositary institute DSMZ on December 20th, 2011 , using
a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 67 and a 3 '-primer as shown in SEQ ID NO: 68 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 69 and a 3 '-primer as shown in SEQ ID NO: 70 for amplification of a second binding domain.
The invention also provides in a preferred embodiment an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 using
a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
The invention also provides an expression vector comprising a nucleic acid molecule encoding an antibody of the invention (or binding molecule or antibody fragment) wherein said nucleic acid molecule is located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab3pBhl deposited under accession number DSM 25531 with the depositary institute DSMZ on December 20th, 2011 , using
a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 75 and a 3 '-primer as shown in SEQ ID NO: 76 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 77 and a 3 '-primer as shown in SEQ ID NO: 78 for amplification of a second binding domain.
As used herein "first binding domain" means, in relation to each DNA contained in each of the deposited vectors DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl, the VL domain, and "second binding domain" means, in relation to the same DNA, the VH domain.
The person skilled in the art is ready in a position to isolate the coding nucleic acid molecules from the vectors described herein and deposited with DSMZ/Germany on December 20th, 2011. Such routine methods include recombinant technologies, as also provided in Sambrook "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory.
The use of primers for molecular technologies, like PCR for the amplification of relevant nucleic acid molecules, is also well known in the art. The conditions for PCR may be stringent conditions.
Exemplified, but not binding conditions, for amplification of the relevant portions of the above described nucleic acid molecules comprise the following: A PCR reaction of a 20 μΐ volume which comprises the following components: 2μ1 of a 10X Taq Polymerase Buffer reaction, a final concentration of MgC of 1.5mM (to be supplemented in case the Buffer reaction does not include it already), 0.5pMol of each of the below indicated primers pairs, 2μ1 of 25mM dNTPs, 2 units of Taq Polymerase and 10 to 20 ng of the deposited plasmid D A.
For amplification of a first binding domain of vector DDRl-ablpBhl with the primer pair having SEQ ID NO: 67 and SEQ ID NO: 68 or for amplification of a first binding domain of vector DDRl -ab2pBhl with the primer pair having SEQ ID NO: 71 and SEQ ID NO: 72 or for amplification of a first binding domain of vector DDRl-ab3pBhl with the primer pair having SEQ ID NO: 75 and SEQ ID NO: 76 the following cycle conditions can be used:
A denaturation first step of 10 minutes at 95°C; a denaturation step of 45 seconds at 95°C, an annealing step of 40 seconds at 62°C, an extension step of 40 seconds at 72°C, the last three steps will be repeated for 30-35 cycles. After the last cycle, a last step of 5 minutes at 72°C can be added. After that the PCR reaction will be complete and the PCR product can be loaded on a 1.5% agarose gel for checking.
For amplification of a second binding domain of vector DDRl-ablpBhl with the primer pair having SEQ ID NO: 69 and SEQ ID NO: 70 the following cycle conditions can be used: A denaturation first step of 10 minutes at 95°C; a denaturation step of 45 seconds at 95°C, an annealing step of 40 seconds at 58°C, an extension step of 40 seconds at 72°C, the last three steps will be repeated for 30-35 cycles. After the last cycle, a last step of 5 minutes at 72°C can be added. After that the PCR reaction will be complete and the PCR product can be loaded on a 1.5% agarose gel for checking.
For amplification of a second binding domain of vector DDRl-ab2pBhl with the primer pair having SEQ ID NO: 73 and SEQ ID NO: 74 or for amplification of a second binding domain of vector DDRl-ab3pBhl with the primer pair having SEQ ID NO: 77 and SEQ ID NO: 78, the following cycle conditions can be used: A denaturation first step of 10 minutes at 95°C; a denaturation step of 45 seconds at 95°C, an annealing step of 40 seconds at 60°C, an extension step of 40 seconds at 72°C, the last three steps will be repeated for 30-35 cycles. After the last cycle, a last step of 5 minutes at 72°C can be added. After that the PCR reaction will be complete and the PCR product can be loaded on a 1.5% agarose gel for checking.
A "recombinant host" may be any prokaryotic or eukaryotic cell that contains a cloning vector, expression vector, or other heterologous nucleic acid sequences. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell. The term "operably linked" is used to describe the connection between regulatory elements and a gene or its coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene or coding region is said to be "operably linked to" or "operatively linked to" or "operably associated with" the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element.
The terms "isolated" "purified" or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. The antibodies provided herein (as well as the nucleic acids encoding them, the herein provided vectors and hosts) are preferably "isolated" "purified" or "biologically pure" as defined herein. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or antibody of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or antibody gives rise to essentially one band in an electrophoretic gel. For an antibody that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. Various levels of purity may be applied as needed according to this invention in the different methodologies set forth herein. The customary purity standards known in the art may be used if no standard is otherwise specified.
An "isolated nucleic acid molecule" can refer to a nucleic acid molecule, depending upon the circumstance, which is separated from the 5' and 3' coding sequences of genes or gene fragments contiguous in the naturally occurring genome of an organism. The term "isolated nucleic acid molecule" also includes nucleic acid molecules which are not naturally occurring, for example, nucleic acid molecules created by recombinant DNA techniques. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide - nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively, modified variants thereof (for example, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with suitable mixed base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res, 19:081 ; Ohtsuka et al., J. Biol. Chem. (1985) 260:2600-2608 Rossolini et al. (1994) Mol. Cell Probes, 8:91-98). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
A "host cell" is a naturally occurring cell or a transformed cell or a transfected cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells, for example, E. coli, or eukaryotic cells, for example, yeast, insect, amphibian, or mammalian cells, for example, Vero, CHO, HEK293, HeLa, and others. In one embodiment, the present invention relates to a vector encoding an antibody/ binding molecule as defined herein deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively. Accordingly, the present invention relates to an antibody/binding molecule obtained or obtainable by the expression of a coding nucleic acid molecule comprised in a vector as deposited under these accession numbers. Preferably, the present invention relates to an antibody/binding molecule obtained or obtainable by the expression of a coding nucleic acid molecule comprised in a vector as deposited under DSM accession number 25530. Also provided herein is an antibody as defined herein above, prepared by a process comprising (a) providing a cell line capable of producing the antibody; and
(b) culturing the cell line (or a cell line derived therefrom) under conditions that provide for the production of the antibody by the cell line and allow for the recovering of the antibody from the culture,
whereby the cell line comprises a vector as deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 2011 under the designation DDRl-ablpBhl , DDRl-ab2pBhl, and DDRl-ab3pBhl , respectively. In one embodiment, the present invention relates to a vector encoding a binding molecule as defined herein deposited under accession number DSM 25529, preferably DSM 25530, and/or DSM 25531 with the depositary institute DSMZ, Braunschweig, GERMANY on December 20th, 2011 under the designation DDRl-ablpBhl , DDRl-ab2pBhl and DDRl-ab3pBhl , respectively. Accordingly, the present invention relates to an antibody obtained or obtainable by the expression of a vector as deposited under these accession numbers.
As discussed herein above, the present invention also relates to anti-DDRl binding molecules/antibodies that comprise CDRs and/or variable regions that are at least 75% identical (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) to the amino acid sequence of these (individual) CDRs or said variable regions of the sequences disclosed herein or as obtainable from the vectors as deposited under accession number DSM 25529, DSM 25530, or DSM 25531 with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively. Accordingly, the methods of preparation of these binding molecules/antibodies are also provided herein and as laid down herein above.
It is evident that the present invention also relates to antibody/binding molecules that show in their amino acid sequences of their individual CDRs and/or their variable regions at least 75% identity (e.g. 80 %, more preferably 85 %, 90 %, most preferably 95 %, 96 %, 97 %, 98 %, 99 % or more) to the antibody molecules/binding molecules defined herein by their sequences as obtainable from the deposited vectors deposited under accession number DSM 25529, preferably DSM 25530, or DSM 25531 with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDR1- ab3pBhl, respectively. Therefore, the present invention also relates to antibodies/binding molecules that bind to and/or recognize the same epitope on the extracellular domain of DDR1 and/or that have the same functional properties as the antibodies/binding molecules obtainable from the deposited nucleic acid vectors DDRl-ablpBhl , preferably DDRl-ab2pBhl and DDR1- ab3pBhl . The ability of an antibody or binding molecule to bind specifically to DDR1 can be determined using well known assays. Affinity or specificity can be determined experimentally by methods known in the art such as Flow Cytometry (FC), Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Other methods include the use of siRNA agents against DDR1. The appended Examples disclose in more detail some of such methods.
The invention, therefore, also provides pharmaceutical compositions for use in the treatment of cancer (and/or proliferative disorders, etc) comprising an antibody/binding molecule as disclosed herein or having essentially the same biological activity of an antibody/binding molecule obtained or obtainable by expression of the nucleic acid molecule comprised in any of the vectors as deposited with the DSMZ, Braunschweig, GERMANY on December 20th, 201 1 under the designation DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively.
The present invention also relates to pharmaceutical compostions and, as discussed herein below, to diagnostic compositions that comprise the herein disclosed antibodies/binding molecules directed against/binding to the extracellular domain of DDR1 , whereby said pharmaceutical composition and said diagnostic composition have their medical use in the medical amelioration of cancer (and/or proliferative disorders, etc) and in the diagnostic of e.g. cancer (and/or proliferative disorders, etc). In one embodiment, said cancer is , epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In another embodiment, said cancer is epidermoid cancer (also known as squamous cell cancer) or endometrial cancer. In a further embodiment, the present invention relates to a composition comprising the antibody/binding molecule directed against/specifically binding to DDR1 and as defined herein or as produced by the above described process, a nucleic acid molecule as described herein, a vector as described herein and/or a host as described herein. Preferably, the composition comprises the antibody/binding molecule as defined and provided herein. The composition may further comprise (a) secondary antibody/antibodies that is/are specifically binding to the primary antibody as defined and provided in the present invention, whereby said secondary antibody/antibodies is/are conjugated to a therapeutic agent as defined above (in particular an anticancer/cyto toxic agent or a toxin such as Saporin) or a diagnostic agent as defined and explained herein above. The primary antibody is preferably an IgG antibody. The secondary antibody may be a goat anti-human IgG secondary antibody. The secondary antibody may also be any of the antibody types as described herein above in context of the anti-DDRl antibodies provided herein.
In a preferred embodiment, the herein above described composition is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s) like, inter alia, stabilizers or carriers. Corresponding excipients are also provided herein below as non-limiting examples. In accordance with this embodiment, the antibody as provided herein, or the antibody as produced by the herein above described process, the nucleic acid molecule described herein, the vector described herein, the host as described herein and/or the composition (in particular the pharmaceutical composition) is for use in medicine. Preferably, the antibody as provided herein (optionally contained in the composition as defined above) is for use in medicine. In one embodiment, the antibody is conjugated to a therapeutic agent or to a diagnostic agent (like a label etc.). In a further embodiment, the present invention relates to the use of the antibody as defined or provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for the preparation of a pharmaceutical composition for the treatment of e.g., cancer and/or a tumorous disease or proliferation disorder. Preferably, the present invention relates to the use of the antibody as defined or provided herein for the preparation of a pharmaceutical composition for the treatment of cancer and/or a tumorous disease or a proliferative disorder. In an alternative embodiment, the present invention relates to the antibody as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for use in the treatment of cancer etc. In one embodiment, the invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, ovarian cancer. The present invention relates to the antibody as defined and provided herein for use in the treatment of cancer selected from the group consisting of epidermoid cancer (squamous cell cancer), endometrial cancer, bladder cancer, colon cancer, stomach cancer, lung cancer, breast cancer, urothelial cancer, prostate cancer, head and neck cancer and skin cancer. More preferably, the present invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer (also known as squamous cell cancer) or bladder cancer. In one embodiment, the present invention relates to the antibody as defined and provided herein for use in the treatment of epidermoid cancer or endometrial cancer. .
Also a method for the treatment of cancer, a proliferative disorder, etc. is subject of the present invention, said method comprising the administration of the antibody/binding molecule as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein to a subject in need of such a treatment. A "patient" or "subject" for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the subject is a mammal, and in the most preferred embodiment the subject is a human.
A "cancer" in an animal refers to the presence of cells possessing one or more characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers. Preferably, the cancer to be treated is selected from epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer and ovarian cancer. In one embodiment, the cancer to be treated is epidermoid cancer or endometrial cancer.
A "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The term "precancerous" refers to cells or tissues having characteristics relating to changes that may lead to malignancy or cancer.
A "proliferative disorder" comprises, but is not limited to cancer and tumorous, cancerous or pre-cancerous disorders. A proliferative disorder may also comprise autoimmune or inflammatory (particularly chronic inflammatory) disorders. One such inflammatory disorder is atherosclerosis.
The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing a disease related to an insufficient immune response from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
"Treating" or "treatment" does not necessarily require a complete cure. It means that the symptoms of the underlying disease are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that reduced, as used in this context, means relative to the state of the disease, including the molecular state of the disease, not just the physiological state of the disease. In a more specific embodiment, the invention provides a method of treating an individual having cancer, tumorous disease and/or a proliferative disorder comprising administering to said individual a therapeutically effective amount of an antibody or fragment thereof to DDRl . In one aspect of this embodiment, the antibody reduces levels of activity of DDRl . In one aspect, the antibody or fragment thereof induces internalization or aggregation of DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl inhibits or reduces proliferation; causes cytotoxicity; inhibits or reduces metastasis; modulates, inhibits or reduces cell adhesion; modulates, inhibits or reduces migration; or modulates, inhibits or reduces invasion of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl inhibits or reduces proliferation of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl causes cytotoxicity to cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits metastasis of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits cell adhesion of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits invasion of cancer or cancer cells expressing DDRl . In one aspect of this embodiment, the antibody or fragment thereof to DDRl reduces or inhibits migration of cancer or cancer cells expressing DDRl . In one embodiment, said cancer is endometrial cancer, epidermoid cancer (also known as squamous cell cancer), bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. . In another embodiment, said cancer is epidermoid cancer or endometrial cancer
Testing anti-DDRl Antibody for anti-cancer properties can be done using standard assays, well known in the art. For example, a cancer cell line (e.g. an epidermoid cancer cell line or an endometrial cancer cell line) is grown and propagated in culture according to methods well known to one of ordinary skill in the art. Various dosages of potentially therapeutic antibodies or fragments thereof or conjugates thereof according to the invention are applied to various cultures of the (cancer) cell line. The treated cultures and control cultures (treated with a sham antibody or fragment) are then followed over time and scored for reduction in proliferation; reduction in cellular growth; reduction in colony formation; appearance of cytotoxicity; reduction in cell-adhesion; reduction of cell invasion; reduction of degradation of the extracellular matrix; or reduction in cell migration or reduction in cell inavtion through differents extracelular matrix proteins. In vivo, the antibodies/binding molecules of the invention or conjugates thereof can be tested in animals having a tumor and/or an animal that have had a cancer cell line implanted subcutaneusly (see for example Talmadge et al. Am J Pathol 2007 Mar 170(3), Cespedes et al. Clin Transl Oncol 2006 May, 8(5)) or ortotopically (Liu et al. Oncol Rep 2011 Nov 22, Vahle et al. Cancer Lett 2011 Nov 25, Bhattacharya et al. Carcinogenesis 2011 Nov 30, Otto et al Br J Cancer 2011 Dec 6, Wenner et al. Int J Oncol 2011 Dec 12) and allowed to proliferate to form a tumor. A brief description of subcutaneus models can be done injecting 1 to lOxlO6 cells of a human cancer cell line into the right or left flank of a immunosuppresed mice. Some examples of immunosupresed mouse strains are Balbc nu/nu, NOD SCID, Fox Chase SCID, Athymic nude or Swiss nude . Cells are allowed to grow until the generated tumor reachs a diameter of about a 100mm3. At this point, the mice are treated with the antibodies to be tested. Routes of antibody administration into mice include intravenous or intraperitoneal administration. Various dosages of potentially therapeutic antibodies or fragments thereof according to the invention (or combinations of a mix of antibodies or combination of the antibodies with chemotherapy) can be tested in in vivo models. The treated animals and control animals (treated with a sham antibody or fragment or no antibody at all) are then followed over time and scored for reduction in tumor size, reduction in tumor weight, reduction in tumor cell proliferation, reduction in tumor cellular growth; appearance of cytotoxicity; reduction in tumor cell-adhesion; reduction in metastasis, reduction in tumor cell invasion, reduction in tumor cell migration or increase in survival.
The appended examples also provide for relevant lists of the inventive antibodies/binding molecules.
In one aspect of this embodiment, the antibody to DDR1 induces, enhances, or mediates ADCC (antibody dependent cellular cytotoxicity) against cells to which it binds. ADCC is one of the mechanism by which an antibody can have a therapeutic effect. ADCC is a cell mechanism where an effector cell of the immune system, mainly Natural Killer cells (NK), lyses a target cell which has been previously bound by specific antibodies. NK cells have specific receptors such as FcyRIIIa which recognize the Fc fragment of immunoglobulins and are responsible for the ADCC response. To test if the antibodies of the invention have a therapeutic effect through a ADCC mechanism, an in vitro assay can be performed in which target cells will be incubated with different antibodies and natural killer cells from human or mouse origin. The effect of the antibodies on the cells can be measured by the occurred lyses.
In one aspect of this embodiment, the antibody to DDR1 induces, enhances, or mediates CDC (complement dependent cytotoxicity) against cells to which it binds. CDC is another immune mechanism to exert cytotoxicity on tumor cells. CDC is a cytolytic cascade mediated by complement proteins in the serum. CDC is initiated by the binding of CI q to the constant region of cell bound antibody molecule.
In another embodiment, the antibody to DDR1 is conjugated to another molecule. In a more specific aspect, the antibody is conjugated to a therapeutic agent, such as a toxin, a radioactive agent, inhibitory peptide, or an anti-tumor drug. The antibody (or fragment thereof) of this embodiment can be provided as a pharmaceutical composition comprising the antibody (or fragment thereof) conjugated to the therapeutic agent and a pharmaceutically acceptable excipient. Pharmaceutical compositions of this invention also can be administered in combination therapy ("cotherapy"), i.e., combined with other agents. For example, the combination therapy can include an anti-DDRl antibody of the present invention combined with at least one other therapeutic agent (e.g. anti-cancer agent); "combined" as used herein means that the at least one other therapeutic agent is not conjugated (as defined above) to the herein provided anti-DDRl antibody (however, the antibody used in cotherapy with one or more other therapeutic agents may, in itself, be conjugated to one or more of the therapeutic agents as defined herein above). The administration of the other therapeutic agent can be prior to, concurrent to or after the administration of the antibody of the invention. The antibody of the invention and the one or more other therapeutic agents may also be combined into a single dosage unit. Furthermore, the invention includes a pharmaceutical composition comprising two or more antibodies to DDR1. Without wishing to be bound by theory, it may be believed that treatment with two or more therapeutic antibodies to DDR1 can have synergistic effects in terms of therapy. Examples of therapeutic agents that can be used in combination therapy are described in greater detail below. In one aspect of this embodiment, the method comprises identifying a patient having a risk factor for cancer and/or a proliferative disorder, obtaining a sample from said patient having a risk factor for such a disorder, and determining the level of DDR1 in said sample wherein a patient having an increased level of DDR1 is treated with an antibody that binds to or modulates DDR1. In one aspect of this embodiment, the risk factor for cancer and/or a proliferative disorder is chosen from age, ethnicity, family history of cancer and/or a proliferative disorder, or genetic predisposing gene or variant thereof. Risk factors for cancer and/or a proliferative disorder are known to the skilled artisan. In one specific aspect the risk factor for cancer and/or a proliferative disorder is one or more SNPs that indicated a higher risk of having cancer and/or a proliferative disorder.
In one embodiment of the invention the subject or patient to be treated was previously treated or is currently being treated with radiation therapy. In a more specific embodiment, the invention provides a method of treatment of cancer and/or a proliferative disorder in a patient wherein said patient was previously treated or is currently being treated with radiation therapy. In one aspect of this embodiment, the treatment comprises identifying a patient previously treated or is currently being treated with radiation therapy and administering to said patient a DDR1 therapeutic antibody as defined herein. Radiation therapy for cancer and/or a proliferative disorder is generally classified as external or internal. External radiation therapy usually involves the focusing of high energy beams of energy (e.g., x-rays) on the affected area. Internal radiation therapy involves implanting a radioactive substance or composition comprising a radioactive substance near or inside the cancer (also referred to as brachytherapy, internal radiation therapy, and/or radiation brachytherapy).
