NZ624534B2 - Anti-fgfr2 antibodies and uses thereof - Google Patents

Anti-fgfr2 antibodies and uses thereof Download PDF

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NZ624534B2
NZ624534B2 NZ624534A NZ62453412A NZ624534B2 NZ 624534 B2 NZ624534 B2 NZ 624534B2 NZ 624534 A NZ624534 A NZ 624534A NZ 62453412 A NZ62453412 A NZ 62453412A NZ 624534 B2 NZ624534 B2 NZ 624534B2
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seq
presented
antibody
heavy chain
light chain
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NZ624534A
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NZ624534A (en
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Sandra Bruder
Frank Dittmer
Ricarda Finnern
Jurgen Franz
Sven Golfier
Simone Greven
Stefanie Hammer
Axel Harrenga
Charlotte Christine Kopitz
Lars Linden
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Bayer Intellectual Property Gmbh
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Priority claimed from PCT/EP2012/073325 external-priority patent/WO2013076186A1/en
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Publication of NZ624534B2 publication Critical patent/NZ624534B2/en

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    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

Disclosed is an isolated antibody or antigen-binding fragment thereof specifically binding to the extracellular N-terminal epitope (RPSFSLVEDTTLEPE) of FGFR2 as presented by SEQ ID NO:63. Also disclosed is the use of said antibody or antigen-binding fragment in the manufacture of a medicament for the treatment of cancer. e treatment of cancer.

Description

GFR2 Antibodies and Uses Thereof The present invention provides recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for the fibroblast growth factor or 2 (FGFR2).
The antibodies, accordingly, can be used to treat tumors and other disorders and conditions associated with expression of FGFR2. The ion also provides c acid sequences encoding the foregoing antibodies, s containing the same, pharmaceutical compositions and kits with instructions for use.
BACKGROUND OF THE INVENTION Antibody-based therapy is proving very effective in the ent of various cancers, ing solid tumors. For example, HERCEPTIN® has been used successfully to treat breast cancer and RITUXAN® is effective in B-cell related cancer types. Central to the pment of a successful antibody—based therapy is isolation of antibodies against cell-surface proteins found to be preferentially expressed on tumor cells.
Fibroblast growth factor receptors are tyrosine receptor kinases (RTKs), from which four are known (FGFRl, FGFR2, FGFR3, FGFR4) in mammals. As s 22 human fibroblast growth factors (FGFs) are identified (Eswarakumar and Schlessinger, Cytokine & Growth Factor Reviews 2005, 16:139-149; Shimada et al., Proc Natl Acad Sci USA 2001, 98:6500-6505). FGFRs consist of three extracellular immunoglobulin (Ig)-like domains, D1-D3, y domains 2 and 3 are required for ligand binding, a single transmembrane domain and a cytoplasmic domain containing the catalytic protein tyrosine kinase core (for a schematic representation see Figure 1). The extracellular part harbors in addition the acidic box (AB) and the heparin binding site (HBS) (see Figure 1). An important hallmark of the FGFR family of RTKs is that a variety of alternatively spliced variants exist. Full length FGFR2 is called FGFR2 alpha, while the m lacking D1 is WO 76186 _ 2 _ termed FGFR2 beta e 1). Alternative splicing in domain 3 results in two ent variants namely FGFR2 IIIb, harboring exons 7 and 8, and FGFR2 IIIc, containing exons 7 and 9 (Figure 1). The latter splicing affects ligand binding, resulting in the specificity pattern. FGFR2 file is mainly expressed by mesenchymal cells, FGFR2 IIIb mainly by epithelial cells. FGF7 also known as keratinocyte growth factor (KGF) only binds to FGFR2 IIIb, which is therefore also termed KGFR. Upon binding of FGFs to their receptors, subsequently dimerization and phosphorylation of FGFRs and downstream signaling via FRS-GRB2 g protein complex to RAS-MAPK signaling cascade and PI3K-AKT signaling cascade occurs. The first signaling cascade is implicated in cell growth and differentiation, the latter in cell survival and fate determination (Katoh and Katoh, Int J Oncol 2006, 29:163-168).
Orchestrated signaling of all four ors (FGFRI to FGFR4) and their splice variants via the ent FGFs is required for proper organogenesis during embryogenesis (Ornitz et al., Genome Biol 2001, 223005). In case of FGFR2, lack of all FGFR2 variants results in defects in ta and limb bud formation and consequently results in lethality in E105. Specific knock-out of FGFR2 IIIb also results in lethality (in P0), associated with agenesis of lungs, anterior pituitary, thyroid, teeth and limbs, while disruption ofthe FGFRZ IIIc variant is viable showing delayed ossification, proportionate dwarfism, and synostosis of skull base (Eswarakumar and Schlessinger, 2005). Germline activating mutations of FGFR2 in humans lead to severe deformities during enbryogenesis, such as coronal- and craniosynostosis in Apert or Pfeiffer syndromes (Robin et al., in Gene Reviews, NCBI Bookshelf Washington, edts. Pagon et al., 1993). In the adult, FGFR2 signaling is involved in wound healing, epithelial repair and otection of skin and mucosa (Braun et al., Phil Trans R Soc Lond B 2004, 359:753-757) and in regeneration of injured liver (Steiling et al., Oncogene 2003, 22:4380-4388; Bohm, dissertation, Swiss Federal Institute of Technology Zurich, 2009). A role of FGFR2 signaling in migration of epicardial derived cells (EPDCs) into the heart after tion is under sion, since during embryogenesis FGFIO/FGFR2 signaling is necessary for migration of EPDCs in the compact dium, a s required for intact heart development (Vega-Hernandez et 2012/073325 _ 3 _ al., Development 2011:3331—3340; Winter and De Groot, Cell M01 Life Sci 2007, 64:692- 703) Increased, germline-independent ing through FGFR2 is involved in different pathologies, such as acne (Katoh, J of Invest Dermatol 2009, 129:1861-1867), psoriasis (Finch et al., Am J Pathol 1997, 15121619-1628; Xu et al., J Invest Dermatol 2011;131:1521-1529) periodontitis (Li et al., J Peridontal Res 2005, 40:128-138), solar lentigines (Lin et al., Journal Dermatol Sci 2010, 59:91-97), bowel disease (Brauchle et al., J Pathol 1996, 149:521-529), endometriosis (Taniguchi et al., Fertil Steril 2008, 89:478- 480), cholesteatoma (Yamamoto-Fukuda et al., Eur Arch Otorhinolaryngol (2008) 265211737] 178; d'Alessandro et al., Otol Neurotol. 2010 Sep;3l(7):ll63-9), cholesteatomatous chronic otitis media (Yamamoto-Fukuda et al., Otol Neurotol. 2010 Jul;3](5):745-5] ), atherosclerosis (Che et al Am J l Heart Circ Physiol 300: H1547 Hl61, 2011) and cancer (see below).
Several studies are hed emphasizing a strong association of FGFR2 expression and poor outcome of cancer patients: Overexpression of FGFR2 and/or KGF is associated with expansive growth of gastric cancer and, shorter survival ofpatients (Matsunobu et al., Int J Cancer 2006, 28:307- 314; Toyokawa et al., Oncol Reports 2009, 212875-880). Overexpression ofFGFR2 was thereby detected in 3] -36.5% of all c cancer samples tested (Matsunobu et al., Int J Cancer 2006, 28:307—314; Toyokawa et al., Oncol Reports 2009, 2] 2875-880). arcinoma (70% of all gastric cancer) are further divided into two ct pathological types, namely the intestinal- and the diffuse-type gastric . Interestingly, the first, less aggressive type is ated with an ted ErbB2 oncogenic pathway, while the latter, more aggressive phenotype harbors aberrations in the FGFR2/PI3K pathway (Yamashita et al., Surg Today 2011, 41:24-38). Approximately 60% of gastric adenocarcinoma belong to the diffiise-type, the ing 40% to the intestinal type (Werner et al., J Cancer Res Clin Oncol 2001, 1272207-216). FGFR2 overexpression was found, in 53% of diffuse-type gastric cancer samples (Yamashita et al., Surg Today 2011, 3 8). Taking all data together, HER2 and FGFR2 expression seem to occur in two _ 4 _ distinct patient populations. Possibly, expression of FGFR2 partly results from gene amplification as in approximately 7-10% of primary c cancers amplification of FGFR2 can be found (Kunii et al. Cancer Res 2008, 68:232348). Furthermore, FGFR2 expression was not only found in metastases, but was even er than in primary tumors (Yamashita et al., Surg Today 2011, 41:24-38).
In breast cancer, FGFR2 IIIb expression was found in 57% of tumor samples but hardly in y tissue (Tamaru et al. 2004, 84:1460-1471). KGF (FGF7) was found in 45% of samples, generally coincided with FGFR2 IIIb. Co-Expression of FGF7 and its only receptor FGFR2 IIIb was associated with a cantly reduced number of apoptotic cells within the primary tumor as compared to primary breast cancers neither expressing FGF7 nor FGFR2 IIIb u et al. 2004, 84:1460-1471). As in gastric cancer, also in breast cancer gene amplification was found: in 4% of triple negative breast cancer (TNBC) (Turner et al. Oncogene 2010, 29:2013—2023). In breast cancer several small nuclear polymorphisms (SNPs) were identified, which are associated with increased breast cancer risk (Hunter et al. Nature Genetics 2007, 62870-874). If SNPs are localized, within intron 2, it results in transcriptional up-regulation of FGFR2 (Katoh Expert s 2010, 1021375- 1379). Interestingly, FGFRI is preferentially upregulated in ER-positive, while FGFR2 in ER-negative breast cancers (Katoh, Expert Reviews 2010, 10:1375-1379) In pancreatic cancer, overexpression of FGFR2 IIIb and/or FGF7 is strongly correlated with venous on (Cho et al., Am J Pathol 170:1964-1974), y co— expression of FGFR2 and FGF7 was found in tumor cells, but even more abundant in the l cells adjacent to tumor cells (Ishiwata et al., Am J Pathol 1998, 153:2]3-222).