In one embodiment of the invention, the subject or patient will be treated or is currently being treated with a chemotherapy or a radiotherapy. In a more specific embodiment, the invention provides a method for treating cancer and/or a proliferative disorder in a patient wherein said patient had discontinued a prior treatment due to disease progression. In one aspect, disease progression occurred due to the cancer developed chemoresistance to the prior treatment. In one aspect, said cancer chemoresistance was or is correlated to increased DDRl expression or activation. In a specific embodiment the antibodies to DDRl confer chemosensitivity to chemoresistant cancer or cancer cells, or increase chemosensitivity of the cancer or cancer cells. The ability of an antibody of the invention to confer or increase chemosensitivity to chemoresistant cancer cells can be tested as follows. Chemoresistant target cells (e.g, expressing DDRl or overexpressing DDRl) are plated on 96 well plates and incubated with the DDRl antibodies to be tested (e.g., of the invention) with and without a chemotherapeutic agent under conditions sufficient for cell growth and proliferation. The effect of the treatments on cell proliferation will be measured by an Alamar Blue assay or similar assays as described herein e.g., cytotoxicity For example, a cell line from epidermoid cancer, endometrial cancer, bladder cancer, colon cancer, lung cancer, breast cancer,or prostate cancer overexpressing DDRl and which is resistant to a chemotherapeutic agent is tested in the presence of (1) DDRl antibody (as described herein) and (2) the presence of DDRl antibody (as described herein) and chemotherapeutic agent and (3) chemotherapeutic agent, wherein an increase in sensitivity to the chemotherapeutic agent in the presence of DDRl antibody indicates said antibody increases chemosensitivity or overcomes chemoresistance. Without wishing to be bound by theory, it is believed that cancer cells expressing or overexpressing DDRl when treated with the antibodies of the invention to DDRl will increase chemosensitivity or restore chemosensitivity or overcome chemoresistance.
In one embodiment of the invention, the subject or patient to be treated has hormone dependent endometrial cancer. In one aspect of this embodiment, the treatment comprises identifying a patient having hormone dependent endometrial cancer and administering to said patient a DDRl antibody. In a further aspect, the method comprises administering to said patient having hormone dependent endometrial cancer a DDRl antibody and another therapeutic agent which is hormone therapy.
In one embodiment of the invention, the subject or patient to be treated has hormone -refractory or resistant endometrial cancer. In one aspect of this embodiment, the therapy comprises identifying a patient having treatment-refractory endometrial cancer and administering to said patient a DDR1 antibody. In a further aspect, the treatment comprises administering to said patient having treatment-refractory endometrial cancer a DDR1 antibody and another therapeutic agent which is hormone therapy.
As mentioned previously, the invention also relates to a pharmaceutical composition comprising an antibody or binding molecule of the invention, as described herein, optionally further comprising one or more pharmaceutically acceptable excipient(s). As used herein, "pharmaceutically acceptable excipient" relates to any component of a pharmaceutical composition other than the active ingredient and includes any and all carriers, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the excipient is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The pharmaceutical compounds of this invention may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N.N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A pharmaceutical composition of this disclosure also may include a pharmaceutically acceptable anti-oxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA)5 butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of this disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization micro filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The amount of active ingredient (i.e. the herein provided antibody, nucleic acid molecules etc.) which can be combined with a excipient to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a excipient to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with (a) pharmaceutically acceptable excipient(s).
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient. The specification for the dosage unit forms of this disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. For administration of the antibody, the dosage typically ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 10 mg/kg, of the host body weight. Typically, when the antibody is administered as an ADC, the ADC will be administered at a dose of less than 1 mg/kg. Antibody/binding molecules etc. can also be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A "therapeutically effective dosage", "therapeutically effective amount" or "effective amount" of an anti-DDRl antibody (or nucleic acid etc.) of this invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of DDR1+ tumors, a "therapeutically effective dosage" preferably inhibits cell growth or tumor growth by at least about 5%, more preferably by at least about 10%, even more preferably by at least about 20%, and still more preferably by at least about 60% relative to untreated subjects (or cells in cell based studies). The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth. Such inhibition can be measured in vitro by assays known to the skilled practitioner (cell proliferation, metastasis, cytotoxicty, invasion, migration, etc.). A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. A composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for antibodies of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Alternatively, an antibody of this disclosure can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. The active compounds can be prepared with excipients that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, eds, Marcel Dekker, Inc., New York, 1978. Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of this disclosure can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941 ,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486, 194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. In certain embodiments, therapeutic antibodies of this disclosure can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of this disclosure cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,81 1 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); marmosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pi 20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; JJ. Killion; U. Fidler (1994) Immunomethods 4:273. As previously described, when used in therapy, the antibodies of the invention can be coadministered (i.e. administered in combination) with one or more other therapeutic agents. The antibody of the invention may be, as described above, a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment, a F(ab)2-fragment or an epitope-binding fragment, as well as a single-chain antibody and may be a monoclonal antibody, a recombinantly produced antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a synthetic antibody or a cross- cloned antibody, a diabody, a triabody, a tetrabody, a single chain antibody, a bispecific single chain antibody, etc. The antibody of the invention may itself be linked to another agent like an anticancer (i.e. cytotoxic agent), i.e. be a conjugated antibody as described above. When used in the therapy of cancer, examples of chemotherapeutic agents that may be used in combination with the DDR1 antibodies of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, azathioprine, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil, decarbazine, capecitabine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BCNU), lomustine (CCNU), cyclophosphamide, ifosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, procarbazine, altretamine and tetrazines), anthracyclines (e.g., daunorubicin, doxorubicin, valrubicin, idarubicin, epirubicin, and mitoxantrone), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin (AMC)), topoisomerase inhibitors (e.g. irinotecan, topotecan and camptothecin), anti-mitotic agents (e.g., vinca alkaloids such as vincristine and vinblastine, taxanes such as paclitaxel (also known as taxol), cabazitaxel and docetaxel, and other tubulin polimeryzation inhibitors such as monomethyl auristatin E (MMAE), maytansine derivatives like mertansine (also known as DM1) and DM4), and protein kinase inhibitors such as imatinib (gleevec), nilotinib and dasatinib.
For other co-therapeutic approaches for example for the use of the inventive antibodies/binding molecules in anti-inflammatory therapy, the following drugs/agents may be employed: steroids such as Glucocorticoids, Non-Steroidal anti-inflammatory drugs such as aspirin, ibuprofen, naproxen or Immune Selective Anti-Inflammatory Derivatives (ImSAIDs) such as the peptide phenylalanine-glutamine-glycine (FEG). For the treatment of atherosclerosis the antibodies of the invention can be combined with e.g. statins or niacin.
The following relates to antibody dependent and complement dependent cytotoxicity. In one embodiment, the invention relates to a DDR1 antibody that induces, enhances, or mediates antibody-dependent cellular cytotoxicity (ADCC). ADCC as described above is a type of immune reaction in which a target cell is coated with antibodies and killed by certain types of white blood cells, particularly NK cells. The white blood cells bind to the antibodies and release substances that kill the target cells or microbes. Not all antibodies produce ADCC. Thus, in one embodiment, the invention relates to an antibody to DDR1 that can induce, enhance or mediate ADCC. Furthermore, antibodies of the invention to DDR1 can be engineered to have improved, increased or enhanced ADCC. For example an antibody of the invention that does not induce, enhance, or mediate ADCC can be engineered, e.g., by making certain amino acid modifications to the antibody or by producing the antibody in certain strains of cells, to induce, enhance or mediate ADCC or have improved/enhanced ADCC properties. In one aspect of this embodiment, an antibody to DDRl has antibody-dependent cellular cytotoxicity when used in a human subject. One example of an antibody with increased or improved ADCC activity is a antibody to DDRl that is defucosylated. In one aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that lacks or has decreased alpha- 1 ,6-fucosyltransferase activity. In another aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks GDP-fucose transporter activity. In another aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell- line that has reduced or lacks GDP-mannose 4,6-dehydratase activity. In another aspect of this embodiment, an antibody to DDRl having ADCC or increased ADCC is generated by producing the antibody in a cell-line that has reduced or lacks both alpha- 1 ,6-fucosyltransferase activity and GDP-mannose 4,6- dehydratase activity. See e.g., Yamane-Ohnuki et al. (2004) Biotechnol Bioeng. 87(5):614-22; Imai-Nishiya et al. (2007) BMC Biotechnology 7:84.
In another aspect of this embodiment, ADCC can be enhanced or improved by increasing the levels of interleukin-21 (IL-21) in a patient or by treating the patient with IL-21 in combination with the antibody of the invention. See e.g., Watanabe et al. Br J Cancer. 2010, 102(3), 520-9.
In one embodiment, the invention relates to a anti-DDRl antibody/binding molecule that enhances, induces or mediates complement dependent cytotoxicty (CDC). Thus, in one embodiment, the invention relates to an antibody/binding molecule to DDRl that induces, enhances or mediates CDC. Furthermore, antibodies of the invention to DDRl can be engineered to have improved, increased or enhanced CDC. For example an antibody of the invention that does not induce or mediate CDC can be engineered, e.g., by making certain modifications to the antibody like amino acid mutations in Fc or the hinge region thereby improving or enhancing CDC. Another method of producing CDC or enhancing an antibody's CDC is by shuffling IgGl and IgG3 sequences within the heavy chain constant region. See e.g., Natsume et al. (2008) Cancer Res. 68:3863-3872.
In a further embodiment, the composition provided herein is a diagnostic composition further comprising, optionally, means and methods for detection. In accordance with the present invention, a method for diagnosing cancer and/or a proliferative disorder is described, comprising detecting or assaying DDRl in a biological sample of an individual suspected of suffering from cancer and/or a proliferative disorder using the antibody as defined herein, in particular the antibody conjugated with a detectable label as described above. Suitable detectable labels or markers include, but are not limited to, a radioisotope, a nanoparticle, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. In general, a "label" or a "detectable moiety" is a compound that when linked with the antibody of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens. In one aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In one specific aspect, said cancer is epidermoid cancer or endometrial cancer.
The usefulness of the antibodies/binding molecules to DDRl or fragments thereof, such as those identified or created by any of the Examples disclosed herein, in the diagnosis of cancer (or a proliferative disorder) and/or increased risk for developing cancer (or a proliferative disorder) can be tested as follows:
A cohort of subjects is identified and a sample collected from each subject. The sample is tested for levels of DDRl using the antibodies or fragments thereof to DDRl . All subjects may be further tested for the presence of cancer and/or said proliferative disorder using techniques standard in the art. All subjects may be followed and periodically tested using the inventive antibodies/binding molecules or fragments thereof to DDRl and further tested for the presence of cancer and/or said proliferative disorder using techniques standard in the art. After each round of testing, the levels of DDRl are correlated with the presence of cancer and/or said proliferative disorder and/or increased risk for developing cancer. Accordingly, the present invention relates to the use of the antibody as defined and provided herein, the antibody as produced by the herein above described process, the nucleic acid molecule as described above, the vector as described herein and/or the host as described herein for the preparation of a diagnostic composition for the diagnosis of cancer (and/or a proliferative disorder). Preferably, the present invention relates to the use of the antibody as defined and provided herein for the preparation of a diagnostic composition for the diagnosis of cancer (and/or a proliferative disorder). In one aspect, said cancer is epidermoid (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In one specific embodiment, said cancer is epidermoid cancer or endometrial cancer.
In an alternative embodiment, the present invention relates to the antibody/binding molecule as defined and provided herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for use in the diagnosis of cancer (and/or a proliferative disorder). Preferably, the present invention relates to the antibody as defined and provided herein for use in the diagnosis of cancer (and/or a proliferative disorder). In one aspect, said cancer is epidermoid (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In one specific embodiment, said cancer is epidermoid cancer or endometrial cancer.
In one embodiment, the present invention relates to the use of the antibody/binding molecule as defined and provided herein, the antibody/binding molecule as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, and/or the host as described herein for the preparation of a diagnostic kit for the diagnosis of cancer (and/or a proliferative disorder). In a specific aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In one specific embodiment, said cancer is epidermoid cancer or endometrial cancer.
As used herein in context of diagnostic purposes, "DDR1" (or "DDR1 biomarker") refers to a "DDR1 nucleic acid" or a "DDR1 protein" that can be specifically detected for diagnostic purposes. Definitions of DDR1 (especially of DDR1 proteins and nucleic acids encoding DDR1) have been provided herein above. These explanations apply, mutatis mutandis, in this context. In accordance with these explanations, a DDR1 nucleic acid can be a R A molecule, DNA molecule, or other nucleic acid that corresponds to the DDR1 gene or a fragment thereof. For example, a DDR1 gene may correspond to a human DDR1 gene. For example, a DDR1 nucleic acid can be a cDNA, or fragment thereof, corresponding to a DDRl mRNA molecule. A DDRl protein refers to a protein (or fragment thereof) encoded or expressed by the DDRl gene.
In general, a "DDRl gene" can be a region on the genome that is capable of being transcribed to an RNA that encodes a DDRl protein as well as the regulatory sequences associated with or operably linked to the coding region. The skilled artisan will appreciate that the present invention encompasses all encoding transcripts that may be found, including splice variants, allelic variants and transcripts that occur because of alternative promoter sites or alternative polyadenylation sites of DDRl . A "full-length" gene or RNA therefore encompasses any naturally occurring splice variants, allelic variants, other alternative transcripts, splice variants generated by recombinant technologies which bear the same function as the naturally occurring variants, and the resulting RNA molecules. A "fragment" of a gene, can be any portion from the gene, which may or may not represent a functional domain, for example, a catalytic domain, a DNA binding domain, etc. A fragment may preferably include nucleotide sequences that encode for at least 25 contiguous amino acids, and preferably at least about 30, 40, 50, 60, 65, 70, 75 or more contiguous amino acids or any integer thereabout or therebetween.
The phrase "detecting cancer (and/or a proliferative disorder)" or "diagnosing cancer (and/or a proliferative disorder)" refers to determining the presence or absence of cancer or a precancerous condition in an subject, preferably in a human. "Detecting cancer (and/or a proliferative disorder)" or "diagnosing cancer (and/or a proliferative disorder)" also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the subject or assessing the predisposition of a subject to the development of cancer (and/or a proliferative disorder). Detecting cancer (and/or a proliferative disorder) can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the subject. The "diagnosis of cancer (and/or a proliferative disorder)" may, in particular, comprise (i) the diagnosis for curative purposes stricto sensu representing the deductive medical or veterinary decision phase as a purely intellectual exercise, (ii) the preceding steps which are constitutive for making that diagnosis, and (iii) the specific interactions with the human or animal body (or biological sample, like a blood sample or a tissue sample) which occur when carrying those out among these precedings steps which are of a technical nature.
Thus, in one aspect, the invention provides a method for diagnosis of cancer (and/or a proliferative disorder) by determining the level or activity of a DDRl biomarker in a biological sample from an individual wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder), whereby the DDRl biomarker (preferably the DDRl protein as defined herein) is detected with an anti-DDRl antibody (i.e. the antibody/binding molecule specifically binding to DDRl) of the invention as provided and described herein. In one aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer In a specific embodiment, said cancer is epidermoid cancer or endometrial cancer.
The invention therefore relates to a method for the diagnosis of cancer (and/or a proliferative disorder) by contacting an antibody/binding molecule of the invention that specifically binds to DDRl and that is capable of detecting a DDRl biomarker with a biological sample from an individual and determining the level of the DDRl biomarker, wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder). In a specific aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer In one specific embodiment, said cancer is epidermoid cancer or endometrial cancer.
In a specific aspect, the invention provides a method for the diagnosis of cancer (and/or a proliferative disorder) by contacting the herein provided antibody/binding molecule to DDRl that is capable of detecting a DDRl biomarker with a tissue, tumor, blood, serum, plasma, body fluid or urine sample from an individual and determining the level of the DDRl biomarker, wherein an altered level of DDRl biomarker in the biological sample as compared to a control or normal value is diagnostic of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder). In an even more specific aspect, the biological sample is a blood, serum, plasma, body fluid or urine. In a preferred aspect, the biological sample is a blood, serum, or plasma sample. In a more preferred aspect, the biological sample is a plasma sample. In a preferred aspect, an increased or elevated level of DDRl biomarker as compared to a control or normal value is indicative of cancer (and/or a proliferative disorder) or an increased likelihood of cancer (and/or a proliferative disorder).
In one aspect of this embodiment, the invention includes a method of diagnosing cancer (and/or a proliferative disorder) disease associated with DDRl expression comprising:
(a) obtaining or providing a biological sample from an individual and
(b) determining the level of DDRl biomarker in the biological sample using the herein provided antibodies to DDRl,
wherein an increased level of DDRl biomarker compared to control or a normal value indicates said disease associated with DDRl expression or an increased likelihood of cancer (and/or a proliferative disorder). In a more specific aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In another more specific aspect, said cancer is epidermoid cancer or endometrial cancer.
In a preferred aspect of this method, the sample is a serum, blood, body fluid, endometrial fluid aspirate, plasma, or urine sample. In a more preferred aspect of this method, the sample is a plasma sample. In another specific aspect, in a method of diagnosing endometrial cancer, the sample is an endometrial fluid aspirate. In one aspect, the DDRl biomarker is a nucleic acid biomarker. In one aspect, the DDRl biomarker is a protein biomarker.
In one embodiment, the present invention provides a method for characterizing a sample obtained from a patient for prognostic, diagnostic and/or pharmaco genomic uses in respect of cancer (and/or a proliferative disorder).
Characterization of a sample obtained from a patient by determining the level of a DDRl biomarker can be used to provide information regarding diagnosis of cancer (and/or a proliferative disorder), prognosis of cancer (and/or a proliferative disorder), disease progression, diagnosis of cancer type (and/or subtype), and selection of an appropriate therapeutic treatment for the cancer (and/or a proliferative disorder). According to the method of the invention, a biological sample is obtained from an individual. The individual can be a healthy person, an individual diagnosed with cancer (and/or a proliferative disorder), an individual suspected of having endometrial cancer, an individual displaying one or more symptoms of cancer (and/or a proliferative disorder) and/or an individual desiring screening for cancer (and/or a proliferative disorder). The method comprises the step of determining the level of a DDRl biomarker in a sample obtained for a patient wherein the DDRl biomarker is a protein or nucleic acid biomarker. Preferably the DDRl biomarker is a DDRl protein biomarker. In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding prognosis of the cancer (and/or a proliferative disorder). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding disease progression of the cancer (and/or a proliferative disorder). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information diagnosis of cancer (and/or a proliferative disorder) cell type (and/or subtype). In one specific aspect, characterization of a sample obtained from a patient by determining the protein or activity level of DDRl can be used to provide information regarding selection of an appropriate therapeutic, for example, chemotherapy and/or an antibody of the invention (for example antibodies to DDRl that are coupled or conjugated with a therapeutic agent).
"Subject" as used herein in context of diagnosis of cancer (and/or a proliferative disorder) refers in particular to a biological subject that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDRl . In some embodiments, the subject may be a mammalian subject, preferably a human.
"Biological sample" as used herein refers to a sample obtained from a subject, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDRl . A biological sample also includes samples from a region of a biological subject containing or suspected of containing precancerous or cancer (and/or a proliferative disorder) cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from mammals including humans. Biological samples also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes. A biological sample can be obtained using commonly employed clinical practices (e.g. fine needle biopsy, fluid aspirate, blood from a blood draw, serum or plasma derived from blood, tumor sections, circulating tumor cells and the like).
A biological sample, as described herein, can be a "control" or a "control sample" or a "test sample". A "control" refers to a representative of healthy, cancer-free biological subject or information obtained from a different individual or a normalized value, which can be based on baseline data obtained from a population or other acceptable sources. A control also can refer to a given level of DDR1, representative of the cancer- free population, that has been previously established based on measurements from normal, cancer- free animals. A control also can be a reference data point in a database based on data obtained from control samples representative of a cancer-free population. Further, a control can be established by a specific age, sex, ethnicity or other demographic parameters. In some situations, the control is implicit in the particular measurement.
A "control sample" refers especially to a sample of biological material representative of healthy, cancer- free animals or a normal biological subject obtained from a cancer- free population. The level of DDR1, in a control sample is desirably typical of the general population of normal, cancer-free animals of the same species. This sample either can be collected from an animal for the purpose of being used in the methods described in the present invention or it can be any biological material representative of normal, cancer-free animals suitable for use in the methods of this invention. A control sample also can be obtained from normal tissue from the animal that has cancer or is suspected of having cancer.