In epithelial ovarian cancer in 80% of tested cases up-regulation of FGFR2 as compared to normal tissue and in 70% FGF7 in the ascetic fluid was found (Steele et al., Oncogene 205878-5887).
FGFR2 protein was found in all tested ve al cancers with strong expression at the invasive front of tumors (Kawase et al., Int J Oncol 2010, 36:331-340).
In lung adenocarcinoma, co-expression of FGF7 and FGFR2 was found in 51.6% of tested cases and correlates with lower differentiation grades, higher proliferation rate, _ 5 _ lymph node metastasis and shorter 5-year survival (Yamayoshi et al., J Pathol 2004, 204:110-118).
In trial cancer, most frequently activating mutations of FGFR2 are found in approximately 16% of endometrial cancer ck et al., Oncogene 2007, 26:7158-7162).
In geal carcinoma (EC), co—expression of FGF7 and FGFR2 in cancer cells was found in 26% of patients associated with a trend for shorter survival (Yoshino et al., Int J Oncol 2007, 31:721-728).
In cellular carcinoma, FGFR2 expression was up-regulated by 4.7 times in poorly differentiated tumors. This expression is associated with incidence of portal vein invasion and lower disease free survival times (Harimoto et al., Oncology 2010, 78:361- 368) Several ations with experimental in vitro and in ViVO data demonstrate a causal relationship of aberrant FGFR2-signaling and tumor progression: Knock-down and/or inhibition of FGFR2 in gastric (Takeda et al., Clin Cancer Res 2007; 13:3051-3057; Kunii et al., Cancer Res 2008; 68:2340-2348), breast (Turner et al.
Oncogene 2010, 29:2013-2023), n (Cole et al., Cancer Biol Ther 2010, 10:495—504) and head and neck squamous cell (Marshall et al., Clin Cancer Res 2011, 17:5016-5025) carcinoma cells resulted in reduced proliferation and/or increased apoptosis of tumor cells.
Also in tumor xenografts, knock-down of FGFR2 as well as inhibition of FGFR2 in tumor cell lines over-expressing FGFR2, growth inhibition was shown for gastric (Takeda et al., Clin Cancer Res 2007; 1-3057) and ovarian (Cole et al., Cancer Biol Ther 2010, :495-504) cancer cell lines. Additionally, FGF7, which solely activates FGFR2, increases proliferation of gastric (Shin et al., J Cancer Res Clin Oncol 2002, 12825967602), breast (Zhang et al., Anticancer Res 1998, 18:2541-2546) and ovarian (Cole et al., Cancer Biol Ther 2010, 10:495—504) cancer cell lines in vitro and in vivo. Furthermore, knock—down of FGFR2 in endometrial cancer cell lines harboring FGFR2 with activating mutations also resulted in cell cycle arrest and induction of cell death (Byron et al., Cancer Res 2008, 68:6902-6907). _ 6 _ FGFR2 signaling promotes migration and invasion of gastric (Shin et al., J Cancer Res Clin Oncol 2002, 12825967602), breast (Zhang et al., Anticancer Res 1998, 1822541— 2546) and pancreatic cancer cell lines in vitro (Nomura et al., Br J Cancer 2008, 99:305— 313; Niu et al., J Biol Chem 2007, 282:6601-6011).
In esophageal carcinoma, FGFR2 is the highest up-regulated gene in tumor— associated fibroblasts. ed tumor-associated fibroblasts released, a soluble factor that promotes eration of geal cancer cells (Zhang et al., hum Cancer Biol 2009, :4017-4022), demonstrating that also FGFR2 expressed by stromal cells can promote tumor progression.
Only a limited number of anti FGFR2 antibodies have been ed. Fortin et al.
(J. Neurosci. 2005, 25: 479) describe a blocking anti FGFR2 antibody. Wei et al.
(Hybridoma 2006, 25: 115-124) showed antibodies specific only for FGFR2 IIIb that inhibits KGF induced cell proliferation. In W02007/144893 inhibitory antibodies that bind FGFR2 and FGFR3 are disclosed. In WO2010/054265 and Zhao et al. (Clin Cancer Res. 2010,1625750-5758) antibodies inhibiting FGF binding are disclosed. Bai et al. (Cancer Res. 2010, 70:7630-7639) describe antibodies specific for FGFR2 IIIb. R&D Systems markets anti-FGFR2 antibodies that neutralize activity in their assays.
In y, several FGFR2 splice variants are known. Furthermore, it is known that FGFR2-related diseases are due to aberrant expression, e.g. overexpression or amplification of FGFR2, or due to various mutated FGFR2 proteins. However, a therapy is lacking which addresses a plurality of different FGFR2 d diseases.
SUMMARY OF THE ION The present invention is directed to the ion of dies, or antigen-binding antibody fragments thereof, or variants thereof which reduce the cell surface expression of FGFR2 after binding to FGFR2 in both cells overexpressing FGFR2 and cells expressing 2012/073325 _ 7 _ mutated FGFRZ. Also ed are antibody-based therapies for FGFRZ—related diseases or conditions such as cancer, in particular for FGFR2 expressing tumors, such as gastric , breast cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, prostate cancer, ovarian , cervical cancer, lung , non-small-cell lung cancer (NSCLC), endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma and bladder cancer.
The invention is also related to polynucleotides encoding the antibodies of the invention, or antigen—binding fragments thereof, cells expressing the antibodies of the ion, or antigen-binding fragments thereof, methods for producing the antibodies of the invention, or n-binding fragments thereof, methods for inhibiting the growth of dysplastic cells using the dies of the invention, or antigen-binding fragments thereof, and methods for treating and detecting cancer using the antibodies of the invention, or antigen-binding fragments thereof.
The invention describes antibodies that are distinguished from existing FGFR2 antibodies in that they reduce the e expression of FGFR2 after binding to FGFRZ in cells overexpressing FGFR2 as well as in cells expressing mutated FGFRZ. An embodiment of the invention is an antibody or antigen-binding fragment f that binds to the extracellular N—terminal epitope (IRPSFSLVEDTTLEPEIS) of FGFR2 (SEQ ID N0263).
The antibodies or antigen—binding fragment thereof of the invention a) activate FGFR2 on the short term, b) induce internalization of FGFR2 c) resulting in efficient degradation, d) de-sensibilization of the FGFRZ-expressing cancer cells or tumor cells and e) y resulting in an anti-tumor activity of these antibodies in in vivo tumor experiments. These and other objects of the invention are more fully described herein.
An antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be modified. For e, an antibody could be conjugated to a cytotoxic agent, immunotoxin, ore or radioisotope to potentially r increase efficacy.
The invention further provides antibodies which constitute a tool for diagnosis of ant or dysplastic conditions in which FGFR2 expression is elevated compared to normal tissue or where FGFR2 is shed from the cell surface and becoming detectable in serum. Provided are anti-FGFR2 antibodies conjugated to a detectable marker. Preferred markers are a radiolabel, an enzyme, a phore or a scer.
The invention is also related to polynucleotides ng the antibodies of the invention, or antigen-binding fragments thereof, cells expressing the dies of the invention, or antigen-binding fragments thereof, methods for producing the antibodies of the invention, or antigen-binding fragments thereof, methods for ting the growth of stic cells using the antibodies of the invention, or n-binding fragments thereof, and methods for treating and detecting cancer using the antibodies of the invention, or antigen-binding fragments thereof.
The invention also is related to isolated nucleic acid sequences, each of which can encode an aforementioned antibody or antigen-binding fragment thereof that is specific for an epitope of FGFR2.
Nucleic acids of the invention are suitable for recombinant production of antibodies or antigen-binding antibody fragments. Thus, the invention also relates to vectors and host cells containing a nucleic acid sequence of the invention.
Compositions of the invention may be used for therapeutic or prophylactic applications. The invention, therefore, includes a pharmaceutical composition comprising an inventive antibody or antigen-binding nt thereof and a ceutically acceptable r or excipient therefore. In a related aspect, the ion provides a method for treating a disorder or condition associated with the undesired presence of FGFR2 expressing cells. In a preferred embodiment the aforementioned disorder is cancer. Such method ns the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an inventive antibody as described or contemplated herein. (followed by 8A) The invention also includes the use of an antibody or n-binding fragment, an dydrug conjugate, a pharmaceutical composition or a combination as described herein in the manufacture of a medicament for treating a disorder or condition associated with the undesired presence of FGFR2.
The ion also provides for the use of an antibody or antigen-binding fragment, an antibody-drug conjugate, a pharmaceutical composition or a combination as described herein in the manufacture of a medicament for the treatment of cancer.
The invention also provides ctions for using an antibody library to isolate one or more members of such library that binds specifically to FGFR2. (followed by 9) PTION OF THE S Figure 1: Schematic diagram of the structure of FGFRZ. Alpha (SEQ ID NO:61) and beta (SEQ ID NO:62) splice variants are shown in comparison. The diagram shows the three Ig— like domains (D1, D2 and D3), the transmembrane domain (TM), and the intracellular kinase domain. The heparin binding site (HBS), acidic box (AB), and the alternative IIIb/IIIc partial domains are indicated. The amino terminus is marked by an N, the carboxy terminus by an C. The binding epitope of the antibodies of this invention is depicted striped.
Figure 2: Induction of phosphorylated FGFRZ (P-FGFRZ) levels after short term (15 min) incubation with anti FGFR2 antibodies at 10ug/ml in MFM223 cells. Y is “% of untreated control cells”. As shown antibodies M048-D01-hlgG] and M047-D08-hIgG] increase the ELISA signal of P-FGFRZ by a factor greater 4 fold compared with untreated l cells.