A "test sample" as used herein refers especially to a biological sample, including sample of biological tissue or fluid origin, obtained, reached, or collected in vivo, ex-vivo, or in situ, that contains or is suspected of containing nucleic acids or polypeptides corresponding to DDR1. A test sample also includes biological samples containing or suspected of containing precancerous or cancer cells or tissues. A test sample also may include sections of the biological sample including tissues, for example, frozen sections taken for histologic purposes.
"Providing a sample, a biological sample, or a test sample" means to obtain from a subject a sample, in vivo, ex-vivo, or in situ, including tissue or cell sample for use in the methods described in the present invention. Most often, this will be done by removing a sample of cells from an animal, but also can be accomplished in vivo, ex-vivo, or in situ, or by using previously isolated cells (for example, isolated from another subject, at another time, and/or for another purpose).
"Data" includes, but is not limited to, information obtained that relates to "biological sample," "test sample." "control sample," and/or "control," as described above, wherein the information is applied in generating a test level for diagnostics, prevention, monitoring or therapeutic use. The present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper formats. Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk; tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above.
"Overexpression" of a gene or an "increased," or "elevated," level of a ribonucleotide or protein refers to a level of the gene, ribonucleotide or polypeptide that, in comparison with a control level of gene, ribonucleotides or polypeptide, is detectably (preferably statistically significantly) higher. Comparison may be carried out by statistical analyses on numeric measurements of the expression; or, it may be done through visual examination of experimental results by qualified researchers.
A level of ribonucleotide or polypeptide, that is "expected" in a control sample refers to a level that represents a typical, cancer-free sample, and from which an elevated, or diagnostic, presence of the polypeptide or polynucleotide, can be distinguished. Preferably, an "expected" level will be controlled for such factors as the age, sex, medical history, etc., of the mammal, as well as for the particular biological subject being tested.
The phrase "functional effects" in the context of an assay or assays for testing compounds that modulate a particular gene's activity includes the determination of any parameter that is indirectly or directly under the influence of the gene, for example, a functional, physical, or chemical effect, for example, of the genes activity, activity of a polypeptide encoded by the gene, the ability to induce gene amplification or overexpression in cancer cells, and to aggravate cancer cell proliferation. "Functional effects" include in vitro, in vivo, and ex vivo activities.
"Determining the functional effect" refers to assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of the gene or the polypeptide encoded by the gene, for example, functional, physical, and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, for example, changes in spectroscopic characteristics (for example, fluorescence, absorbance, refractive index), hydrodynamic (for example, shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the gene, measuring binding activity or binding assays (for example, substrate binding, and measuring cellular proliferation), measuring signal transduction, or measuring cellular transformation.
In one aspect of the invention, the present invention relates to methods for comparing and compiling data wherein the data is stored in electronic or paper format. Electronic format can be selected from the group consisting of electronic mail, disk, compact disk (CD), digital versatile disk (DVD), memory card, memory chip, ROM or RAM, magnetic optical disk, tape, video, video clip, microfilm, internet, shared network, shared server and the like; wherein data is displayed, transmitted or analyzed via electronic transmission, video display, telecommunication, or by using any of the above stored formats; wherein data is compared and compiled at the site of sampling specimens or at a location where the data is transported following a process as described above. The data of this embodiment is information regarding the results of the analysis of DDR1. The compounds, targets, assays, tests, inquiries and methodologies described herein can be employed in a variety of contexts, including diagnostic and therapeutic discovery, diagnostic and therapeutic development, safety and efficacy monitoring, compound and treatment regimen potency determination and validation, treatment assessment, comparative studies, marketing and the like. The information provided by the invention can be communicated to regulators, physicians and other healthcare providers, manufacturers, owners, investors, patients, and/or the general public. This information and the like can be used in exploratory research, pre-clinical and clinical settings, labeling, production, advertising, and sales, for example.
In a further embodiment, the present invention relates to a kit comprising the antibody as provided and described herein, the antibody as produced by the herein described process, the nucleic acid molecule as described herein, the vector as described herein, the host and/or the composition as described herein. Preferably, the kit comprises the antibody as provided and described herein. Preferably, the kit is used for the diagnosis of cancer (and/or a proliferative disorder). In a specific aspect, said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer, or ovarian cancer. In one specific embodiment, said cancer is epidermoid cancer orendometrial cancer In a particularly preferred embodiment of the present invention, the kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to diagnose cancer (and/or a proliferative disorder) in accordance with the present invention. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses. The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the expression level of DDR1 as defined herein.
Brief description of the Figures Figure 1 shows the results of a immunofluorescent staining of DDRl-ab2 with a fluorescent Cy2 labeled secondary anti -human antibody on DDR1 overexpressed cells from clone 44 which have been previously incubated with DDRl-ab2 and fixed at different times. The pictures show the different results when the staining is done under permeable (bottom row), or non permeable conditions (top row) (see Example 7). Top row: the staining under non-permeable conditions visualizes the DDRl-ab2 on the outer cell surface. Bottow row: the staining under permeable conditions visualizes the efficient internalization of DDRl-ab2 on outer cell surface towards the inside of the cell. After 5 minutes of co-incubation most of the fluorescent signal is still located at the cell surface, after 15 minutes of co-incubation, internalization has clearly started and by 30 minutes of co-incubation, most of the signal is located with the cell in a spotted pattern typical for endosomes.
Figure 2 shows the results of cell proliferation assays for the indicated cells treated with DDR1- ab2 and a secondary antibody to human IgG carrying saporin. The two cells types are CHO (o) and a CHO line overexpressing DDR1(B) (Clone 44) (see Example 8).
Figure 3 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-abl (white diamond) or DDRl-abl plus a secondary antibody to human IgG carrying saporin (·). (see Example 10). Figure 4 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-ab3 (□) or DDRl-ab3 plus a secondary antibody to human IgG carrying saporin (·). (see Example 10). Figure 5 shows the results of cell proliferation assays for the indicated cells (AN3CA) treated with DDRl-ab2 (white triangle) or DDRl-ab2 plus a secondary antibody to human IgG carrying saporin (·). (see Example 10).
Figure 6 shows the results obtained in an in vivo tumor model in mice with DDRl-ab2-tox, see Example 12.
Figure 7 shows the results obtained with DDRl-ab2-tox an in vivo model of AN3CA human endometrial tumor in mice (see Example 13) Examples
The present invention is additionally described by way of the following illustrative non-limiting examples that provide a better understanding of the present invention and of its many advantages. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques used in the present invention to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) which is incorporated herein by reference in its entirety.
Example 1: Characteristics of DDR1
DDR1 is overexpressed in several types of cancers. For example, DDR1 has been shown to be upregulated in endometrial cancer primary tissue as compared to normal tissue by a microarray experiment and further studies using RT-PCR demonstrated that DDR1 was overexpressed in endometrial cancer tissue as compared to normal tissue. Studies also demonstrated DDR1 was overexpressed in endometrial cancer tissue at the protein level as compared to normal tissue. See WO201 1/009637. In addition, DDR1 is significantly over-expressed in human malignant glioma, breast, colon, ovarian, lung, esophageal, and brain cancers (see e.g., Turashvili et al. (2007) BMC Cancer. 7:55; Yamanaka et al. (2006) Oncogene 25:5994-6002); Yang et al. (2010) 24(2):311-9; Nemoto et al. (1997) Pathobiology 65(4): 195-203; Johansson et al. (2005) Oncogene 24:3896-3905; Heinzelmann-Schwartz et al. (2004) Clin Cane. Res. 10:4427-4436). .
DDR1 is also reported to be overexpressed in injured arteries and has been implicated in additional diseases such as inflammation (Hachehouche et al. (2010) Mol Immunol.;47(9): 1866- 9); cirrhotic liver (Song et al. (2011) Am J Pathol. 178(3): 1134-44.), pulmonary fibrosis , pulmonary fibrosis (C Avivi-Green et al (2006) Am J Respir Crit Care Med, 174(4), 420-27), pituitary adenoma (Yoshida et al. (2007) J. Neuro-Oncol. 82:29-40), congestive heart failure (Andersson et al. (2006) Acta Physiol. 186: 17-27), atherosclerosis (Ahmad et al. (2009) Am J Pathol.l75(6):2686-96; Franco et al.(2008) Circ Res.l02(10): 1202-11), Alport syndrome (a hereditary type IV collagen disease which symptoms include renal inflammation and fibrosis; Gross et al. (2010) Matrix Biol. 29(5):346-56), obstructive nephropathy (Guerrot et al. (2011) Am J Pathol. 179(1):83-91) and lymphangioleiomyomatosis (Ferri et al. (2004) Am. J. Pathol. 5: 1575-1585).
There are 6 isoforms of human DDR1 listed in the NCBI database. Their corresponding accession numbers for the nucleic acid and amino acid sequences are as follows:
Figure imgf000099_0001
Example 2: Preparation of Monoclonal Antibodies
Monoclonal antibodies to DDR1 can be prepared using known hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma a host animal (e.g., mammal) is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent (or the lymphocytes may be immunized in vitro). The immunizing agent will typically include the target protein polypeptide DDRl (or fragment thereof) or a fusion protein thereof or epitope thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against DDRl target protein or epitope. The binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem. 107:220.
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (e.g., U.S. Pat. No. 4,816,567; Morrison et ah, supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Example 3: Humanization of Murine Antibodies
This example sets out a procedure for humanization of a murine anti-DDRl antibody. Design of Genes for Humanized DDR1 Antibody Light and Heavy Chains
The VL and VH amino acid sequences for murine antibodies are known or can be determined using standard molecular biology techniques (e.g., from a monoclonal antibody as prepared according to Example 2 or commercially available hybridomas encoding such an antibody). The sequence of a human antibody identified using the National Biomedical Foundation Protein Identification Resource or similar database can be used to provide the framework of the humanized antibody. To select the sequence of the humanized heavy chain, the murine heavy chain sequence is aligned with the sequence of the human antibody heavy chain. At each position, the human antibody amino acid is selected for the humanized sequence, unless that position falls in any one of four categories defined below, in which case the murine amino acid is selected: (1) The position falls within a complementarity determining region (CDR), as defined by Kabat (1980) J. Immunol. 125:961-969;
(2) The human antibody amino acid is rare for human heavy chains at that position, whereas the murine amino acid is common for human heavy chains at that position;
(3) The position is immediately adjacent to a CDR in the amino acid sequence of the murine heavy chain; or
(4) 3 -dimensional modeling of the murine antibody suggests that the amino acid is physically close to the antigen binding region.
To select the sequence of the humanized light chain, the murine light chain sequence is aligned with the sequence of the human antibody light chain. The human antibody amino acid is selected at each position for the humanized sequence, unless the position again falls into one of the categories described above and repeated below:
(1) CDR's;
(2) murine amino acid more typical than human antibody;
(3) Adjacent to CDR's; or
(4) Possible 3-dimensional proximity to binding region.
The actual nucleotide sequence of the heavy and light chain genes is selected as follows:
(1) The nucleotide sequences code for the amino acid sequences chosen as described above;
(2) 5' of these coding sequences, the nucleotide sequences code for a leader (signal) sequence. These leader sequences are chosen as typical of antibodies;
(3) 3' of the coding sequences, the nucleotide sequences are the sequences that follow the mouse light chain J5 segment and the mouse heavy chain J2 segment, which are part of the murine sequence. These sequences are included because they contain splice donor signals; and
(4) At each end of the sequence is a specific restriction site (e.g., Xba I site) to allow cutting at the restriction site (e.g., Xba I sites and cloning into the restriction site (e.g., Xba I site) of a vector. Construction of Humanized Light and Heavy Chain Genes
The genes encoding the humanized light and heavy chain genes can be prepared by any method. One method involves annealing fragments of the gene together to synthesize the full length genes.
To synthesize the heavy chain, four oligonucleotides are synthesized using a DNA synthesizer (e.g., Applied Biosystems 380B DNA synthesizer). Two of the oligonucleotides are part of each strand of the heavy chain, and each oligonucleotide overlaps the next one by about 20 nucleotides to allow annealing. Together, the oligonucleotides cover the entire humanized heavy chain variable region with a few extra nucleotides at each end to allow cutting at the restriction site (e.g., Xba I sites). The oligonucleotides are purified from polyacrylamide gels.
One method for annealing oligonucleotides is as follows: Each oligonucleotide is phosphorylated using ATP and T4 polynucleotide kinase by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). To anneal the phosphorylated oligonucleotides, they are suspended together in 40 μΐ of TA (e.g., 33 mM Tris acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate) at a concentration of about 3.75 μΜ each, heated to 95° C for about 4 min. and cooled slowly to 4° C. The complete gene can be synthesized from the oligonucleotides by synthesizing the opposite strand of each oligonucleotide, the following components are added in a final volume of 100 μΐ:
• 10 ul annealed oligonucleotides
• 0.16 mM each deoxyribonucleotide
· 0.5 mM ATP
• 0.5 mM DTT
• 100 ug/ml BSA 3.5 ug/ml
• T4 g43 protein (DNA polymerase)
• 25 ug/ml T4 g44/62 protein (polymerase accessory protein)
· 25 ug/ml 45 protein (polymerase accessory protein)
The mixture is incubated at 37° C for about 30 min. Then 10 ug of T4 DNA ligase is added and incubation at 37° C is resumed for about 30 min. The polymerase and ligase are inactivated by incubation of the reaction at 70° C for about 15 min. To digest the gene with Xba I, 50 ul of 2xTA containing BSA at 200 ug/ml and DTT at 1 mM, 43 ul of water, and 50 ug of Xba I in 5 ul of buffer is added to the reaction. The reaction is incubated for 3 hr at 37° C and then purified on a gel. The Xba I fragment is purified from a gel and cloned into the Xba I site of the plasmid pUC19 by standard methods. Plasmids are purified using standard techniques and sequenced using the dideoxy method.
Construction of plasmids to express humanized light and heavy chains can be accomplished, - e.g., by isolating the light and heavy chain Xba I fragments from the pUC19 plasmid in which it had been inserted and then inserting it into the Xba I site of an appropriate expression vector which will express high levels of a complete heavy chain when transfected into an appropriate host cell.
Synthesis and Affinity of Humanized Antibody
The expression vectors are transfected into mouse Sp2/0 cells, and cells that integrate the plasmids are selected on the basis of the selectable marker(s) conferred by the expression vectors by standard methods. To verify that these cells secreted antibody that binds to DDR1 , supernatant from the cells are incubated with cells that are known to express DDR1. After washing, the cells they can be, e.g., incubated with fluorescein-conjugated goat anti-human antibody, washed, and analyzed for fluorescence on a FACSCAN cytofluorometer. The cells producing the humanized antibody are cultured in vitro. Humanized antibody is purified to substantial homogeneity from the cell supernatants by passage through an affinity column of Protein A (Pro-Chem. Inc., Littleton, Mass. or equivalent) according to standard techniques. The affinity of the humanized antibody relative to the original murine antibody is determined according to techniques known in the art.
Example 4: Selection of Anti-DDRl protein Fabs and IgGs via phage display
Phage display libraries can be used to identify antibodies against a target of interest. A phage display library is a library of genetically engineered phage (virus that can infect bacteria). The phage library is engineered in such a way that it encodes a diversity of antibodies, single chain antibodies or fragments thereof which are expressed in a bacteriophage library as the form of fusions with the bacteriophage coat protein where the antibodies, single chain antibodies or fragments thereof are displayed on the surface of the phage. Each individual phage corresponds to and displays an antibody, single chain antibody or fragment thereof that corresponds to the genetic sequence within the phage. Phage with specific binding properties can be isolated and the gene for the antibody can be sequenced, cloned or otherwise isolated.
Thus, the phage-display library (e.g., Fab or scFv) is added to a microtiter plate containing the DDR1 target protein or DDR1 epitope and incubated for a length of time. After incubation for a time sufficient to allow phage to bind to the target protein or epitope, the dish is washed. Phage- displayed antibodies that bind with the target protein or epitope remain attached to the dish, while the other phage are washed away. Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant (i.e. non-binding phage) than were present in the initial mixture. The DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.
The following describes the method used to generate the antibodies of the invention:
We have used a commercial Phagemid Antibody Library (Dyax Corp) which repertoire has been isolated from 45 different human donors (35 autoinmune and 10 healthy donors). Cells from a DDRl-overexpressed clone 44 (see below) were incubated with the human antibody phage library during different time periods to allow the antibodies recognise their specific targets on the cell surface. Phages which are not been bound could be eliminated by washing the samples. Previously to the first selection round a preincubation of the phage library with wild type CHO cells was performed in order to eliminate non speficic interactions. This way, antibodies present on the library which are non specific cancer epitopes will be eliminated before incubation with clone 44. Four rounds of selection were performed in order to enrich the phage mix with DDR1 specific antibodies. After the fourth round a cell specific ELISA screening was conducted where more than 5000 individual clones were tested. Phages which recognise cells from clone 44 but did not recognise CHO cells were finally selected. Finally, the variable regions present in the selected phages are characterised by DNA sequencing. The Fab region can then be cloned onto a suitable vector. For example, the Fab region can be cloned onto vectors expressing IgGs, such IgGl , IgG4 or IgG2a, to express a complete IgG in eukaryotic cells, such as pBhl , pBh4 or pBm2a (Dyax). Clone 44 construction:
A DDR1 overexpressed cell line to be used on the selection of anti DDR1 Fab fragments on cells was constructed by routinary techniques.
We used the Gateway® recombination system from Invitrogen to construct an overexpressed DDR1 CHO clone.
The plasmid IOH5763 (Invitrogen) encoding the cDNA sequence of human DDR1 (sequence shown below) was cloned on the eukaryotic plasmid pEF5_FRT_V5-DEST from Invitrogen.
A transient transfection using liposomes was done in order to introduce the plasmid onto the Flpin Cho cell line (Invitrogen). A plasmid able to express the enzyme recombinase once inside a eucariotic cell was co-transfected with the vector pEF5_FRT_V5-DEST incorporating the DDR1 cDNA. The antibiotic Hygromicin was added to the cell culture media 48 hours after transfection. The advantage of Flpin system is that the sequence of interest is not incorporated into the cell genome at random but does so at a particular place, avoiding changes in expression patterns from different clones due to position effects.
Transfected cells were kept on antibiotic media (Hygromycin) for several weeks in order to select stable overexpressing clones. Antibiotic resistant clones were tested by different techniques such as immunofluorescence and western blot with commercial antibodies from Santa Cruz : anti-DDRl C-20 in order to choose the ones which showed higher expression of DDR1. The selected clone was named Clone 44.
SEQ ID No. 61 , DDR1 DNA sequence from plasmid IOH5763 used to establish Clone 44 with DDR1 overexpressed in a CHO cell line:
ATGGGACCAGAGGCCCTGTCATCTTTACTGCTGCTGCTCTTGGTGGCAAGTGGAGATGCT GACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTATGCCCTGGGCATGCAGGACCGG ACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCCACTGCCGCCCGC CACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGTTT CCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTG GGCACCCAGGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTG CGTTACTCCCGGGATGGTCGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTG ATCTCAGGCAATGAGGACCCTGAGGGAGTGGTGCTGAAGGACCTTGGGCCCCCCATGGTT GCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGTCATGAGCGTCTGTCTGCGGGTA GAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACTGCCCCTGTGGGGCAG ACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCGTG GGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGAC TTTAGGAAGAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGAC TATGTGGGATGGAGCAAC CACAGCTTCTCCAGTGGCTATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTC CAGGCTATGCAGGTCCACTGTAACAACATGCACACGCTGGGAGCCCGTCTGCCTGGCGGG GTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTGGGAGGGGGAGCCCATGCGCCAC AACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTGCCCCTTGGCGGC CGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCAGC GAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTC
CCGCCAGCCCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTG GAGCCCAGAGGCCAGCAGCCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATC GGCTGCCTGGTGGCCATCATCCTGCTCCTGCTGCTCATCATTGCCCTCATGCTCTGGCGG CTGCACTGGCGCAGGCTCCTCAGCAAGGCTGAACGGAGGGTGTTGGAAGAGGAGCTGACG GTTCACCTCTCTGTCCCTGGGGACAC TATCCTCATCAACAACCGCCCAGGTCCTAGAGAG CCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTGTC CCCAATGGCTCTGCCTACAGTGGGGACTATATGGAGCCTGAGAAGCCAGGCGCCCCGCTT CTGCCCCCACCTCCCCAGAACAGCGTCCCCCATTATGCCGAGGCTGACATTGTTACCCTG CAGGGCGTCACCGGGGGCAACACCTATGCTGTGCCTGCACTGCCCCCAGGGGCAGTCGGG GATGGGCCCCCCAGAGTGGATTTCCCTCGATCTCGACTCCGCTTCAAGGAGAAGCTTGGC GAGGGCCAGTTTGGGGAGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGT CTTGATTTCCCCCTTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTA CGGCCAGATGCCACCAAGAATGCCAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCG AGGCTCAAGGACCCAAACATCATTCGGCTGCTGGGCGTGTGTGTGCAGGACGACCCCCTC TGCATGATTACTGACTACATGGAGAACGGCGACCTCAACCAGTTCCTCAGTGCCCACCAG CTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTGCGCAGGGGCCCACC ATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTATCTG GCCACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAAT TTCACCATCAAAATCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGAC TATTAC CGTGTGCAGGGCCGGGCAGTGCTGCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATG GGGAAGTTCACGACTGCGAGTGACGTGTGGGCCTTTGGTGTGACCCTGTGGGAGGTGCTG ATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACCGACGAGCAGGTCATCGAGAACGCG GGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGCCGCCTGCCTGCCCG CAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACCACCC TTTTCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGTAG
Using the above described method, the following 3 antibodies, designated DDRl-abl, DDR1- ab2 and DDRl-ab3, were isolated. Their CDRs and variable sequences are listed below. Vectors encoding these 3 antibodies designated DDRl-ablpBhl, DDRl-ab2pBhl and DDRl-ab3pBhl , respectively, were deposited on December 20, 201 1 with the depositary institute DSMZ (Braunschweig, Germany) under deposit numbers DSM 25529, DSM 25530 and DSM 25531.
DDRl-abl
This antibody has the following CRD sequences:
SEQ ID No. 2: Amino acid sequence of VL_CDR1_ DDRl -ab l
RASQGISSWLA
SEQ ID No. 4: Amino acid sequence of VL_CDR2_DDRl -abl
AASSLES
SEQ ID No. 6: Amino acid sequence of VL_CDR3_DDRl -abl
QQANSFPLT
SEQ ID No. 8 : Amino acid sequence of VH CDR1 DDR1 -ab 1
FTFSYYWMW
SEQ ID No. 10: Amino acid sequence of VH_CDR2_DDRl -ab l : VIGPSGGHTYYADSV
SEQ ID No. 12: Amino acid sequence of VH_CDR3_DDRl -abl
EGITGISAAFDI This antibody has the following variable L and H region sequences:
SEQ ID No. 38: Amino acid sequence of VL DDRl -ab l
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVE
SEQ ID No. 40: Amino acid sequence of VH DDRl -abl
QLLESGGGLVQPGGSLRLSCAASGFTFSYYWMWWVRQAPGKGLEWVSVIGPSGG HTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGITGISAAFDIW GQGTMVTVSS
DDRl-ab2
This antibody has the following CRD sequences: SEQ ID No. 14: Amino acid sequence of VL_CDR1_ DDRl -ab2
SGSSSNIGTNTVN
SEQ ID No. 16: Amino acid sequence of VL_CDR2_DDRl -ab2
SNNQRPS
SEQ ID No. 18: Amino acid sequence of VL_CDR3_DDRl -ab2
AVWDDSLSVPV
SEQ ID No. 20: Amino acid sequence of VH_CDRl_DDRl -ab2
FTFSSYVMV
SEQ ID No. 22: Amino acid sequence of VH_CDR2_DDRl -ab2
SIGPSGGHTSYADSV SEQ ID No. 24: Amino acid sequence of VH_CDR3_DDRl -ab2
LGIFGYMDV
This antibody has the following variable L and H region sequences:
SEQ ID No. 42: Amino acid sequence of VL_DDRl -ab2 LTQPPSASGPPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIDSNNQRPSGVP DRFSGSKSGTSASLAIGGLQSEDEADYYCAVWDDSLSVPVFGGGTKLT
SEQ ID No. 44: Amino acid sequence of VH_DDRl -ab2
QLLESGGGLVQPGGSLRLSCAASGFTFSSYVMVWVRQAPGKGLEWVSSIGPSGGH TSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGIFGYMDVWGKG TTVTVSS
DDRl-ab3
This antibody has the following CRD sequences:
SEQ ID No. 26: Amino acid sequence of VL_CDR1_ DDRl -ab3
RASQSISSYLN SEQ ID No. 28: Amino acid sequence of VL_CDR2_DDRl -ab3
SASSLQS
SEQ ID No. 30: Amino acid sequence of VL_CDR3_DDRl -ab3
QQSYIIPLT
SEQ ID No. 32: Amino acid sequence of VH_CDRl_DDRl -ab3
FTFSEYFMA
SEQ ID No. 34: Amino acid sequence of VH_CDR2_DDRl -ab3
VISPGGWTSYADSV
SEQ ID No. 36: Amino acid sequence of VH_CDR3_DDRl -ab3
HVFQDRHFDY
This antibody has the following variable L and H region sequences: SEQ ID No. 46: Amino acid sequence of VL_DDRl -ab3
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVLIHSASSLQSG VPSRFSGSGSGTEFTLIITSLQPEDFATYYCQQSYIIPLTFGGGTKVE
SEQ ID No. 48: Amino acid sequence of VH_DDRl -ab3
QLLESGGGLVQPGGSLRLSCAASGFTFSEYFMAWVRQAPGKGLEWVSVISPGGW TSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHVFQDRHFDYWGQ GTLVTVSS Example 5: antiDDRl-antibodies recognize DDRl specifically
In order to demonstrate that the antibodies of the invention recognize DDRl and not any other protein expressed as a result of DDRl overexpression, an experiment was performed using the small interfering R A technique (siRNAs). siRNA was used to modulate expression levels of DDRl and demonstrate specificity of binding of the antibodies of the invention.
More specifically, the level of DDRl protein detected by flow cytometry in the original CHO cell was compared to the level of protein detected in the same cell line overexpressing DDRl protein (Clone 44). Clone 44 cells were transiently transfected with a siRNA control or a siRNA which blocked the expression of DDRl (s2298, Ambion) and labelled with the antibodies described herein. Briefly, cells were plated on 6 well plate dishes and transiently transfected with lipofectamine 2000 with the corresponding siRNA. Cell were transfected at 70-80% confluence with 50 pmoles of siRNA per well for two consecutive days. 48 hours after the second transfection, the expression of cell surface DDRl was evaluated by flow cytometry. Cells were incubated with the corresponding anti-DDRl antibody directly on the well at 37 C for an hour. The antibody was used at 1 :200 dilution from a stock of lmg/ml concentration. After that, cells were washed with PBS three times and incubated for an hour at 4C with a goat anti-human secondary antibody conjugated to cyanine dye Cy2. Cells were harvested and suspended in 500ul of PBS/1% FBS and analyzed by flow cytometry. In a parallel study, we analyzed if our antibodies could recognize DDRl on the human endometrial cancer cell line Heel A. This cell line was isolated from a 71 years old female patient with stage 1A endometrioid endometrial cancer in G2 (ref. Kuramoto H. Acta Obstet. Gynaecol. Jpn. 19: 47-58, 197 Hum Cell. 2002 Jun;15(2):81-95). Results are summarized in Table 1, which shows the signal detected on CHO cells (negative for DDRl expression), Clone 44 transfected with a siRNA control, Clone 44 transfected with a siRNA selected to reduce DDRl expression (s2298, Ambion), Heel A with a siRNA control, and Heel A transfected with a siRNA selected to reduce DDRl expression:
Table 1 Clon 44 Clon 44 HeclA HeclA
IgG tested CHO
(si control) (si DDR1) (si control) (si DDR1)
DDRl_abl 0.11% 92.98% 0.54% 80.60% 23.99%
DDRl_ab2 0.04% 93.91% 14.11% 61.63% 8.53%
DDRl_ab3 0.10% 92.56% 17.96% 77.66% 14.00% As shown on Table 1, the signal detected with the antibodies of the invention is drastically reduced when Clone 44 cells were analyzed 48 hours after transfection with siRNA against DDR1. These results show that the antibodies of the invention recognize DDR1 in a specific manner.
Similar results were obtained when the siRNA control and the siRNA against DDR1 were transfected in HeclA cells. Columms 5 and 6 on Table 1 show how the signal percentage obtained in HeclA transfected with the siRNA against DDR1 (siDDRl) were significantly lower than the signal detected on the same cell line when the cell line was transfected with the siRNA control. Example 6: Antibodies of invention are able to internalize after collagen stimulation once bound to DDR1.
It is been described in the literature that DDR1 internalizes on cells upon collagen stimulation (see e.g. Mihai et al, 2009, J Mol Biol, 385, 432-445). We wanted to test if the antibodies of the invention could penetrate inside cells once bound to DDR1. We used collagen stimulation to induce DDR1 internalization.
Clone 44 cells were plated on 6 well plates. Once the cells were seed, cells were serum starved for a day prior incubation with the antibodies. The antibody of interest was incubated on live cells at 37C for an hour before stimulating the cells with lOug/ml rat tail collagen type I at different times in order to promote DDR1 internalization. After collagen treatment, cells were washed with PBS to remove all traces of collagen and incubated directly with a goat anti-human secondary antibody conjugated to cyanine dye Cy2 at 4C in order to detect the amount of anti- DDR1 antibodies that remained on the cell surface. To stop internalization, the incubation with the secondary antibody was done at 4C which will slow down any natural cell process. Cells were then harvested in PBS /1%FBS and the remaining DDR1 on plasma membrane was measured by flow cytometry in order to test any differences due to collagen treatment. A IgG which does not specifically bind to DDRl was used as a control.
The results obtained are shown in table 2.
Table 2
Figure imgf000112_0001
If the signal detected on the cell surface of unstimulated cells using the antiDDRl antibodies is compared with the signal detected upon collagen stimulation, we see that the longer the stimulation with collagen, the lower is the detected signal on the cell surface. This indicates that once the antibody binds to DDRl, the complex DDRl -antibody internalizes inside the cells upon collagen stimulation. The internalization process seems to be faster for DDRl_ab2 than for the DDRl abl or DDRl ab3.
Example 7: Antibodies of invention are able to internalize without collagen stimulation
We wanted to test if the antibodies of the invention could internalize inside a DDRl expressing cell even in the absence of collagen stimulation.
Cells from Clone 44 were plated onto coverslips with a complete growth media suplemented with 10% serum and left at 37C and 5% C02. After 24 hours DDRl_ab2 was added onto live cells and was left for different periods of time from 5 minutes to 30 minutes to allow for antibody binding and internalization. After that, cells were fixed with 4%PFA (paraformaldehide) .
To find out where the antibodies localized after the different incubation times, an immunofluorescence assay was performed. The coverslips with the fixed cells were incubated with a goat anti-human antibody coupled with the fluorochrome Cy2 under permeable and non- permeable conditions. In non-permeable conditions, this Cy2-coupled secondary antibody is not able to go through the plasma membrane, so only the anti-DDRl antibodies localized outside the plasma membrane will be detected. On the contrary, under permeable conditions, the secondary antibody will be able to detect not only the primary antibodies outside cells but also the ones that could have been internalized.
As shown in figure 1 , the longer the time of incubation with the antiDDRl antibodies, the higher the detected signal with the anti-DDRl antibody inside the cell. The dots inside the cells correspond to endosomes.
Antibodies DDRl-abl and DDRl-ab3 were also tested in this assay and exhibited similar properties to DDRl-ab2.
In summary, we have proven that collagen stimulation was not needed for the internalization of the antibodies of the invention. Only the binding of the antibodies to the DDR1 was enough for the antibody's internalization. Example 8: Antibodies of the invention are useful as carriers
The antibodies of the invention internalized inside cells which express DDR1 on the plasma membrane, as shown in Examples 6 and 7. The following assay shows these antibodies can be used as carriers to introduce inside cells conjugates or bound agents such as toxins and thus are able to work as immunotoxins.
An indirect cell proliferation assay was performed with Clone 44. Alamar Blue was used to measure the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability— and cytotoxicity. Measure of Alamar Blue is proportional to the number of living cells and corresponds to the cells metabolic activity. Cells were incubated with serial dilutions of the isolated antibodies of the invention with a goat anti-human IgG secondary antibody conjugated to saporin. Saporin is a ribosome-inactivating protein which comes from the seeds of the plant Saponaria officinali. Once inside the cells Saporin causes inhibition of protein synthesis which leads to cell death. Recognition and internalization of the primary antibody thus results in delivery of the saporin-antibody complex to the cell interior followed by cell killing.
Cells from Clone 44 or control cell line CHO were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, serial dilutions of the antibodies to be tested were added to the corresponding well, from 10~6 to 10~13 M. The antibodies were added in combination with a human secondary antibody coupled to the toxin Saporin, the Hum-ZAP secondary antibody from Advanced Targeting Systems. This Hum-ZAP is a chemical conjugate of a purified goat anti-human IgG and the ribosome-inactivating protein, saporin. The secondary antibody was used at a constant concentration of 100 ng per well. 72 hours after the addition of the antibodies, an Alamar Blue® assay was performed. AlamarBlue® (Invitrogen) is a proven cell viability indicator that uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin. The active ingredient of AlamarBlue® (resazurin) is a nontoxic, cell permeable compound that is blue in color and virtually nonfluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces very bright red fluorescence. Viable cells continuously convert resazurin to resorufin, thereby generating a quantitative measure of viability— and cytotoxicity. Basically, AlamarBlue® was added to the cells and after 4 hours incubation fluorescence signal was read with an spectrophotometer. The amount of fluorescence is proportional to the number of living cells and corresponds to the cells metabolic activity. Damaged and nonviable cells have lower innate metabolic activity and thus generate a proportionally lower signal than healthy cells.
An EC50 value of 7.1 nM for DDRl-ab2 was determined using clone 44, indicating potent reduction in proliferation. See also Fig 2.
Antibodies DDRl-abl and DDRl-ab3 were also tested in this assay and exhibited an EC50 value of 6.35 nM and 26 nM, respectively.
No effects on cell proliferation were observed with the tested antibodies in the control cell line CHO.
The above data show that the antibodies of the invention have excellent internalization properties, which makes them particularly suitable for use in antibody-drug conjugate (ADC) therapy to introduce drugs such as toxins into cells, like cancer cells.
In order to show the advantages of the antibodies of the invention based on their excellent capacity to internalize and incorporate toxins into cells, a similar internalization assay to the one disclosed above was performed testing two commercially available antiDDRl antibodies raised against the extracellular domain of human DDR1, according to the information provided by the supplier (R&D Systems, Antibody # AF2396, raised against human DDR1 Asp21-Thr416 Accession Number Q08345, and Antibody # MAB2396, raised against human DDRl Asp 19- Thr416 Accession Number Q08345, respectively). As positive control, an antibody of the invention conjugated to saporin was used, DDRl-abl-tox (see example 11 for further details). Cells from Clone 44 or control cell line CHO were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, different concentrations of the antibodies to be tested (as indicated in table 3 below) were added to the corresponding well. The antibodies were added in combination with an appropriate secondary antibody coupled to Saporin, as indicated below. No secondary antibody is required for DDRl-abl-tox since the antibody is conjugated to saporin.
Figure imgf000115_0001
The secondary antibody was used at a constant concentration of 100 ng per well. 72 hours after the addition of the antibodies, an Alamar Blue® assay was performed and the proliferation rate was determined. The results obtained using clone 44 are shown in table 3, below. No effects on proliferation were observed with any of the tested antibodies in the DDRl -negative control cell line, CHO.
Table 3:
Test antibody Primary Ab Concentration % Proliferation Rate
10"7M 103.7
Test Ab #l + Goat-ZAP 10"8M 97.6
10_9M 102.1
10_7M 99.5
Test Ab #2 + Mab-ZAP 10_8M 97.9
10_9M 98.8
DDRl-abl-tox 10_8M 22.6
Medium 100.0 No effects on cell proliferation of clone 44 are observed with Test Ab #1 and Test Ab#2 at any of the concentrations tested, which shows that these antibodies are not able to enter DDR1- expressing cells. By contrary, antibodies of the invention have been shown to exhibit a potent effect in this assay. The results in table 3 thus show that while the antibodies of the invention exhibit an excellent capacity to internalize into cells, other antibodies directed to the extracellular domain of human DDR1 like antibodies Test Ab #1 and Test Ab#2 do not internalize into the cells. This highlights the advantages of the antibodies of the invention for use in antibody-drug conjugates.
Example 9: Testing Anti-DDRl antibodies in different cancer cell lines
The antibodies of the invention can be used to identify cell lines which express DDR1. This assay is performed as follows:
0.5 millions of cells were incubated with lOug/ml of different primary antibodies (DDRl-abl ; DDRl-ab2 or DDRl-ab3) in 200 ul of PBS/1 %FBS for an hour at 4C. After the incubation, cells were washed twice with PBS/ 1%FBS and they were incubated for an hour at 4C with a goat anti-human IgG Cy2 lallebed antibody. Signal was detected using a flow cytometer. The experiment was done in no-permeabilized cells so the levels of DDR1 detected correspond to the plasma membrane protein.
Tables 4 and 5 show different human cancer cell lines that were found to express DDR1. Table 4: Expression level of DDR1 detected in different human endometrial cancer cell lines
Cancer Type Cell Line DDRl abl DDRl_ab2 DDRl abS
HeclA +++ +++ +++
ft TndometriaC cancer AN 3 CA +++ +++ +++
Ishikawa +++ +++ +++ Table 5:
Figure imgf000117_0001
Score for expression level:
(+++) strong signal detection, which means high expression levels of DDRl at the membrane surface.
(++) medium signal detection, which means regular expression levels of DDRl at the membrane surface.
(+) Low signal detection, which means minimum expression levels of DDRl at the membrane surface.
(— ) No signal detection, which means no expression levels of DDRl at the membrane surface.
Without wishing to be bound by theory, it is believed the different signal levels detected on the distinct cell types could indicate:
1) The cell line expresses DDRl or not
2) The cell line express different isoforms of DDRl which show different binding affinities for the described antibodies 3) Because DDR1 could be present on cells as a monomer or as a dimer, the described antibodies could have different affinities for the monomer or the dimer of DDR1 or even be able to recognise only one the two aspects of DDR1. So the different signals detected upon cell for the three antibodies could indicate distinct amounts of monomer and dimer present on the surface of specific cells.
Example 10: The isolated antibody is used to target different cell lines which express DDR1.
The isolated antibody is used to introduce a toxin inside DDR 1 -expressing cells and inhibit cell proliferation.
A further proliferation assay was done in different human cancer cell lines.
Several human cancer cell lines with different expression levels of DDR1 were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, the cells were incubated with the antiDDRl antibodies plus an anti-human IgG antibody which is coupled to Saporin (Hum-ZAP, ATSBio). The antiDDRl antibodies were tested at a concentration of 10"9 M and lOOng of Hum-ZAP was added per well. 72 hours after the addition of the antibodies, an Alamar Blue assay was performed on the plates to check cell viability, following the procedure disclosed in Example 8. A summary of the results obtained with DDRl-abl, DDRl-ab2 and DDRl-ab3 are shown in table 6, below:
Table 6:
Figure imgf000119_0001
Table 6 shows the expression level of DDR1 detected on the indicated cell line with each antibody (column 4, corresponding to the same data shown in Example 9) and the corresponding proliferation rate obtained after treating the cells with the antibody plus the Hum-ZAP for 72 hours (shown in column 5). The lower the proliferation, the higher is the effect of the antibody upon cell proliferation inhibition.
The cell line U87MG was used as a negative control to prove that the effect upon cell proliferation is due to the use of DDR1 antibodies. Since U87MG does not express DDR1, no effect on cell proliferation is observed when cells are incubated with the antibodies.
The data on Table 6 show that the level of cell proliferation inhibition observed is correlated with the level of DDR 1 expression in the tested cell line.
A further proliferation assay was done in human endometrial adenocarcinoma AN3 CA cell line. Cells were incubated with serial dilutions of the three isolated antibodies DDRl -abl , DDRl -ab2 and DDRl -ab3 with or without a goat anti-human IgG secondary antibody conjugated to saporin.