In st neither the l IgG antibody nor anti FGFRZ antibodies commercially available from R&D (MAB665, MAB684, MAB6843) showed any significant effect on P- FGFRZ levels after short—term tion. These results reveal an agonistic effect of anti FGFR2 antibodies described within this invention on FGFRZ after term incubation.
Figure 3: itizing of MFM223 cells against FGF7 (25ng/ml, 15min) ed induction of P-FGFRZ levels after long term (24h) incubation with anti FGFRZ antibodies at 10ug/ml. Y is “% of untreated control cells”. As shown the antibodies M048-D01-hIgGl and M047-D08-hIgG1 reduce the level of P-FGFRZ which can be achieved after FGF7 stimulation very pronounced. In cells treated without antibody treatment as well as in cells treated with e control IgG stimulation with FGF7 lead to an about 4fold increase of P- FGFRZ levels. In contrast, in samples pretreated with anti FGFRZ antibodies for 24h, FGF7 only induced P-FGFRZ levels by l.37-l.4 fold. _ 10 _ Taken together these results show that ged incubation of cells with anti FGFR2 antibodies of this invention leads to desensitization towards stimulation with FGF7.
Figure 4: Downregulation of FGFR2 surface expression in cell lines with FGFR2 overexpression (MFM223, SNU16) or FGFRZ ons (AN3-CA, MFE-296) 4.5 h after incubation with anti FGFRZ antibodies at 10ug/ml measured by FACS is. Y is “% of control cells”. As shown antibodies M048-D01-hlgG1 and, 08-hIgG1 are the only antibodies that reduce FGFR2 surface expression with FGFR2 overexpressing cell lines (MFM223, SNU16) and cells lines having FGFRZ mutations (AN3-CA, MFE-296).
Antibodies like MAB684 and MAB6843 (R&D) only reduce FGFR2 surface expression with cell lines which do not overexpress FGFRZ. Antibodies like GAL-FR21 do not reduce FGFRZ surface expression with cell lines having FGFR2 mutations.
Figure 5: Downregulation of total FGFRZ levels after long term (96h) incubation with anti FGFR2 antibodies in SNU16 cells. Y is “% of control cells”. X is “Antibody concentration [pg/mu”. As shown antibodies M048-D01-hIgGl (white) and M047-D08-hIgG] (striped) decrease the total FGFR2 levels significantly after 96h in a dose dependent manner. A non- g control antibody (black) does not show any effects. These results indicate that anti FGFRZ antibodies M048-D01—hIgGl and M047-D08—hIgGl do not only lead, to a short term decrease in e FGFR2 levels but also a long term reduction of total FGFRZ levels.
Figure 6: Microscopic tion of the time course of specific internalization of M048- DOl-hIgGl and M047-D08-hIgG1 upon binding to endogenous FGFR2 expressing cells. Y is “granule counts per cell”. X is “time [min]’.7 Internalization of antibodies was igated on breast cancer cell line SUM 52PE. The e counts per cell were measured in a kinetic fashion. As shown antibodies M048-D01—hIgG1 (black s and solid line) and M047-D08—hIgGl (black triangles and dashed line) show a rapid _ 11 _ internalization as indicated by increasing granule count per cell. An isotype control antibody (stars and dashed line) does not show any internalization.
Figure 7: Internalization of M048-D01-hIgGl (A, B) and M047-D08-hIgGl (C, D) in SUM 52PE cells showed co-staining as indicated with Rab 7 (A, C) and not with Rab 11 (B, D). Internalization of GAL-FR21 (E, F) and GAL-FR22 (G,H) in SUM 52PE cells showed co-staining as ted with Rab 11 (F, H) and not with Rab 7 (E, G).
Figure 8: Growth of subcutaneous SNU-16 xenografts under intraperitoneal treatment with 2 mg/kg of MOl7-B02-hIgG1 (open triangles, solid line) in ison to PBS (filled circles, solid line) and control IgG treatment (filled triangles, solid line). Mean + rd deviation are plotted. X is “time after tumor inoculation [days]“. Y is “tumor area [mm2]“. ent with M017-B02-hIgG1 resulted in a very significant tumor growth inhibition.
Figure 9: Growth of subcutaneous SNU-16 xenografts under intraperitoneal treatment with 2 mg/kg of M021-HO2-hIgGl (open triangles, solid line) in comparison to PBS (filled circles, solid line) and control IgG ent (filled triangles, solid line). Mean + standard deviation are plotted. X is “time afier tumor inoculation [days]“. Y is “tumor area [mm2]“.
Treatment with M021 -HO2-hIgGl resulted in a very significant tumor growth inhibition.
Figure 10: Growth of subcutaneous SNU—16 xenografts under intraperitoneal treatment with 2 mg/kg of M048-DOl-hIgG1 (open triangles, solid line) in comparison to PBS (filled s, solid line) and control IgG treatment (filled triangles, solid line). Mean + standard deviation are plotted. X is “time after tumor ation [days]“. Y is “tumor area [mm2]“.
Treatment with 0] -hIgG1 resulted in a very significant tumor growth tion.
Figure 11: Growth of subcutaneous SNU—16 xenografts under intraperitoneal treatment with 2 mg/kg of M054-A05-hIgGl (open triangles, solid line) in comparison to PBS (filled _ 12 _ circles, solid line) and control IgG treatment (filled les, solid line). Mean + standard deviation are plotted. X is “time after tumor inoculation [days]“. Y is “tumor area [mnfl‘i Treatment with M054-A05-hIgGl resulted in a very cant tumor growth inhibition.
Figure 12: Growth of subcutaneous SNU—l6 xenografts under intraperitoneal treatment with 2 mg/kg of M054-D03-hlgGl (open triangles, solid line) in comparison to PBS (filled circles, solid line). Mean + standard deviation are plotted. X is “time after tumor inoculation [days]“. Y is “tumor area [mm2]“. Treatment with M054-D03—hIgGl resulted in a very significant tumor growth inhibition.
Figure 13: Growth of subcutaneous SNU—16 xenografts under intraperitoneal treatment with 2 mg/kg of M047-D08-hlgGl (open les, solid line) in comparison to PBS (filled circles, solid line). Mean + standard deviation are plotted. X is “time after tumor inoculation [days]“. Y is “tumor area . ent with M047-D08—hlgGl resulted in a very significant tumor growth inhibition.
Figure 14: Dot plots of the tumor area of subcutaneous 4T1 tumors at day 13 after tumor cell inoculation, the last time point before tumors became necrotic. At this time point mice recived treatment with PBS alone (A), 5 mg/kg of M048-D01-hIgGl twice weekly i.v. (B), 100 mg/kg Lapatinib 13.0. (C) or with 5 mg/kg of M048-D01-hIgGl twice weekly iv and 100 mg/kg Lapatinib p.o. (D). Y is tumor area [mmz] at day 13, dotted lines indicate the mean values, solid lines indicate the medians. Treatment with M048-D01-hIgGl alone resulted in a significant reduction of tumor area, while nib alone did not significantly affect tumor area. Combination of M048-D01-hIgGl with Lap atinib resulted in a cantly additive anti—tumor activity.
Figure 15: Dot plots of the tumor area of subcutaneous 4T1 tumors at day 13 after tumor cell inoculation, the last time point before tumors became necrotic. At this time point mice 2012/073325 _ 13 _ recived treatment with PBS alone (A), 5 mg/kg of M048—D01—hIgGl twice weekly i.v. (B), 24 mg/kg Taxol once weekly i.v. (C) or with 5 mg/kg of M048—D01-hlgG1 twice weekly iv. and 24 mg/kg Taxol once weekly i.v. (D). Y is tumor area [mmz] at day 13, dotted lines indicate the mean values, solid lines indicate the medians. ent with M048—D01— hIgGl alone resulted in a significant reduction of tumor area, while Taxol alone did not significantly affect tumor area. Combination of M048-DOl-hIgG1 with Taxol resulted in a significantly additive anti-tumor activity.
Figure 16: Growth of subcutaneous patient-derived GC10-0608 xenografts under intraperitoneal treatment with 5 mg/kg (filled triangles, solid line), 2 mg/kg (filled circles, dashed line) and 1 mg/kg (filled squares, dotted line) of M048-D01-hlgGl in comparison to PBS (open diamonds, solid line). Mean i standard error of the means are plotted. X is “time under treatment [days]“. Y is “tumor volume [mm3]“. ent with all three doses of M048-D01—hIgGl resulted in a significant tumor growth inhibition.
Figure 17: Growth of subcutaneous patient-derived, 811 xenografts under intraperitoneal treatment with 5 mg/kg (filled triangles, solid line), 2 mg/kg (filled circles, dashed line) and 1 mg/kg (filled s, dotted line) of M048-D01-hlgGl in comparison to PBS (open diamonds, solid line). Mean i standard error of the means are plotted. X is “time under treatment [days]“. Y is “tumor volume [mm3]“. ent with doses of 5 and 1 mg/kg 01-hIgGl resulted in a significant tumor growth inhibition.
Figure 18: Downregulation of total FGFR2 [total FGFR2] and phosphorylated FGFR2 [P- FGFR2] afier long term ent of SNU16 xenografts with anti FGFR2 antibodies M048— DOl—hIgGl and M047-D08—hIgGl in comparison with a control antibody (Zing/kg, twice weekly, i.p., samples were taken 24h after the last dose). As shown after treatment with M048-D01-hIgGl and M047-D08—hIgGl total FGFR2 [total FGFR2] and phosphorylated _ 14 _ FGFRZ RZ] were reduced significantly in comparison with ent with control IgGl. Actin served as loading control.
Figure 19: Sequences of the invention DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery of novel antibodies that have a specific affinity for FGFR2 and can r a therapeutic benefit to a subject. The antibodies of the invention, which may be human, zed or chimeric, can be used in many ts, which are more fully described herein.
Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning ly tood by one of ordinary skill in the art to which this invention belongs. The following references, however, can provide one of skill in the art to which this invention pertains with a general definition ofmany of the terms used in this ion, and can be referenced and used so long as such definitions are consistent the g commonly understood in the art. Such references include, but are not limited to, Singleton et ah, Dictionary of Microbiology and lar Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 198 8); Hale & Marham, The Harper Collins Dictionary of Biology (199] ); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. Saunders Company. Any additional technical resources available to the person of ordinary skill in the art providing definitions of terms used herein having the meaning commonly understood in the art can be consulted. For the purposes of the present invention, the following terms are further defined. Additional terms are defined _ 15 _ elsewhere in the description. As used herein and in the appended , the singular forms ”a," "and," and "the" include plural reference unless the t clearly dictates otherwise.
Thus, for example, reference to "a gene" is a reference to one or more genes and es equivalents thereofknown to those skilled in the art, and so forth.
A “human” antibody or antigen-binding fragment thereof is hereby defined as one that is not chimeric (e.g., not “humanized”) and not from (either in whole or in part) a non- human species. A human antibody or antigen-binding fragment thereof can be d from a human or can be a synthetic human antibody. A “synthetic human dy” is defined herein as an antibody having a sequence derived, in whole or in part, in silico from synthetic sequences that are based on the analysis ofknown human antibody sequences. In silico design of a human antibody sequence or fragment thereof can be achieved, for example, by analyzing a database of human antibody or antibody fragment sequences and devising a ptide sequence utilizing the data obtained there from. Another example of a human antibody or antigen-binding fragment thereof is one that is d by a nucleic acid isolated from a y of antibody sequences of human origin (e.g.., such library being based on antibodies taken from a human natural source). Examples of human antibodies include antibodies as described in Soderlind et al., Nature Biotech. 2000, 18:853-856.
A “humanized antibody” or humanized antigen-binding fragment thereof is defined herein as one that is (i) derived from a non-human source (e.g., a transgenic mouse which bears a heterologous immune system), which antibody is based on a human ne ce; (ii) where amino acids of the framework regions of a non human antibody are partially exchanged to human amino acid sequences by genetic engineering or (iii) CDR- grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more orks of the variable domain are of human origin and the nt domain (if any) is of human origin.
A “chimeric antibody” or antigen-binding fragment thereof is defined herein as one, wherein the variable domains are derived from a non-human origin and, some or all constant domains are d from a human origin. _ 16 _ The term "monoclonal dy" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring ons, that may be present in minor amounts. Thus, the term lonal" indicates the character of the antibody as not being a e of discrete antibodies. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an n. In addition to their specificity, monoclonal dy preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The term "monoclonal” is not to be construed as to require production of the dy by any particular method. The term monoclonal antibody specifically includes chimeric, humanized and human antibodies.
As used herein, an antibody “binds specifically to”, is “specific ” or “specifically recognizes” an antigen of interest, e. g. a tumor-associated ptide antigen target (here, FGFRZ), is one that binds the antigen with sufficient affinity such that the dy is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins or does not significantly cross- react with proteins other than ogs and variants (e. g. mutant forms, splice variants, or proteolytically truncated forms) of the aforementioned antigen target. The term ”specifically recognizes" or "binds specifically to" or is ”specific to/for" a ular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by an antibody, or antigen-binding fragment thereof, having a monovalent KD for the antigen of less than about 10‘4 M, alternatively less than about 10'5 M, alternatively less than about '6 M, alternatively less than about 10'7 M, alternatively less than about 10'8 M, alternatively less than about 10'9 M, alternatively less than about 10'10 M, alternatively less than about 10'11 M, alternatively less than about 10'12 M, or less. An antibody “binds specifically to,” is fic to/for” or “specifically recognizes” an antigen if such antibody is able to minate between such antigen and one or more reference antigen(s). In its most general form, “specific binding”. “binds specifically to”, is “specific to/for” or WO 76186 _ 17 _ “specifically recognizes” is referring to the y of the dy to minate between the antigen of interest and an unrelated antigen, as determined, for example, in accordance with one of the following methods. Such methods se, but are not limited to Western blots, ELISA-, RIA—, ECL—, IRMA-tests and peptide scans. For example, a rd ELISA assay can be carried out. The scoring may be carried out by standard color development (e. g. secondary antibody with horseradish peroxidase and tetramethyl ine with hydrogen peroxide). The on 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 positive/negative is more than 5- fold, 10-fold, 50-fold, and preferably more than 100-fold. Typically, determination of binding specificity is med by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
"Binding affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule and its binding partner. Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g. an antibody and an antigen). The dissociation constant “K9” is commonly used to describe the affinity between a le (such as an antibody) and its binding partner (such as an n) i.e. how tightly a ligand binds to a particular protein. -protein affinities are influenced by non— covalent intermolecular interactions between the two molecules Affinity can be measured by common methods known in the art, including those described herein. In one embodiment, the "KB" or "KD value" according to this invention is measured by using surface plasmon resonance assays using a Biacore T100 instrument (GE Healthcare Biacore, Inc.) according to Example 7. In brief, antibodies were immobilized onto a CMS sensor chip through an indirect capturing reagent, anti-human IgG Fc. Reagents from the “Human Antibody Capture Kit” (BR39, GE Healthcare Biacore, Inc.) were used as bed by the manufacturer. Approximately 5000 resonance units (RU) monoclonal mouse uman IgG (Fc) antibody were immobilized per cell. Anti FGFRZ antibodies were ed to reach a capturing level of approximately 200 to 600 RU. Various _ 18 _ concentrations of human, murin, rat, dog and of other species derived FGFR2 es containing amino acids 1—15 were ed over immobilized anti-FGFRZ antibodies.