Cells from the endometrial cell line AN3 CA were plated on 96 well plates (2000 cells per well) in complete growth media. The day after, serial dilutions of the tested antibodies were added to the corresponding well. The antibodies were added alone or in combination with a human secondary antibody coupled to the toxin Saporin (hum-ZAP, ATSBio). The secondary antibody was used on a constant concentration of l OOng per well of a 96 well plate. 72 hours after the addition of the antibodies an Alamar Blue assay was performed on the plates to check cell viability, following the procedure disclosed in Example 8. The results obtained with DDRl -abl , DDRl -ab2 and DDR1 - ab3 are shown in Figures 3 to 5. EC50 values of 1.46, 2.47 and 1.34 nM were obtained for DDRl-abl, DDRl-ab3 and DDRl-ab2, respectively, indicating potent cell proliferation inhibitory activity. In summary, the above data further show that the antibodies of the invention can be used as carriers of a therapeutic agent such as a toxin into DDR1 -expressing cells and inhibit proliferation of said cells.
Example 11: Preparation of antibody-toxin conjugates
Antibodies of the invention can be conjugated to toxins for use in therapy using any of the conjugation procedures available in the art.
Antibodies DDRl-abl , DDRl-ab2 and DDRl-ab3 were conjugated to saporin using Advanced Targeting Systems technology for conjugation to saporin. The corresponding Ab-saporin conjugates are designated DDRl-abl-tox, DDRl-ab2-tox and DDRl-ab3-tox.
Starting from 15 mg of purified antibodies DDRl-abl , DDRl-ab2, DDRl-ab3, 5 miligrams of
DDRl-ab2-tox was obtained with a ratio moles saporin/mole antibody=2.4 and an average molecular weight=232kDa, 1.9 miligrams of DDRl-ab3-tox was obtained with a ratio moles saporin/mole antibody=2.5 and an average molecular weight=235kDa, and 1.6 miligrams of
DDRl-abl-tox was obtained with a ratio moles saporin/mole antibody=2.3 and an average molecular weight=229kDa. Example 12: Anti-tumor effects in a human epidermoid cancer model in mice
An A431 Human Epidermoid tumor model in mice was used to evaluate the anticancer activity of DDRl-ab2, administered as a conjugate with the toxin saporin (DDRl-ab2-tox, see example 11).
Briefly, 5xl06cells were s.c. injected in Balb lc nude mice (6-8-week-old, females) on the lower back on day 0. On day 7, mice were randomized and divided into control group (group 1 , 10 animals) and treatment group (group 2, DDRl-ab2-tox, 9 animals). Six 100 μΐ i.v injections were given to each group on days 7, 8, 9, 12, 15, 18 at the doses specified: group 1 : PBS alone, group 2: 250μg/kg ab2_DDRl-tox.
Tumor size was measured twice weekly in two dimensions using a caliper, and the volume was
3 · 2
expressed in mm using the formula: V = 0.5 a x b , where a and b are the long and short diameters of the tumor, respectively. On day 25 primary tumours were removed from all mice.
Treatment with DDRl-ab2-tox at 250 g/kg produced significant reduction in tumor volume, as shown in Fig 6.
Example 13: Anti-tumor effects in a human endometrial cancer model in mice
An AN3CA human endometrial tumor model in mice was used to evaluate the anticancer effects of DDRl-ab2, administered as a conjugate with the toxin saporin (ab2-DDRl-tox, see example 11). Briefly, 5xl06cells were s.c injected in Balb/c nude mice (6-8-week-old females) on the lower back on day 0. On day 12, mice were randomized and divided into control group (15 animals) and treatment group (DDRl-ab2-tox, 12 animals). Eight 100 μΐ i.v injections were given to each group on days 13, 14, 15, 17, 19, 21 ,23,25 at the doses specified: group 1 : PBS alone, group 2: 250μg/kg ab2-DDRl-tox. Primary tumors were measured twice weekly. On day 26 primary tumors were removed from all mice. The results of the change in tumour weight are shown in Figure 7.
Treatment with DDRl-ab2-tox at 250 μg/k produced a statistically significant difference (p<0.05) in the reduction of tumour weight compared to the group control.
Example 14: Hemotoxicity study
An in vitro study was conducted to test the hemotoxicity of the antibodies of the invention. Preparation of red blood cells (RBC):
Erythrocytes and mononuclear cells were isolated from 20 tubes with 10 ml of human whole blood. Histopaque 1077 (Sigma 10771) was used for a density gradient separation. 3 ml of Histopaque 1077 and 3 ml blood were mixed. After 30 minutes of centrifugation at 300g separate pools were obtained for mononuclear cells and erythrocytes. Subsequently, successive washings were performed with PBS. The erythrocytes were resuspended en 4ml of PBS and divided in 4 tubes (concentrated RBC). Each tube was diluted with 19 mL of PBS to obtain the RBC working solution.
Hemolysis assay:
All antibodies were tested at the same concentrations, 150, 75, 37.5, 18.75, 9.38, 4.69 and 2.34 ug / mL. Concentrations were prepared by mixing appropriate amount of each of the Ab in a solution containing 200 uL of working RBC solution and 2 ml of PBS. After leaving the antibodies act for 10 minutes under constant stirring, the solution was centrifuged at 3000 rpm for 2 min. The supernatant was recovered and read the absorbance at 541 nm. Optical densities (OD) allowed us to calculate percentage of hemolysis using the following equation:
% Hemolysis = 100 * (OD sample-OD negative control) / (OD Total - OD negative control) Where:
OD Total is the maximum OD of positive control hemolysis.
Positive control was performed with 7 concentrations of Sodium dodecyl sulfate (SDS), at 80, 70, 60, 50, 40, 30, 20 ug / mL, and a negative control with PBS
The results obtained at the highest dose tested for each antibody are shown in the table below:
Figure imgf000122_0001
No hemolysis was observed with any of the antibodies under the conditions tested (< 1.5% hemolysis). Example 15: In vivo toxicity study
An initial assessment of in vivo toxicity was conducted by administration of a single dose into Swiss nude mice (8-week-old females). A 200 μΐ s.c injection was given to each group on day 1 at the following doses: group 1 (control): PBS alone, group 2: 30mg/kg ab2_DDRl , group 3: 30mg/kg ab3_DDRl . Mouse body weight, behaviour, survival and overall health were monitored every day. Animals were sacrificed at 15 days post-administration.
No significant differences were found in body weight between the treatment groups and the control group. No toxicity alerts were identified in any of the parameters assessed. An additional in vivo toxicity study was performed by administration of multiple doses to Balb/c nude mice (6-8-week-o\d females). Five 200 μΐ i.p injections were given to each group on days 1, 2, 3, 7, 11, at the doses specified: group 1 : PBS alone, group 2: 250μg/kg DDRl-ab2-tox, group 3: 50μg/kg DDRl-ab2-tox, group 4: 50μg/kg DDRl-ab3-tox, group 5: 250μg/kg DDR1- ab3-tox, group 6: 30 mg/kg DDRl-ab2, and group 7: 30 mg/kg DDRl-ab3. Mouse weight and overall health were monitored every day. Animals were sacrificed at 14 days post- administration. Blood samples and major organs (liver, spleen, kidney, mesenteric ganglia) were collected. Hematological study (hemoglobin content, RBC, WBC count) and biochemical analysis (AST, ALT, Creatinin, Total Protein) were examined as well as histopathological analysis of the major organs. No significant differences were found between the control and treated animals.
Example 16: Flow Cytometry
Flow cytometry experiments were undertaken with antibodies of the invention.
Flow cytometry is a technique that allows measuring certain physical and chemical characteristics of cells or microscopic particles as they pass in a fluid stream by a beam of laser light. Millions of cells can be analyzed by staining their proteins with specific fluorescent antibodies. Antibody staining of cell membrane proteins and its analysis by flow cytometry is the best way to characterize cell populations. A major application is to separate cells according to subtype or epitope expression for further biological studies. This process is called cell sorting or FACS. Other interesting applications are detection of protein expression levels, comparation of membrane protein levels, monitorization of internalization of receptors or even studies of antibody affinities and specificities. See e.g., Handbook of Flow Cytometry Methods by J. Paul Robinson, et al. ISBN 0471596345.
The results of studies with various cell lines and various antibodies of the invention disclosed in Example 5, 6 and 9 show that flow cytometry can be used to detect the binding of anti-DDRl antibodies of the invention to DDR1 to measure DDR1 protein expression levels.
Example 17: ELISA
An ELISA assay can be undertaken with antibodies of the invention. Enzyme-linked immunosorbent assay (ELISA) is a plate -based technique designed for detecting and quantifying substances such as peptides, proteins, antibodies or hormones using specific labeled antibodies. The antigen must be immobilized to a solid surface, usually a polystyrene microtiter plate, either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). If the antigen is in a live cell, this can be growth in the same place where they are going to be detected with a specific antibody. The detection requires using a specific fluorescent or covalently linked to an enzyme antibody that is applied over the antigen coated surface of the plates. The most commonly used enzyme labels horseradish peroxidase (HRP) and alkaline phosphatase (AP). If the detection antibody is is biotin labeled a secondary antibody linked to a protein such as streptavidin is required. Therefore the assay combines the specificity of antibodies with the sensitivity of simple enzyme assays or fluorescence intensity signal. The ELISA is very useful as a diagnostic tool in basic research, medicine.
Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA).". Clin. Chem. 51 (12): 2415-8. doi: 10.1373/clinchem.2005.051532. PMID 16179424 The present invention refers to the following nucleotide and amino acid sequences:
Some sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih.gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such "variants" are genetic variants.
SEQ ID No. 1 :
Nucleotide sequence encoding VL_CDR1_ DDRl -abl
cgggcgagtcagggtattagcagctggttagcc SEQ ID No. 2:
Amino acid sequence of VL_CDR1_ DDRl -abl
RASQGISSWLA
SEQ ID No. 3:
Nucleotide sequence encoding VL_CDR2_DDRl -abl
gctgcatccagtttggaaagt
SEQ ID No. 4:
Amino acid sequence of VL_CDR2_DDRl -ab l
AASSLES
SEQ ID No. 5:
Nucleotide sequence encoding VL_CDR3_DDRl -abl
caacaggctaacagtttcccgctcact
SEQ ID No. 6:
Amino acid sequence of VL_CDR3_DDRl -ab l
QQANSFPLT SEQ ID No. 7: Nucleotide sequence encoding VH_CDRl_DDRl -abl ttcactttctcttattactggatgtgg
SEQ ID No. 8:
Amino acid sequence of VH_CDRl_DDRl -abl FTFSYYWMW
SEQ ID No. 9:
Nucleotide sequence encoding VH_CDR2_DDRl -abl gttatcggtccttctggtggccatacttattatgctgactccgtt
SEQ ID No. 10:
Amino acid sequence of VH_CDR2_DDRl -abl : VIGPSGGHTYYADSV
SEQ ID No. 1 1 :
Nucleotide sequence encoding VH_CDR3_DDRl -abl gaaggtataactggaattagtgctgcttttgatatc SEQ ID No. 12:
Amino acid sequence of VH_CDR3_DDRl -abl EGITGISAAFDI
SEQ ID No. 13:
Nucleotide sequence encoding VL_CDR1_ DDRl -ab2 tctggaagcagctccaatatcggaactaatactgtaaac
SEQ ID No. 14:
Amino acid sequence of VL_CDR1_ DDRl -ab2 SGSSSNIGTNTVN
SEQ ID No. 15:
Nucleotide sequence encoding VL_CDR2_DDRl -ab2 agtaataatcagcggccctca SEQ ID No. 16:
Amino acid sequence of VL_CDR2_DDRl -ab2 SNNQRPS SEQ ID No. 17:
Nucleotide sequence encoding VL_CDR3_DDRl -ab2 gcagtatgggatgacagcctgagtgttccggtg
SEQ ID No. 18:
Amino acid sequence of VL_CDR3_DDRl -ab2 AVWDDSLSVPV
SEQ ID No. 19:
Nucleotide sequence encoding VH_CDRl_DDRl -ab2 ttcactttctcttcttacgttatggtt
SEQ ID No. 20:
Amino acid sequence of VH_CDRl_DDRl -ab2 FTFSSYVMV
SEQ ID No. 21 :
Nucleotide sequence encoding VH_CDR2_DDRl -ab2 tctatcggtccttctggtggccatacttcttatgctgactccgtt SEQ ID No. 22:
Amino acid sequence of VH_CDR2_DDRl -ab2 SIGPSGGHTSYADSV
SEQ ID No. 23:
Nucleotide sequence encoding VH_CDR3_DDRl -ab2 cttgggatttttgggtacatggacgtc
SEQ ID No. 24:
Amino acid sequence of VH_CDR3_DDRl -ab2 LGIFGYMDV SEQ ID No. 25:
Nucleotide sequence encoding VL_CDR1_ DDRl -ab3 cgggcaagtcagagcattagcagctatttaaat
SEQ ID No. 26:
Amino acid sequence of VL_CDR1_ DDRl -ab3 RASQSISSYLN SEQ ID No. 27:
Nucleotide sequence encoding VL_CDR2_DDRl -ab3 agtgcatccagtttgcaaagt
SEQ ID No. 28:
Amino acid sequence of VL_CDR2_DDRl -ab3 SASSLQS
SEQ ID No. 29:
Nucleotide sequence encoding VL_CDR3_DDRl -ab3 caacagagttacattataccgctcact
SEQ ID No. 30:
Amino acid sequence of VL_CDR3_DDRl -ab3 QQSYIIPLT
SEQ ID No. 31 :
Nucleotide sequence encoding VH_CDRl_DDRl -ab3 ttcactttctctgagtactttatggct SEQ ID No. 32:
Amino acid sequence of VH_CDRl_DDRl -ab3 FTFSEYFMA
SEQ ID No. 33:
Nucleotide sequence encoding VH_CDR2_DDRl -ab3 gttatctctcctggtggctggacttcttatgctgactccgtt
SEQ ID No. 34:
Amino acid sequence of VH_CDR2_DDRl -ab3
VISPGGWTSYADSV
SEQ ID No. 35:
Nucleotide sequence encoding VH_CDR3_DDRl -ab3
catgtatttcaagaccggcactttgactac
SEQ ID No. 36:
Amino acid sequence of VH_CDR3_DDRl -ab3
HVFQDRHFDY SEQ ID No. 37:
Nucleotide sequence encoding VL DDRl -abl gacatccagatgacccagtctccatcttccgtgtctgcatctgtaggagacagagtcaccatca cttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaaccagggaaagc ccctaagctcctgatctatgctgcatccagtttggaaagtggggtcccatcaagattcagcggc agtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaactt actattgtcaacaggctaacagtttcccgctcactttcggcggagggaccaaggtggag
SEQ ID No. 38:
Amino acid sequence of VL DDRl -abl
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVE SEQ ID No. 39:
Nucleotide sequence encoding VH DDRl -ab l
caattgttagagtctggtggcggtcttgttcagcctggtggttctttacgtctttcttgc gctgcttccggattcactttctcttattactggatgtggtgggttcgccaagctcctggt aaaggtttggagtgggtttctgttatcggtccttctggtggccatacttattatgctgac tccgttaaaggtcgcttcactatctctagagacaactctaagaatactctctacttgcag atgaacagcttaagggctgaggacacggccgtgtattactgtgcgagagaaggtataact ggaattagtgctgcttttgatatctggggccaagggacaatggtcaccgtctcaagc SEQ ID No. 40:
Amino acid sequence of VH DDRl -ab l
QLLESGGGLVQPGGSLRLSCAASGFTFSYYWMWWVRQAPGKGLEWVSVIGPSGG HTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGITGISAAFDIW GQGTMVTVSS
SEQ ID No. 41 :
Nucleotide sequence encoding VL_DDRl-ab2
ttgactcagccaccctcagcgtctgggccccccgggcagagggtcaccatctcttgttctggaagcagctccaatatcggaactaatactgt aaactggtaccagcagctcccaggaacggcccccaaactcctcatcgatagtaataatcagcggccctcaggggtccctgaccgattctct ggctccaagtctggcacctcagcctccctggccatcggtgggctccagtctgaggatgaggctgactattattgtgcagtatgggatgacag cctgagtgttccggtgttcggcggagggaccaagctgacc
SEQ ID No. 42:
Amino acid sequence of VL_DDRl -ab2
LTQPPSASGPPGQRVTISCSGSSSNIGTNTVNWYQQLPGTAPKLLIDSNNQRPSGVP DRFSGSKSGTSASLAIGGLQSEDEADYYCAVWDDSLSVPVFGGGTKLT
SEQ ID No. 43:
Nucleotide sequence encoding VH_DDRl-ab2
caattgttagagtctggtggcggtcttgttcagcctggtggttctttacgtctttcttgcgctgcttccggattcactttctcttcttacgttatggttt gggttcgccaagctcctggtaaaggtttggagtgggtttcttctatcggtccttctggtggccatacttcttatgctgactccgttaaaggtcgct tcactatctctagagacaactctaagaatactctctacttgcagatgaacagcttaagggctgaggacacggccgtatattactgtgcgagac ttgggatttttgggtacatggacgtctggggcaaagggaccacggtcaccgtctcaagc
SEQ ID No. 44:
Amino acid sequence of VH_DDRl -ab2
QLLESGGGLVQPGGSLRLSCAASGFTFSSYVMVWVRQAPGKGLEWVSSIGPSGGH TSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLGIFGYMDVWGKG TTVTVSS
SEQ ID No. 45:
Nucleotide sequence encoding VL_DDRl-ab3
gacatccagatgacccagtctccatcatccctgtctgcatctgttggagacagagtcaccatcacttgccgggcaagtcagagcattagcag ctatttaaattggtatcagcagaaaccagggaaagcccctaaagtcctgatccatagtgcatccagtttgcaaagtggggtcccatcaaggtt cagtggcagtggatctgggacagagttcactctcatcatcaccagtcttcaacctgaagattttgcaacttactactgtcaacagagttacatta taccgctcactttcggcggagggaccaaggtggag
SEQ ID No. 46:
Amino acid sequence of VL_DDRl -ab3
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKVLIHSASSLQSG VPSRFSGSGSGTEFTLIITSLQPEDFATYYCQQSYIIPLTFGGGTKVE
SEQ ID No. 47:
Nucleotide sequence encoding VH_DDRl-ab3
caattgttagagtctggtggcggtcttgttcagcctggtggttctttacgtctttcttgcgctgcttccggattcactttctctgagtactttatggctt gggttcgccaagctcctggtaaaggtttggagtgggtttctgttatctctcctggtggctggacttcttatgctgactccgttaaaggtcgcttca ctatctctagagacaactctaagaatactctctacttgcagatgaacagcttaagggctgaggacacggccgtgtattactgtgcgagacatg tatttcaagaccggcactttgactactggggacagggcaccctggtcaccgtctcaagc
SEQ ID No. 48:
Amino acid sequence of VH_DDRl -ab3
QLLESGGGLVQPGGSLRLSCAASGFTFSEYFMAWVRQAPGKGLEWVSVISPGGW
TSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHVFQDRHFDYWGQ
GTLVTVSS
SEQ ID No. 49: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 1 , mRNA with the NCBI Reference Sequence: NM_001954.4
GTCTTCCCCTCGTGGGCCCTGAGCGGGACTGCAGCCAGCCCCCTGGGGCGCCAGCTTTGGAGGCCCCCGA CAGCTGCTCTCGGGAGCCGCCTCCCGACACCCGAGCCCCGCCGGCGCCTCCCGCTCCCGGCTCCCGGCTC CTGGCTCCCTCCGCCTCCCCCGCCCCTCGCCCCGCCGCCGAAGAGGCCCCGCTCCCGGGTCGGACGCCTG GGTCTGCCGGGAAGAGCGATGAGAGGTGTCTGAAGGTGGCTATTCACTGAGCGATGGGGTTGGACTTGAA GGAATGCCAAGAGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCT GTCATCTTTACTGCTGCTGCTCTTGGTGGCAAGTGGAGATGCTGACATGAAGGGACATTTTGATCCTGCC AAGTGCCGCTATGCCCTGGGCATGCAGGACCGGACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCT GGTCAGATTCCACTGCCGCCCGCCACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGC AGGGTCGGTGTTTCCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTG GTGGGCACCCAGGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTGCGTTACT CCCGGGATGGTCGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTGATCTCAGGCAATGAGGA CCCTGAGGGAGTGGTGCTGAAGGACCTTGGGCCCCCCATGGTTGCCCGACTGGTTCGCTTCTACCCCCGG GCTGACCGGGTCATGAGCGTCTGTCTGCGGGTAGAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGT CTTACACCGCCCCTGTGGGGCAGACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGA CGGACATACCGTGGGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGAT GACTTTAGGAAGAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGAC TATGTGGGATGGAGCAACCACAGCT TCTCCAGTGGCTATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTCCAGGCTATGCAGGTCCA CTGTAACAACATGCACACGCTGGGAGCCCGTCTGCCTGGCGGGGTGGAATGTCGCTTCCGGCGTGGCCCT
GCCATGGCCTGGGAGGGGGAGCCCATGCGCCACAACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGG CTGTCTCAGTGCCCCTTGGCGGCCGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTG GTTACTCTTCAGCGAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACC TTCCCGCCAGCCCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTGGAGCCCA GAGGCCAGCAGCCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATCGGCTGCCTGGTGGCCAT CATCCTGCTCCTGCTGCTCATCATTGCCCTCATGCTCTGGCGGCTGCACTGGCGCAGGCTCCTCAGCAAG GCTGAACGGAGGGTGTTGGAAGAGGAGCTGACGGTTCACCTCTCTGTCCCTGGGGACACTATCCTCATCA ACAACCGCCCAGGTCCTAGAGAGCCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTC CGCTCCCTGTGTCCCCAATGGCTCTGCCTACAGTGGGGACTATATGGAGCCTGAGAAGCCAGGCGCCCCG CTTCTGCCCCCACCTCCCCAGAACAGCGTCCCCCATTATGCCGAGGCTGACATTGTTACCCTGCAGGGCG TCACCGGGGGCAACACCTATGCTGTGCCTGCACTGCCCCCAGGGGCAGTCGGGGATGGGCCCCCCAGAGT GGATTTCCCTCGATCTCGACTCCGCTTCAAGGAGAAGCTTGGCGAGGGCCAGTTTGGGGAGGTGCACCTG TGTGAGGTCGACAGCCCTCAAGATCTGGTTAGTCTTGATTTCCCCCTTAATGTGCGTAAGGGACACCCTT TGCTGGTAGCTGTCAAGATCTTACGGCCAGATGCCACCAAGAATGCCAGGAATGATTTCCTGAAAGAGGT GAAGATCATGTCGAGGCTCAAGGACCCAAACATCATTCGGCTGCTGGGCGTGTGTGTGCAGGACGACCCC CTCTGCATGATTACTGACTACATGGAGAACGGCGACCTCAACCAGTTCCTCAGTGCCCACCAGCTGGAGG ACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTGCGCAGGGGCCCACCATCAGCTACCCAATGCT GCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTATCTGGCCACACTCAACTTTGTACATCGGGAC CTGGCCACGCGGAACTGCCTAGTTGGGGAAAATTTCACCATCAAAATCGCAGACTTTGGCATGAGCCGGA ACCTCTATGCTGGGGACTATTACCGTGTGCAGGGCCGGGCAGTGCTGCCCATCCGCTGGATGGCCTGGGA GTGCATCCTCATGGGGAAGTTCACGACTGCGAGTGACGTGTGGGCCTTTGGTGTGACCCTGTGGGAGGTG CTGATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACCGACGAGCAGGTCATCGAGAACGCGGGGGAGT TCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGCCGCCTGCCTGCCCGCAGGGCCTATATGAGCT GATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACCACCCTTTTCCCAGCTGCATCGGTTCCTGGCA GAGGATGCACTCAACACGGTGTGAATCACACATCCAGCTGCCCCTCCCTCAGGGAGCGATCCAGGGGAAG CCAGTGACACTAAAACAAGAGGACACAATGGCACCTCTGCCCTTCCCCTCCCGACAGCCCATCACCTCTA ATAGAGGCAGTGAGACTGCAGGTGGGCTGGGCCCACCCAGGGAGCTGATGCCCCTTCTCCCCTTCCTGGA CACACTCTCATGTCCCCTTCCTGTTCTTCCTTCCTAGAAGCCCCTGTCGCCCACCCAGCTGGTCCTGTGG ATGGGATCCTCTCCACCCTCCTCTAGCCATCCCTTGGGGAAGGGTGGGGAGAAATATAGGATAGACACTG GACATGGCCCATTGGAGCACCTGGGCCCCACTGGACAACACTGATTCCTGGAGAGGTGGCTGCGCCCCCA GCTTCTCTCTCCCTGTCACACACTGGACCCCACTGGCTGAGAATCTGGGGGTGAGGAGGACAAGAAGGAG AGGAAAATGTTTCCTTGTGCCTGCTCCTGTACTTGTCCTCAGCTTGGGCTTCTTCCTCCTCCATCACCTG AAACACTGGACCTGGGGGTAGCCCCGCCCCAGCCCTCAGTCACCCCCACTTCCCACTTGCAGTCTTGTAG CTAGAACTTCTCTAAGCCTATACGTTTCTGTGGAGTAAATATTGGGATTGGGGGGAAAGAGGGAGCAACG GCCCATAGCCTTGGGGTTGGACATCTCTAGTGTAGCTGCCACATTGATTTTTCTATAATCACTTGGGGTT TGTACATTTTTGGGGGGAGAGACACAGATTTTTACACTAATATATGGACCTAGCTTGAGGCAATTTTAAT CCCCTGCACTAGGCAGGTAATAATAAAGGTTGAGTTTTCCACAAAAAAAAAAAAAAAAAA
SEQ ID No. 50 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDR1 ), variant 1 , amino acid sequence with the NCBI Reference Sequence NP 001945.3
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPD SDI SASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGT
QGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMV
ARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGH
TVGGLQYGGLGQLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDR
LRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAV
SVPLGGRVARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPP TN SSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLLI IALMLWRLHWRRLLSKA
ERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPRGNPPHSAPCVPNGSAYSG DYMEPEKPGAPLLPPPPQNSVPHYAEADIVTLQGVTGGNTYAVPALPPGAVGDGPPRV DFPRSRLRFKEKLGEGQFGEVHLCEVDSPQDLVSLDFPLNVRKGHPLLVAVKILRPDA TKNARNDFLKEVKIMSRLKDP I IRLLGVCVQDDPLCMITDYMENGDLNQFLSAHQLE DKAAEGAPGDGQAAQGPTI SYPMLLHVAAQIASGMRYLATLNFVHRDLATRNCLVGEN FTIKIADFGMSRNLYAGDYYRVQGRAVLPIRWMAWECILMGKFTTASDVWAFGVTLWE VLMLCRAQPFGQLTDEQVIENAGEFFRDQGRQVYLSRPPACPQGLYELMLRCWSRESE QRPPFSQLHRFLAEDALNTV
SEQ ID No. 51 : Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDR1), transcript variant 2, mRNA with the NCBI Reference Sequence: NM_013993.2
TGCTCTCGGGAGCCGCCTCCCGACACCCGAGCCCCGCCGGCGCCTCCCGCTCCCGGCTCCCGGCTCCTGG CTCCCTCCGCCTCCCCCGCCCCTCGCCCCGCCGCCGAAGAGGCCCCGCTCCCGGGTCGGACGCCTGGGTC TGCCGGGAAGAGCGATGAGAGGTGTCTGAAGGTGGCTATTCACTGAGCGATGGGGTTGGACTTGAAGGAA TGCCAAGAGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCA TCTTTACTGCTGCTGCTCTTGGTGGCAAGTGGAGATGCTGACATGAAGGGACATTTTGATCCTGCCAAGT GCCGCTATGCCCTGGGCATGCAGGACCGGACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTC AGATTCCACTGCCGCCCGCCACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGG TCGGTGTTTCCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTGG GCACCCAGGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTGCGTTACTCCCG GGATGGTCGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTGATCTCAGGCAATGAGGACCCT GAGGGAGTGGTGCTGAAGGACCTTGGGCCCCCCATGGTTGCCCGACTGGTTCGCTTCTACCCCCGGGCTG ACCGGGTCATGAGCGTCTGTCTGCGGGTAGAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTA CACCGCCCCTGTGGGGCAGACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGA CATACCGTGGGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGACT TTAGGAAGAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGACTATGTGGGATGGAGCAACCACAGCTTCTC CAGTGGCTATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTCCAGGCTATGCAGGTCCACTGT AACAACATGCACACGCTGGGAGCCCGTCTGCCTGGCGGGGTGGAATGTCGCTTCCGGCGTGGCCCTGCCA TGGCCTGGGAGGGGGAGCCCATGCGCCACAACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGT CTCAGTGCCCCTTGGCGGCCGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTA CTCTTCAGCGAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTCC CGCCAGCCCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTGGAGCCCAGAGG CCAGCAGCCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATCGGCTGCCTGGTGGCCATCATC CTGCTCCTGCTGCTCATCATTGCCCTCATGCTCTGGCGGCTGCACTGGCGCAGGCTCCTCAGCAAGGCTG AACGGAGGGTGTTGGAAGAGGAGCTGACGGTTCACCTCTCTGTCCCTGGGGACACTATCCTCATCAACAA CCGCCCAGGTCCTAGAGAGCCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCT CCCTGTGTCCCCAATGGCTCTGCGTTGCTGCTCTCCAATCCAGCCTACCGCCTCCTTCTGGCCACTTACG CCCGTCCCCCTCGAGGCCCGGGCCCCCCCACACCCGCCTGGGCCAAACCCACCAACACCCAGGCCTACAG TGGGGACTATATGGAGCCTGAGAAGCCAGGCGCCCCGCTTCTGCCCCCACCTCCCCAGAACAGCGTCCCC CATTATGCCGAGGCTGACATTGTTACCCTGCAGGGCGTCACCGGGGGCAACACCTATGCTGTGCCTGCAC TGCCCCCAGGGGCAGTCGGGGATGGGCCCCCCAGAGTGGATTTCCCTCGATCTCGACTCCGCTTCAAGGA
GAAGCTTGGCGAGGGCCAGTTTGGGGAGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGT CTTGATTTCCCCCTTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTACGGCCAGATG CCACCAAGAATGCCAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCGAGGCTCAAGGACCCAAACAT CATTCGGCTGCTGGGCGTGTGTGTGCAGGACGACCCCCTCTGCATGATTACTGACTACATGGAGAACGGC GACCTCAACCAGTTCCTCAGTGCCCACCAGCTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGC AGGCTGCGCAGGGGCCCACCATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCAT GCGCTATCTGGCCACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAAT TTCACCATCAAAATCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGACTATTACCGTGTGCAGG GCCGGGCAGTGCTGCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATGGGGAAGTTCACGACTGCGAG TGACGTGTGGGCCTTTGGTGTGACCCTGTGGGAGGTGCTGATGCTCTGTAGGGCCCAGCCCTTTGGGCAG CTCACCGACGAGCAGGTCATCGAGAACGCGGGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGT CCCGGCCGCCTGCCTGCCCGCAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCA GCGACCACCCTTTTCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGTGAATCACACAT CCAGCTGCCCCTCCCTCAGGGAGCGATCCAGGGGAAGCCAGTGACACTAAAACAAGAGGACACAATGGCA CCTCTGCCCTTCCCCTCCCGACAGCCCATCACCTCTAATAGAGGCAGTGAGACTGCAGGTGGGCTGGGCC CACCCAGGGAGCTGATGCCCCTTCTCCCCTTCCTGGACACACTCTCATGTCCCCTTCCTGTTCTTCCTTC CTAGAAGCCCCTGTCGCCCACCCAGCTGGTCCTGTGGATGGGATCCTCTCCACCCTCCTCTAGCCATCCC TTGGGGAAGGGTGGGGAGAAATATAGGATAGACACTGGACATGGCCCATTGGAGCACCTGGGCCCCACTG GACAACACTGATTCCTGGAGAGGTGGCTGCGCCCCCAGCTTCTCTCTCCCTGTCACACACTGGACCCCAC TGGCTGAGAATCTGGGGGTGAGGAGGACAAGAAGGAGAGGAAAATGTTTCCTTGTGCCTGCTCCTGTACT TGTCCTCAGCTTGGGCTTCTTCCTCCTCCATCACCTGAAACACTGGACCTGGGGGTAGCCCCGCCCCAGC CCTCAGTCACCCCCACTTCCCACTTGCAGTCTTGTAGCTAGAACTTCTCTAAGCCTATACGTTTCTGTGG AGTAAATATTGGGATTGGGGGGAAAGAGGGAGCAACGGCCCATAGCCTTGGGGTTGGACATCTCTAGTGT AGCTGCCACATTGATTTTTCTATAATCACTTGGGGTTTGTACATTTTTGGGGGGAGAGACACAGATTTTT ACACTAATATATGGACCTAGCTTGAGGCAATTTTAATCCCCTGCACTAGGCAGGTAATAATAAAGGTTGA GTTTTCCACAAAAAAAAAAAAAAAAAA
SEQ ID No. 52 Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDR1), variant 2, amino acid sequence with the NCBI Reference Sequence:NP_054699.2
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPD SDI SASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGT QGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMV ARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGH TVGGLQYGGLGQLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDR LRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAV SVPLGGRVARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPP TNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLLI IALMLWRLHWRRLLSKA ERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPRGNPPHSAPCVPNGSALLL SNPAYRLLLATYARPPRGPGPPTPAWAKPTNTQAYSGDYMEPEKPGAPLLPPPPQNSV PHYAEADIVTLQGVTGGNTYAVPALPPGAVGDGPPRVDFPRSRLRFKEKLGEGQFGEV HLCEVDSPQDLVSLDFPLNVRKGHPLLVAVKILRPDATKNARNDFLKEVKIMSRLKDP NIIRLLGVCVQDDPLCMITDYMENGDLNQFLSAHQLEDKAAEGAPGDGQAAQGPTISY
PMLLHVAAQIASGMRYLATLNFVHRDLATRNCLVGENFTIKIADFGMSRNLYAGDYYR VQGRAVLPIRWMAWECILMGKFTTASDVWAFGVTLWEVLMLCRAQPFGQLTDEQVIEN
AGEFFRDQGRQVYLSRPPACPQGLYELMLRCWSRESEQRPPFSQLHRFLAEDALNTV
SEQ ID No. 53 : Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 3, mRNA with the NCBI Reference Sequence: NM_013994.2
AGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCATCTTTAC TGCTGCTGCTCTTGGTGGCAAGTGGAGATGCTGACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTA TGCCCTGGGCATGCAGGACCGGACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCC ACTGCCGCCCGCCACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGT TTCCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTGGGCACCCA GGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTGCGTTACTCCCGGGATGGT CGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTGATCTCAGGCAATGAGGACCCTGAGGGAG TGGTGCTGAAGGACCTTGGGCCCCCCATGGTTGCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGT CATGAGCGTCTGTCTGCGGGTAGAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACCGCC CCTGTGGGGCAGACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCG TGGGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGACTTTAGGAA GAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGACTATGTGGGATGGAGCAACCACAGCTTCTCCAGTGGC TATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTCCAGGCTATGCAGGTCCACTGTAACAACA TGCACACGCTGGGAGCCCGTCTGCCTGGCGGGGTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTG GGAGGGGGAGCCCATGCGCCACAACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTG CCCCTTGGCGGCCGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCA GCGAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTCCCGCCAGC CCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTGGAGCCCAGAGGCCAGCAG CCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATCGGCTGCCTGGTGGCCATCATCCTGCTCC TGCTGCTCATCATTGCCCTCATGCTCTGGCGGCTGCACTGGCGCAGGCTCCTCAGCAAGGCTGAACGGAG GGTGTTGGAAGAGGAGCTGACGGTTCACCTCTCTGTCCCTGGGGACACTATCCTCATCAACAACCGCCCA GGTCCTAGAGAGCCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTG TCCCCAATGGCTCTGCGTTGCTGCTCTCCAATCCAGCCTACCGCCTCCTTCTGGCCACTTACGCCCGTCC CCCTCGAGGCCCGGGCCCCCCCACACCCGCCTGGGCCAAACCCACCAACACCCAGGCCTACAGTGGGGAC TATATGGAGCCTGAGAAGCCAGGCGCCCCGCTTCTGCCCCCACCTCCCCAGAACAGCGTCCCCCATTATG CCGAGGCTGACATTGTTACCCTGCAGGGCGTCACCGGGGGCAACACCTATGCTGTGCCTGCACTGCCCCC AGGGGCAGTCGGGGATGGGCCCCCCAGAGTGGATTTCCCTCGATCTCGACTCCGCTTCAAGGAGAAGCTT GGCGAGGGCCAGTTTGGGGAGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGTCTTGATT TCCCCCTTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTACGGCCAGATGCCACCAA GAATGCCAGCTTCTCCTTGTTCTCCAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCGAGGCTCAAG GACCCAAACATCATTCGGCTGCTGGGCGTGTGTGTGCAGGACGACCCCCTCTGCATGATTACTGACTACA TGGAGAACGGCGACCTCAACCAGTTCCTCAGTGCCCACCAGCTGGAGGACAAGGCAGCCGAGGGGGCCCC TGGGGACGGGCAGGCTGCGCAGGGGCCCACCATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATC GCCTCCGGCATGCGCTATCTGGCCACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAG TTGGGGAAAATTTCACCATCAAAATCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGACTATTA CCGTGTGCAGGGCCGGGCAGTGCTGCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATGGGGAAGTTC ACGACTGCGAGTGACGTGTGGGCCTTTGGTGTGACCCTGTGGGAGGTGCTGATGCTCTGTAGGGCCCAGC CCTTTGGGCAGCTCACCGACGAGCAGGTCATCGAGAACGCGGGGGAGTTCTTCCGGGACCAGGGCCGGCA GGTGTACCTGTCCCGGCCGCCTGCCTGCCCGCAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGG GAGTCTGAGCAGCGACCACCCTTTTCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGT GAATCACACATCCAGCTGCCCCTCCCTCAGGGAGCGATCCAGGGGAAGCCAGTGACACTAAAACAAGAGG ACACAATGGCACCTCTGCCCTTCCCCTCCCGACAGCCCATCACCTCTAATAGAGGCAGTGAGACTGCAGG TGGGCTGGGCCCACCCAGGGAGCTGATGCCCCTTCTCCCCTTCCTGGACACACTCTCATGTCCCCTTCCT
GTTCTTCCTTCCTAGAAGCCCCTGTCGCCCACCCAGCTGGTCCTGTGGATGGGATCCTCTCCACCCTCCT CTAGCCATCCCTTGGGGAAGGGTGGGGAGAAATATAGGATAGACACTGGACATGGCCCATTGGAGCACCT GGGCCCCACTGGACAACACTGATTCCTGGAGAGGTGGCTGCGCCCCCAGCTTCTCTCTCCCTGTCACACA CTGGACCCCACTGGCTGAGAATCTGGGGGTGAGGAGGACAAGAAGGAGAGGAAAATGTTTCCTTGTGCCT GCTCCTGTACTTGTCCTCAGCTTGGGCTTCTTCCTCCTCCATCACCTGAAACACTGGACCTGGGGGTAGC CCCGCCCCAGCCCTCAGTCACCCCCACTTCCCACTTGCAGTCTTGTAGCTAGAACTTCTCTAAGCCTATA CGTTTCTGTGGAGTAAATATTGGGATTGGGGGGAAAGAGGGAGCAACGGCCCATAGCCTTGGGGTTGGAC ATCTCTAGTGTAGCTGCCACATTGATTTTTCTATAATCACTTGGGGTTTGTACATTTTTGGGGGGAGAGA CACAGATTTTTACACTAATATATGGACCTAGCTTGAGGCAATTTTAATCCCCTGCACTAGGCAGGTAATA ATAAAGGTTGAGTTTTCCACAAAAAAAAAAAAAAAAAA
SEQ ID No. 54: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 3, amino acid sequence with the NCBI Reference Sequence NP 054700.2
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPD SDI SASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGT QGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMV ARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGH TVGGLQYGGLGQLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDR LRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAV SVPLGGRVARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPP TNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLLI IALMLWRLHWRRLLSKA ERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPRGNPPHSAPCVPNGSALLL SNPAYRLLLATYARPPRGPGPPTPAWAKPTNTQAYSGDYMEPEKPGAPLLPPPPQNSV PHYAEADIVTLQGVTGGNTYAVPALPPGAVGDGPPRVDFPRSRLRFKEKLGEGQFGEV HLCEVDSPQDLVSLDFPLNVRKGHPLLVAVKILRPDATKNASFSLFSRNDFLKEVKIM SRLKDP I IRLLGVCVQDDPLCMITDYMENGDLNQFLSAHQLEDKAAEGAPGDGQAAQ GPTISYPMLLHVAAQIASGMRYLATLNFVHRDLATRNCLVGENFTIKIADFGMSRNLY AGDYYRVQGRAVLPIRWMAWECILMGKFTTASDVWAFGVTLWEVLMLCRAQPFGQLTD EQVIENAGEFFRDQGRQVYLSRPPACPQGLYELMLRCWSRESEQRPPFSQLHRFLAED ALNTV
SEQ ID No. 55 : Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 4, mRNA with the NCBI Reference Sequence: NM_001202521.1 AGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCATCTTTAC
TGCTGCTGCTCTTGGTGGCAAGTGGAGATGCTGACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTA TGCCCTGGGCATGCAGGACCGGACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCC ACTGCCGCCCGCCACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGT TTCCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTGGGCACCCA GGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTGCGTTACTCCCGGGATGGT CGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTGATCTCAGGCAATGAGGACCCTGAGGGAG TGGTGCTGAAGGACCTTGGGCCCCCCATGGTTGCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGT CATGAGCGTCTGTCTGCGGGTAGAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACCGCC CCTGTGGGGCAGACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCG TGGGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGACTTTAGGAA GAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGACTATGTGGGATGGAGCAACCACAGCTTCTCCAGTGGC TATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTCCAGGCTATGCAGGTCCACTGTAACAACA TGCACACGCTGGGAGCCCGTCTGCCTGGCGGGGTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTG GGAGGGGGAGCCCATGCGCCACAACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTG CCCCTTGGCGGCCGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCA GCGAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTCCCGCCAGC CCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTGGAGCCCAGAGGCCAGCAG CCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATCGGCTGCCTGGTGGCCATCATCCTGCTCC TGCTGCTCATCATTGCCCTCATGCTCTGGCGGCTGCACTGGCGCAGGCTCCTCAGCAAGGCTGAACGGAG GGTGTTGGAAGAGGAGCTGACGGTTCACCTCTCTGTCCCTGGGGACACTATCCTCATCAACAACCGCCCA GGTCCTAGAGAGCCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTG TCCCCAATGGCTCTGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGTCTTGATTTCCCCC TTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTACGGCCAGATGCCACCAAGAATGC CAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCGAGGCTCAAGGACCCAAACATCATTCGGCTGCTG GGCGTGTGTGTGCAGGACGACCCCCTCTGCATGATTACTGACTACATGGAGAACGGCGACCTCAACCAGT TCCTCAGTGCCCACCAGCTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTGCGCAGGG GCCCACCATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTATCTGGCC ACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAATTTCACCATCAAAA TCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGACTATTACCGTGTGCAGGGCCGGGCAGTGCT GCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATGGGGAAGTTCACGACTGCGAGTGACGTGTGGGCC TTTGGTGTGACCCTGTGGGAGGTGCTGATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACCGACGAGC AGGTCATCGAGAACGCGGGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGCCGCCTGC CTGCCCGCAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACCACCCTTT TCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGTGAATCACACATCCAGCTGCCCCTC CCTCAGGGAGCGATCCAGGGGAAGCCAGTGACACTAAAACAAGAGGACACAATGGCACCTCTGCCCTTCC CCTCCCGACAGCCCATCACCTCTAATAGAGGCAGTGAGACTGCAGGTGGGCTGGGCCCACCCAGGGAGCT GATGCCCCTTCTCCCCTTCCTGGACACACTCTCATGTCCCCTTCCTGTTCTTCCTTCCTAGAAGCCCCTG TCGCCCACCCAGCTGGTCCTGTGGATGGGATCCTCTCCACCCTCCTCTAGCCATCCCTTGGGGAAGGGTG GGGAGAAATATAGGATAGACACTGGACATGGCCCATTGGAGCACCTGGGCCCCACTGGACAACACTGATT CCTGGAGAGGTGGCTGCGCCCCCAGCTTCTCTCTCCCTGTCACACACTGGACCCCACTGGCTGAGAATCT GGGGGTGAGGAGGACAAGAAGGAGAGGAAAATGTTTCCTTGTGCCTGCTCCTGTACTTGTCCTCAGCTTG GGCTTCTTCCTCCTCCATCACCTGAAACACTGGACCTGGGGGTAGCCCCGCCCCAGCCCTCAGTCACCCC CACTTCCCACTTGCAGTCTTGTAGCTAGAACTTCTCTAAGCCTATACGTTTCTGTGGAGTAAATATTGGG ATTGGGGGGAAAGAGGGAGCAACGGCCCATAGCCTTGGGGTTGGACATCTCTAGTGTAGCTGCCACATTG ATTTTTCTATAATCACTTGGGGTTTGTACATTTTTGGGGGGAGAGACACAGATTTTTACACTAATATATG GACCTAGCTTGAGGCAATTTTAATCCCCTGCACTAGGCAGGTAATAATAAAGGTTGAGTTTTCCACAAAA AAAAAAAAAAAAAA
SEQ ID No. 56: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 4, amino acid sequence with the NCBI Reference Sequence: NP 001 189450.1
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPD SDI SASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGT
QGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMV ARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGH TVGGLQYGGLGQLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDR LRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAV SVPLGGRVARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPP TNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLLI IALMLWRLHWRRLLSKA ERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPRGNPPHSAPCVPNGSGAPV
SEQ ID No. 57: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 5, mRNA with the NCBI Reference Sequence: NM_001202522.1
AGATGCTGCCCCCACCCCCTTAGGCCCGAGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCATCTTTAC TGCTGCTGCTCTTGGTGGCAAGTGGAGATGCTGACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTA TGCCCTGGGCATGCAGGACCGGACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCC ACTGCCGCCCGCCACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGT TTCCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTGGGCACCCA GGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTGCGTTACTCCCGGGATGGT CGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTGATCTCAGGCAATGAGGACCCTGAGGGAG TGGTGCTGAAGGACCTTGGGCCCCCCATGGTTGCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGT CATGAGCGTCTGTCTGCGGGTAGAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACCGCC CCTGTGGGGCAGACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCG TGGGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGACTTTAGGAA GAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGACTATGTGGGATGGAGCAACCACAGCTTCTCCAGTGGC TATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTCCAGGCTATGCAGGTCCACTGTAACAACA TGCACACGCTGGGAGCCCGTCTGCCTGGCGGGGTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTG GGAGGGGGAGCCCATGCGCCACAACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTG CCCCTTGGCGGCCGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCA GCGAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTCCCGCCAGC CCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTGGAGCCCAGAGGCCAGCAG CCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATCGGCTGCCTGGTGGCCATCATCCTGCTCC TGCTGCTCATCATTGCCCTCATGCTCTGGCGGCTGCACTGGCGCAGGCTCCTCAGCAAGGTCCTAGAGAG CCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTGTCCCCAATGGCT CTGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGTCTTGATTTCCCCCTTAATGTGCGTA AGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTACGGCCAGATGCCACCAAGAATGCCAGGAATGATTT CCTGAAAGAGGTGAAGATCATGTCGAGGCTCAAGGACCCAAACATCATTCGGCTGCTGGGCGTGTGTGTG CAGGACGACCCCCTCTGCATGATTACTGACTACATGGAGAACGGCGACCTCAACCAGTTCCTCAGTGCCC ACCAGCTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTGCGCAGGGGCCCACCATCAG CTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTATCTGGCCACACTCAACTTT GTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAATTTCACCATCAAAATCGCAGACTTTG GCATGAGCCGGAACCTCTATGCTGGGGACTATTACCGTGTGCAGGGCCGGGCAGTGCTGCCCATCCGCTG GATGGCCTGGGAGTGCATCCTCATGGGGAAGTTCACGACTGCGAGTGACGTGTGGGCCTTTGGTGTGACC CTGTGGGAGGTGCTGATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACCGACGAGCAGGTCATCGAGA ACGCGGGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGCCGCCTGCCTGCCCGCAGGG CCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACCACCCTTTTCCCAGCTGCAT CGGTTCCTGGCAGAGGATGCACTCAACACGGTGTGAATCACACATCCAGCTGCCCCTCCCTCAGGGAGCG ATCCAGGGGAAGCCAGTGACACTAAAACAAGAGGACACAATGGCACCTCTGCCCTTCCCCTCCCGACAGC
CCATCACCTCTAATAGAGGCAGTGAGACTGCAGGTGGGCTGGGCCCACCCAGGGAGCTGATGCCCCTTCT CCCCTTCCTGGACACACTCTCATGTCCCCTTCCTGTTCTTCCTTCCTAGAAGCCCCTGTCGCCCACCCAG CTGGTCCTGTGGATGGGATCCTCTCCACCCTCCTCTAGCCATCCCTTGGGGAAGGGTGGGGAGAAATATA GGATAGACACTGGACATGGCCCATTGGAGCACCTGGGCCCCACTGGACAACACTGATTCCTGGAGAGGTG GCTGCGCCCCCAGCTTCTCTCTCCCTGTCACACACTGGACCCCACTGGCTGAGAATCTGGGGGTGAGGAG GACAAGAAGGAGAGGAAAATGTTTCCTTGTGCCTGCTCCTGTACTTGTCCTCAGCTTGGGCTTCTTCCTC CTCCATCACCTGAAACACTGGACCTGGGGGTAGCCCCGCCCCAGCCCTCAGTCACCCCCACTTCCCACTT GCAGTCTTGTAGCTAGAACTTCTCTAAGCCTATACGTTTCTGTGGAGTAAATATTGGGATTGGGGGGAAA GAGGGAGCAACGGCCCATAGCCTTGGGGTTGGACATCTCTAGTGTAGCTGCCACATTGATTTTTCTATAA TCACTTGGGGTTTGTACATTTTTGGGGGGAGAGACACAGATTTTTACACTAATATATGGACCTAGCTTGA GGCAATTTTAATCCCCTGCACTAGGCAGGTAATAATAAAGGTTGAGTTTTCCACAAAAAAAAAAAAAAAA AA
SEQ ID No. 58: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 5, amino acid sequence with the NCBI Reference Sequence NP 001 189451 .1
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPD SDI SASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGT QGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMV ARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYDGH TVGGLQYGGLGQLADGWGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDR LRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAV SVPLGGRVARFLQCRFLFAGPWLLFSEISFISDWNNSSPALGGTFPPAPWWPPGPPP TNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLLI IALMLWRLHWRRLLSKV LESHPRTRSPGLVGIRPTPLPVSPMALVHLCEVDSPQDLVSLDFPLNVRKGHPLLVAV KILRPDATKNARNDFLKEVKIMSRLKDP I IRLLGVCVQDDPLCMITDYMENGDLNQF LSAHQLEDKAAEGAPGDGQAAQGPTI SYPMLLHVAAQIASGMRYLATLNFVHRDLATR NCLVGENFTIKIADFGMSRNLYAGDYYRVQGRAVLPIRWMAWECILMGKFTTASDVWA FGVTLWEVLMLCRAQPFGQLTDEQVIENAGEFFRDQGRQVYLSRPPACPQGLYELMLR CWSRESEQRPPFSQLHRFLAEDALNTV
SEQ ID No. 59: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), transcript variant 6, mRNA with the NCBI Reference Sequence: NM_001202523.1
AGTGCGGAGGAACTGAGCCCCGGGAGGAGGTGCTCCTGTGCAGCCCCACTGAGTCAGCTCATCTATCGCC TGCCCTCCACCTGGCCAGTCCCTGCGGGCATCTAACTGCTAAGCCTCCGCTCAGCCAACACCCAGTTGGT CAGTCTGGTCACAGTCCAGCAAAAAGAGGGACTGCCACTCTAACCCACCAGTGACACCACTCTTCCCGGC TGGATGGTCAATTAGCTCTGGCATGAGAGAATGTCACTGCCGAGATGCTGCCCCCACCCCCTTAGGCCCG AGGGATCAGGAGCTATGGGACCAGAGGCCCTGTCATCTTTACTGCTGCTGCTCTTGGTGGCAAGTGGAGA
TGCTGACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTATGCCCTGGGCATGCAGGACCGGACCATC CCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCCACTGCCGCCCGCCACAGCAGGTTGGAGA GCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGTTTCCCAAGGAGGAGGAGTACTTGCAGGT GGATCTACAACGACTGCACCTGGTGGCTCTGGTGGGCACCCAGGGACGGCATGCCGGGGGCCTGGGCAAG GAGTTCTCCCGGAGCTACCGGCTGCGTTACTCCCGGGATGGTCGCCGCTGGATGGGCTGGAAGGACCGCT GGGGTCAGGAGGTGATCTCAGGCAATGAGGACCCTGAGGGAGTGGTGCTGAAGGACCTTGGGCCCCCCAT GGTTGCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGTCATGAGCGTCTGTCTGCGGGTAGAGCTC TATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACCGCCCCTGTGGGGCAGACAATGTATTTATCTG AGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCGTGGGCGGACTGCAGTATGGGGGTCTGGG CCAGCTGGCAGATGGTGTGGTGGGGCTGGATGACTTTAGGAAGAGTCAGGAGCTGCGGGTCTGGCCAGGC TATGAC TATGTGGGATGGAGCAACCACAGCTTCTCCAGTGGCTATGTGGAGATGGAGTTTGAGTTTGACC GGCTGAGGGCCTTCCAGGCTATGCAGGTCCACTGTAACAACATGCACACGCTGGGAGCCCGTCTGCCTGG CGGGGTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTGGGAGGGGGAGCCCATGCGCCACAACCTA GGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTGCCCCTTGGCGGCCGTGTGGCTCGCTTTC TGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCAGCGAAATCTCCTTCATCTCTGATGTGGT GAACAATTCCTCTCCGGCACTGGGAGGCACCTTCCCGCCAGCCCCCTGGTGGCCGCCTGGCCCACCTCCC ACCAACTTCAGCAGCTTGGAGCTGGAGCCCAGAGGCCAGCAGCCCGTGGCCAAGGCCGAGGGGAGCCCGA CCGCCATCCTCATCGGCTGCCTGGTGGCCATCATCCTGCTCCTGCTGCTCATCATTGCCCTCATGCTCTG GCGGCTGCACTGGCGCAGGCTCCTCAGCAAGGCTGAACGGAGGGTGTTGGAAGAGGAGCTGACGGTTCAC CTCTCTGTCCCTGGGGACACTATCCTCATCAACAACCGCCCAGGTCCTAGAGAGCCACCCCCGTACCAGG AGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTGTCCCCAATGGCTCTGCCTACAGTGGGGA CTATATGGAGCCTGAGAAGCCAGGCGCCCCGCTTCTGCCCCCACCTCCCCAGAACAGCGTCCCCCATTAT GCCGAGGCTGACATTGTTACCCTGCAGGGCGTCACCGGGGGCAACACCTATGCTGTGCCTGCACTGCCCC CAGGGGCAGTCGGGGATGGGCCCCCCAGAGTGGATTTCCCTCGATCTCGACTCCGCTTCAAGGAGAAGCT TGGCGAGGGCCAGTTTGGGGAGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGTCTTGAT TTCCCCCTTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTACGGCCAGATGCCACCA AGAATGCCAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCGAGGCTCAAGGACCCAAACATCATTCG GCTGCTGGGCGTGTGTGTGCAGGACGACCCCCTCTGCATGATTACTGACTACATGGAGAACGGCGACCTC AACCAGTTCCTCAGTGCCCACCAGCTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTG CGCAGGGGCCCACCATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTA TCTGGCCACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAATTTCACC ATCAAAATCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGAC TATTACCGTGTGCAGGGCCGGG CAGTGCTGCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATGGGGAAGTTCACGACTGCGAGTGACGT GTGGGCCTTTGGTGTGACCCTGTGGGAGGTGCTGATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACC GACGAGCAGGTCATCGAGAACGCGGGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGC CGCCTGCCTGCCCGCAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACC ACCCTTTTCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGTGAATCACACATCCAGCT GCCCCTCCCTCAGGGAGCGATCCAGGGGAAGCCAGTGACACTAAAACAAGAGGACACAATGGCACCTCTG CCCTTCCCCTCCCGACAGCCCATCACCTCTAATAGAGGCAGTGAGACTGCAGGTGGGCTGGGCCCACCCA GGGAGCTGATGCCCCTTCTCCCCTTCCTGGACACACTCTCATGTCCCCTTCCTGTTCTTCCTTCCTAGAA GCCCCTGTCGCCCACCCAGCTGGTCCTGTGGATGGGATCCTCTCCACCCTCCTCTAGCCATCCCTTGGGG AAGGGTGGGGAGAAATATAGGATAGACACTGGACATGGCCCATTGGAGCACCTGGGCCCCACTGGACAAC ACTGATTCCTGGAGAGGTGGCTGCGCCCCCAGCTTCTCTCTCCCTGTCACACACTGGACCCCACTGGCTG AGAATCTGGGGGTGAGGAGGACAAGAAGGAGAGGAAAATGTTTCCTTGTGCCTGCTCCTGTACTTGTCCT CAGCTTGGGCTTCTTCCTCCTCCATCACCTGAAACACTGGACCTGGGGGTAGCCCCGCCCCAGCCCTCAG TCACCCCCACTTCCCACTTGCAGTCTTGTAGCTAGAACTTCTCTAAGCCTATACGTTTCTGTGGAGTAAA TATTGGGATTGGGGGGAAAGAGGGAGCAACGGCCCATAGCCTTGGGGTTGGACATCTCTAGTGTAGCTGC CACATTGATTTTTCTATAATCACTTGGGGTTTGTACATTTTTGGGGGGAGAGACACAGATTTTTACACTA ATATATGGACCTAGCTTGAGGCAATTTTAATCCCCTGCACTAGGCAGGTAATAATAAAGGTTGAGTTTTC CACAAAAAAAAAAAAAAAAAA
SEQ ID No. 60: Homo sapiens discoidin domain receptor tyrosine kinase 1 (DDRl), variant 6, amino acid sequence with the NCBI Reference Sequence NP 001 189452.1 MSLPRCCPHPLRPEGSGAMGPEALSSLLLLLLVASGDADMKGHF
DPAKCRYALGMQDRTI PDSDISASSSWSDSTAARHSRLESSDGDGAWCPAGSVFPKEE
EYLQVDLQRLHLVALVGTQGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVIS GNEDPEGWLKDLGPPMVARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQ TMYLSEAVYLNDSTYDGHTVGGLQYGGLGQLADGVVGLDDFRKSQELRVWPGYDYVGW SNHSFSSGYVEMEFEFDRLRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGE PMRHNLGGNLGDPRARAVSVPLGGRVARFLQCRFLFAGPWLLFSE ISFISDWNNSSP
ALGGTFPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAI ILLLLL I IALMLWRLHWRRLLSKAERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPR GNPPHSAPCVPNGSAYSGDYMEPEKPGAPLLPPPPQNSVPHYAEADIVTLQGVTGGNT YAVPALPPGAVGDGPPRVDFPRSRLRFKEKLGEGQFGEVHLCEVDSPQDLVSLDFPLN VRKGHPLLVAVKILRPDATKNARNDFLKEVKIMSRLKDP I IRLLGVCVQDDPLCMIT
DYMENGDLNQFLSAHQLEDKAAEGAPGDGQAAQGPTISYPMLLHVAAQIASGMRYLAT LNFVHRDLATRNCLVGENFTIKIADFGMSRNLYAGDYYRVQGRAVLPIRWMAWECILM GKFTTASDVWAFGVTLWEVLMLCRAQPFGQLTDEQVIENAGEFFRDQGRQVYLSRPPA CPQGLYELMLRCWSRESEQRPPFSQLHRFLAEDALNTV
SEQ ID No 61 : corresponding to plasmid IOH5763 from Invitrogen corresponding to the cDNA sequence of human DDR1
ATGGGACCAGAGGCCCTGTCATCTTTACTGCTGCTGCTCTTGGTGGCAAGTGGAGATGCT GACATGAAGGGACATTTTGATCCTGCCAAGTGCCGCTATGCCCTGGGCATGCAGGACCGG ACCATCCCAGACAGTGACATCTCTGCTTCCAGCTCCTGGTCAGATTCCACTGCCGCCCGC CACAGCAGGTTGGAGAGCAGTGACGGGGATGGGGCCTGGTGCCCCGCAGGGTCGGTGTTT CCCAAGGAGGAGGAGTACTTGCAGGTGGATCTACAACGACTGCACCTGGTGGCTCTGGTG GGCACCCAGGGACGGCATGCCGGGGGCCTGGGCAAGGAGTTCTCCCGGAGCTACCGGCTG CGTTACTCCCGGGATGGTCGCCGCTGGATGGGCTGGAAGGACCGCTGGGGTCAGGAGGTG ATCTCAGGCAATGAGGACCCTGAGGGAGTGGTGCTGAAGGACCTTGGGCCCCCCATGGTT GCCCGACTGGTTCGCTTCTACCCCCGGGCTGACCGGGTCATGAGCGTCTGTCTGCGGGTA GAGCTCTATGGCTGCCTCTGGAGGGATGGACTCCTGTCTTACACTGCCCCTGTGGGGCAG ACAATGTATTTATCTGAGGCCGTGTACCTCAACGACTCCACCTATGACGGACATACCGTG GGCGGACTGCAGTATGGGGGTCTGGGCCAGCTGGCAGATGGTGTGGTGGGGCTGGATGAC TTTAGGAAGAGTCAGGAGCTGCGGGTCTGGCCAGGCTATGACTATGTGGGATGGAGCAAC CACAGCTTCTCCAGTGGCTATGTGGAGATGGAGTTTGAGTTTGACCGGCTGAGGGCCTTC CAGGCTATGCAGGTCCACTGTAACAACATGCACACGCTGGGAGCCCGTCTGCCTGGCGGG GTGGAATGTCGCTTCCGGCGTGGCCCTGCCATGGCCTGGGAGGGGGAGCCCATGCGCCAC AACCTAGGGGGCAACCTGGGGGACCCCAGAGCCCGGGCTGTCTCAGTGCCCCTTGGCGGC CGTGTGGCTCGCTTTCTGCAGTGCCGCTTCCTCTTTGCGGGGCCCTGGTTACTCTTCAGC
GAAATCTCCTTCATCTCTGATGTGGTGAACAATTCCTCTCCGGCACTGGGAGGCACCTTC CCGCCAGCCCCCTGGTGGCCGCCTGGCCCACCTCCCACCAACTTCAGCAGCTTGGAGCTG GAGCCCAGAGGCCAGCAGCCCGTGGCCAAGGCCGAGGGGAGCCCGACCGCCATCCTCATC GGCTGCCTGGTGGCCATCATCCTGCTCCTGCTGCTCATCATTGCCCTCATGCTCTGGCGG CTGCACTGGCGCAGGCTCCTCAGCAAGGCTGAACGGAGGGTGTTGGAAGAGGAGCTGACG GTTCACCTCTCTGTCCCTGGGGACACTATCCTCATCAACAACCGCCCAGGTCCTAGAGAG CCACCCCCGTACCAGGAGCCCCGGCCTCGTGGGAATCCGCCCCACTCCGCTCCCTGTGTC CCCAATGGCTCTGCCTACAGTGGGGACTATATGGAGCCTGAGAAGCCAGGCGCCCCGCTT CTGCCCCCACCTCCCCAGAACAGCGTCCCCCATTATGCCGAGGCTGACATTGTTACCCTG CAGGGCGTCACCGGGGGCAACACCTATGCTGTGCCTGCACTGCCCCCAGGGGCAGTCGGG GATGGGCCCCCCAGAGTGGATTTCCCTCGATCTCGACTCCGCTTCAAGGAGAAGCTTGGC GAGGGCCAGTTTGGGGAGGTGCACCTGTGTGAGGTCGACAGCCCTCAAGATCTGGTTAGT CTTGATTTCCCCCTTAATGTGCGTAAGGGACACCCTTTGCTGGTAGCTGTCAAGATCTTA CGGCCAGATGCCACCAAGAATGCCAGGAATGATTTCCTGAAAGAGGTGAAGATCATGTCG AGGCTCAAGGACCCAAACATCATTCGGCTGCTGGGCGTGTGTGTGCAGGACGACCCCCTC TGCATGATTACTGACTACATGGAGAACGGCGACCTCAACCAGTTCCTCAGTGCCCACCAG CTGGAGGACAAGGCAGCCGAGGGGGCCCCTGGGGACGGGCAGGCTGCGCAGGGGCCCACC ATCAGCTACCCAATGCTGCTGCATGTGGCAGCCCAGATCGCCTCCGGCATGCGCTATCTG GCCACACTCAACTTTGTACATCGGGACCTGGCCACGCGGAACTGCCTAGTTGGGGAAAAT TTCACCATCAAAATCGCAGACTTTGGCATGAGCCGGAACCTCTATGCTGGGGACTATTAC CGTGTGCAGGGCCGGGCAGTGCTGCCCATCCGCTGGATGGCCTGGGAGTGCATCCTCATG GGGAAGTTCACGACTGCGAGTGACGTGTGGGCCTTTGGTGTGACCCTGTGGGAGGTGCTG ATGCTCTGTAGGGCCCAGCCCTTTGGGCAGCTCACCGACGAGCAGGTCATCGAGAACGCG GGGGAGTTCTTCCGGGACCAGGGCCGGCAGGTGTACCTGTCCCGGCCGCCTGCCTGCCCG CAGGGCCTATATGAGCTGATGCTTCGGTGCTGGAGCCGGGAGTCTGAGCAGCGACCACCC TTTTCCCAGCTGCATCGGTTCCTGGCAGAGGATGCACTCAACACGGTGTAG
SEQ ID No. 62: aminoacid sequence corresponding to plasmid IOH5763
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTI PDSDISASSSWSDSTAARHSRLESSDGDGA WCPAGSVFPKEEEYLQVDLQRLHLVALVGTQGRHAGGLGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGN EDPEGVVLKDLGPPMVARLVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYD GHTVGGLQYGGLGQLADGVVGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVEMEFEFDRLRAFQAMQVHCN NMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNLGGNLGDPRARAVSVPLGGRVARFLQCRFLFAGPWLLFS EISFISDWNNSSPALGGTFPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPTAILIGCLVAIILLLLL I IALMLWRLHWRRLLSKAERRVLEEELTVHLSVPGDTILINNRPGPREPPPYQEPRPRGNPPHSAPCVPNGS AYSGDYMEPEKPGAPLLPPPPQNSVPHYAEADIVTLQGVTGGNTYAVPALPPGAVGDGPPRVDFPRSRLRFK EKLGEGQFGEVHLCEVDSPQDLVSLDFPLNVRKGHPLLVAVKILRPDATKNARNDFLKEVKIMSRLKDP I I RLLGVCVQDDPLCMITDYMENGDLNQFLSAHQLEDKAAEGAPGDGQAAQGPTISYPMLLHVAAQIASGMRYL ATLNFVHRDLATRNCLVGENFTIKIADFGMSRNLYAGDYYRVQGRAVLPIRWMAWECILMGKFTTASDVWAF GVTLWEVLMLCRAQPFGQLTDEQVIENAGEFFRDQGRQVYLSRPPACPQGLYELMLRCWSRESEQRPPFSQL HRFLAEDALNTV
SEQ ID No. 63: Epitope corresponding to the extracellular domain of human DDR1 :
MGPEALSSLLLLLLVASGDADMKGHFDPAKCRYALGMQDRTIPDSDISASSSWSD STAARHSRLESSDGDGAWCPAGSVFPKEEEYLQVDLQRLHLVALVGTQGRHAGG LGKEFSRSYRLRYSRDGRRWMGWKDRWGQEVISGNEDPEGVVLKDLGPPMVAR LVRFYPRADRVMSVCLRVELYGCLWRDGLLSYTAPVGQTMYLSEAVYLNDSTYD GHTVGGLQYGGLGQLADGVVGLDDFRKSQELRVWPGYDYVGWSNHSFSSGYVE MEFEFDRLRAFQAMQVHCNNMHTLGARLPGGVECRFRRGPAMAWEGEPMRHNL GGNLGDPRARAVSVPLGGRVARFLQCRFLFAGPWLLFSEISFISDVVNNSSPALGG TFPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPT
SEQ ID No. 64:
Amino acid sequence of an inhibitory peptide (peptide inhibitor of the transcription factor STAT6) to be conjugated to the anti-DDRl antibodies
YARAAARQARAGRGYVSTT (Y represents a phosphotyrosine)
SEQ ID No. 65:
Amino acid sequence of an inhibitory peptide (phosphopeptide which inhibits the activity of the transcription factor STAT3 in vitro and in vivo) to be conjugated to the anti-DDRl antibodies
PYLKTK (Y represents a phosphotyrosine) SEQ ID No. 66:
Amino acid sequence of an inhibitory peptide (part of the N-terminal sequence of pl4ARF that is able to induce apoptosis) to be conjugated to the anti-DDRl antibodies MVRRFLVTLRIRRACGPPRVRV
SEQ ID No. 67:
Nucleotide sequence of 5 '-primer for amplification of a first binding domain of vector
DDRl -ab lpBhl
gacatccagatgacccagtctccatcttccgtg
SEQ ID No. 68:
Nucleotide sequence of 3 '-primer for amplification of a first binding domain of vector
DDRl -ab lpBhl
gccgaaagtgagcgggaaactgttagc
SEQ ID No. 69:
Nucleotide sequence of 5 '-primer for amplification of a second binding domain of vector DDRl -ablpBhl
ttcactttctcttattactggatgtgg SEQ ID No. 70:
Nucleotide sequence of 3 '-primer for amplification of a second binding domain of vector DDRl -ablpBhl
ccagatatcaaaagcagcactaattcc
SEQ ID No. 71 :
Nucleotide sequence of 5 '-primer for amplification of a first binding domain of vector
DDRl -ab2pBhl
ttgactcagccaccctcagcgtct
SEQ ID No. 72:
Nucleotide sequence of 3 '-primer for amplification of a first binding domain of vector
DDRl -ab2pBhl
gccgaacaccggaacactcaggctgtc
SEQ ID No. 73:
Nucleotide sequence of 5 '-primer for amplification of a second binding domain of vector DDRl -ab2pBhl
ttcactttctcttcttacgttatggtt
SEQ ID No. 74:
Nucleotide sequence of 3 '-primer for amplification of a second binding domain of vector DDRl -ab2pBhl
gacgtccatgtacccaaaaatcccaag
SEQ ID No. 75:
Nucleotide sequence of 5 '-primer for amplification of a first binding domain of vector
DDRl -ab3pBhl
gacatccagatgacccagtctccatcatccctg
SEQ ID No. 76:
Nucleotide sequence of 3 '-primer for amplification of a first binding domain of vector
DDRl -ab3pBhl
gccgaaagtgagcggtataatgtaact
SEQ ID No. 77: Nucleotide sequence of 5 '-primer for amplification of a second binding domain of vector DDRl -ab3pBhl
ttcactttctctgagtactttatggct SEQ ID o. 78:
Nucleotide sequence of 3 '-primer for amplification of a second binding domain of vector DDRl -ab3pBhl
gtagtcaaagtgccggtcttgaaatac
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Figure imgf000146_0001
Figure imgf000147_0001
Indications are Made All designations
FOR RECEIVING OFFICE USE ONLY -4 This form was received with the
international application: yes
(yes or no)
-4-1 Authorized officer
Krista Delimon
FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the
international Bureau on:
-5-1 Authorized officer

Claims

An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14, a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16, and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18, or a CDR sequence having 85% or more amino acid identity to one of said CDRs; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-Hl region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24, or a CDR sequence having 85% or more amino acid identity to one of said CDRs.
The antibody of claim 1,
wherein the variable region of the light chain of said antibody comprises a CDR-Ll region having an amino acid sequence as depicted in SEQ ID NO: 14, a CDR-L2 region having an amino acid sequence as depicted in SEQ ID NO: 16, and a CDR-L3 region having an amino acid sequence as depicted in SEQ ID NO.: 18; and
wherein the variable region of the heavy chain of said antibody comprises a CDR-Hl region having an amino acid sequence as depicted in SEQ ID NO: 20, a CDR-H2 region having an amino acid sequence as depicted in SEQ ID NO: 22, and a CDR-H3 region having an amino acid sequence as depicted in SEQ ID NO.: 24.
An antibody that specifically binds to discoidin domain receptor 1 (DDR1),
wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43, or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region; or a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 , or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable Vn-region.
The antibody of claim 3,
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 , or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO: 42, or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.
An antibody that specifically binds to discoidin domain receptor 1 (DDR1 ), wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or a variable Vn-region as encoded by a nucleic acid molecule having 75% or more identity to said variable Vn-region or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO:44 or a variable Vn-region having an amino acid sequence which has 75% or more identity to said variable VH-region; and
wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or a variable VL-region as encoded by a nucleic acid molecule having 75% or more identity to said variable VL-region or a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42 or a variable VL-region having an amino acid sequence which has 75% or more identity to said variable VL-region.
The antibody of claim 5,
wherein said antibody comprises a variable Vn-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:43 or
a variable Vn-region having an amino acid sequence as shown in SEQ ID NO: 44; and wherein said antibody comprises a variable VL-region as encoded by a nucleic acid molecule as shown in SEQ ID NO:41 or
a variable VL-region having an amino acid sequence as shown in SEQ ID NO:42.
7. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), wherein said antibody is DDRl-ab2 as encoded by a nucleic acid molecule comprised in vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 201 1.
8. The antibody of any one of claims 1 to 7, wherein said antibody specifically binds to discoidin domain receptor 1 (DDRl), wherein the antibody is capable of internalizing into cells.
9. The antibody of claim 8, wherein the antibody is capable of internalizing into cells in the absence of collagen.
10. The antibody of any one of claims 8 or 9, wherein said cell is a DDRl expressing cell.
11. The antibody of claim 10, wherein said cell is a cancer cell.
12. The antibody of any one of claims 1 to 11 , wherein said antibody is a full antibody (immunoglobulin), an antibody fragment such as a F(ab)-fragment or a F(ab)2-fragment, a single-chain antibody, a chimeric antibody, a humanized antibody, a human antibody, a fully human antibody, a CDR-grafted antibody, a bivalent antibody-construct, a bispecific single-chain antibody, a synthetic antibody or a cross-cloned antibody.
13. The antibody of claim 12, wherein said antibody is an immunoglobulin selected from the group consisting of IgA, IgD, IgE, IgG or IgM antibody.
14. The antibody of any one of claims 1 to 13, wherein said antibody is conjugated to one or more therapeutic agents.
15. The antibody of claim 14, wherein the therapeutic agent is a toxin.
16. The antibody of claim 14, wherein the therapeutic agent is an anticancer agent.
17. A nucleic acid molecule having a sequence encoding the antibody as defined in any one of claims 1 to 16.
18. A nucleic acid molecule comprised in the vector as defined in claim 7.
19. A vector comprising a nucleic acid molecule according to claim 17 or 18.
20. The vector of claim 19, which further comprises a nucleic acid molecule having a regulatory sequence which is operably linked to said nucleic acid molecule according to claim 17 or 18.
21. The vector of claim 19 or 20, wherein the vector is an expression vector.
22. The vector of any one of claims 19 to 21 , wherein said vector is DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December
20th, 2011.
23. A host transformed or trans fected with a vector according to any of claims 19 to 22.
24. The host of claim 23, wherein said host is a eukaryotic host cell like COS, CHO, HEK293 or a multiple myeloma host cell.
25. A process for the production of the antibody as defined in any one of claims 1 to 16, said process comprising culturing a host of claim 23 or 24 under conditions allowing the expression of the antibody and recovering the produced antibody from the culture.
26. A composition comprising the antibody as defined in any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22 and/or a host of claim 23 or 24.
27. The composition of claim 26, further comprising a secondary antibody that is specifically binding to the primary antibody as defined in any one of claims 1 to 16, whereby said secondary antibody is conjugated to a therapeutic agent.
28. The composition of claim 27, wherein said therapeutic agent is a toxin.
29. The composition of claim 27 or 28, wherein said primary antibody is an IgG antibody and said secondary antibody is a goat anti-human IgG secondary antibody.
30. The antibody of claim 15 or the composition of claim 28 or 29, wherein said toxin is Saporin.
31. The composition of any one of claims 26 to 30 which is a pharmaceutical composition, optionally further comprising one or more pharmaceutically acceptable excipient(s).
32. The antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22 and/or a host of claim 23 or 24 and/or the composition of any one of claims 26 to 31 for use in medicine.
33. Use of the antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22 and/or a host of claim 23 or 24 for the preparation of a pharmaceutical composition for the treatment of cancer.
34. The antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22, a host of claim 23 or 24 and/or the composition of any one of claims 26 to 31 for use in the treatment of cancer.
A method for the treatment of cancer comprising the administration of the antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22, a host of claim 23 or 24 and/or the composition of any one of claims 26 to 31 to a subject in need of such a treatment.
The method of claim 35, wherein said subject is a human.
The use of claim 33, the antibody of claim 11 or 34, the nucleic acid molecule of claim 34, the vector of claim 34, the host of claim 34, the composition of claim 34, or the method of claim 35 or 36, wherein said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer.
An antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in claim 22.
An antibody obtainable by a process comprising culturing a host transfected or transformed with the vector as defined in claim 22 under conditions that provide for the production of the antibody by the host and allow for the recovering of the antibody from the culture.
The composition of claim 26, which is a diagnostic composition further comprising optionally, means and methods for detection.
41. A method for diagnosing cancer, comprising detecting or assaying DDR1 in a biological sample of an individual suspected of suffering from cancer using the antibody according to any one of claims 1 to 16.
42. Use of the antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22 and/or a host of claim 23 or 24 for the preparation of a diagnostic composition for the diagnosis of cancer.
43. The antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22, a host of claim 23 or 24 and/or the composition of claim 26 for use in the diagnosis of cancer.
44. Use of the antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22, a host of claim 23 or 24 and/or the composition of claim 26 for the preparation of a diagnostic kit for the diagnosis of cancer.
45. Kit comprising the antibody of any one of claims 1 to 16 or as produced by the process of claim 25, a nucleic acid molecule of claim 17 or 18, a vector of any one of claims 19 to 22 and/or a host of claim 23 or 24 and/or the composition of any one of claims 26 to 31 or 40.
46. The use of claim 42 or 44, the antibody of claim 43, the nucleic acid molecule of claim 43, the vector of claim 43, the host of claim 43 and/or the composition of claim 43, wherein said cancer is epidermoid cancer (also known as squamous cell cancer), endometrial cancer, bladder cancer, colorectal cancer, breast cancer, lung cancer, stomach cancer, prostate cancer, pancreatic cancer, hepatic cancer, urothelial cancer, head and neck cancer, skin cancer, vaginal cancer, cervix cancer or ovarian cancer.
47. An antibody having essentially the same biological activity of an antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in claim 22.
48. An antibody that binds to the same epitope as an antibody obtained or obtainable by expression of the nucleic acid molecule contained in the vector as defined in claim 22.
49. An antibody that specifically binds to discoidin domain receptor 1 (DDRl), which is encoded by a nucleic acid molecule located on a nucleotide fragment that can be obtained by PCR amplification of DNA of vector DDRl-ab2pBhl deposited under accession number DSM 25530 with the depositary institute DSMZ on December 20th, 2011 using a. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 71 and a 3 '-primer as shown in SEQ ID NO: 72 for amplification of a first binding domain; and/ or
b. a primer pair comprising a 5 '-primer as shown in SEQ ID NO: 73 and a 3 '-primer as shown in SEQ ID NO: 74 for amplification of a second binding domain.
PCT/EP2014/055191 2013-03-15 2014-03-14 Anti-ddr1 internalizing antibodies and their medical use WO2014140330A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003085125A1 (en) * 2002-04-03 2003-10-16 Agy Therapeutics, Inc. Use of biomolecular targets in the treatment and visualization of brain tumors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003085125A1 (en) * 2002-04-03 2003-10-16 Agy Therapeutics, Inc. Use of biomolecular targets in the treatment and visualization of brain tumors

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Title
"Antibody Engineering : Methods and Protocols", vol. 248, 5 December 2003, HUMANA PRESS, totowa NJ, ISBN: 978-1-58829-092-2, article JD MARKS: "Selection of internalizing antibodies for drug delivery", pages: 201 - 208, XP002726191, DOI: 10.1385/1-59259-666-5:201 *

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