Sensograms were generated after in—line reference cell correction followed by buffer sample subtraction. The dissociation equilibrium constant (Kn) was calculated based on the ratio of association (km) and dissociation rated (keg) constants, obtained by fitting sensograms with a first order 1:1 g model using Biacore Evaluation Software. Other suitable devices are BIACORE(R)—2000, a BIACORE (R)—3000 (BIAcore, Inc., Piscataway, NJ), or ProteOn XPR36 instrument (Bio-Rad tories, Inc).
To determine critical residues for binding of the antibodies or antibody fragments epitope fine mapping can be performed, using for e Alanine-scanning of es.
Therefore, each amino acid of the binding epitope is replaced by an Alanine residue and the binding of representative antibodies of the invention is tested in an ELISA-based assay. y, a residue is regarded as critical for binding when the antibody loses more than 50% of its ELISA signal by changing this residue into an Alanine as described in example The term "antibody", as used herein, is intended to refer to immunglobulin molecules, ably comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains which are typically inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise e.g. three domains CHl, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain nt region is comprised of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity ining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically ed of three CDRs and up to four FRs. arranged from amino us to carboxy-terminus e.g. in the following order: FRI, CDR] , FR2, CDRZ, FR3, CDR3, FR4. _ 19 _ As used herein, the term "Complementarity Determining Regions (CDRs; e.g., CDRl, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for n binding. Each le domain typically has three CDR s identified as CDRl, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g. about residues 24-34 (L1), 50-56 (L2) and, 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50—65 (H2) and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. about residues 26—32 (L1), 50-52 (L2) and, 91-96 (L3) in the light chain variable domain and 26— 32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined, according to Kabat and a hypervariable loop.
Depending on the amino acid ce of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes ofintact antibodies: IgA, IgD, IgE, IgG, and IgM, and several ofthese maybe further divided into asses" pes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant s that correspond to the different classes of antibodies are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively. The subunit structures and three-dimensional configurations of different s of immunglobulins are well known. As used herein antibodies are conventionally known antibodies and functional fragments f.
A ional fragment” or “antigen-binding antibody fragment” of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region. An “antigen- binding region” of an antibody typically is found in one or more hyper variable region(s) of an antibody, e.g., the CDRl, -2, and/or ,3 regions; however, the variable “framework” regions can also play an important role in n binding, such as by providing a scaffold for the CDRs. Preferably, the en—binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 113 of VH; numbering according to WO 97/08320). A preferred class of immunoglobulins for use in the present invention is lgG.
“Functional fragments” or “antigen-binding antibody fragments” of the invention include Fab, Fab', 2, and Fv nts; diabodies; single domain antibodies (DAbs), linear antibodies; single-chain antibody molecules (scFv); and pecific, such as bi- and tri-specific, antibodies formed from antibody fragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in lar Biology), Oxford University Press; R.
Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer tory Manual), Springer Verlag). An antibody other than a "multi-specific" or "multi-functional" antibody is understood to have each of its binding sites identical. The 2 or Fab may be ered to minimize or completely remove the intermolecular disulphide interactions that occur between the CH1 and CL domains.
Variants of the antibodies or antigen-binding antibody fragments contemplated in the invention are molecules in which the binding activity of the antibody or antigen-binding antibody fragment for FGFR2 is maintained. g proteins contemplated in the invention are for example antibody cs, such as Affibodies, Adnectins, ,Anticalins, DARPins, Avimers, Nanobodies (reviewed by Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13:245-255; Nuttall S.D. et al., Curr.
Opinion in Pharmacology 2008; 82608—617).
As used herein, the term ‘epitope’ includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptors. Epitopic determinants y consist of chemically active surface ngs of molecules such as amino acids or sugar side chains, or ations thereof and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Two antibodies are said to ‘bind _ 21 _ the same epitope’ if one antibody is shown to compete with the second antibody in a competitive binding assay, by any of the methods well known to those of skill in the art.
An "isolated" antibody is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to greater than 95% by weight of dy as determined e.g. by the Lowry method, UV-Vis spectroscopy or by by pillary Gel electrophoresis (for example on a Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and in further preferred embodiments more than 99% by weight, (2) to a degree sufficient to obtain at least 15 es ofN-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under ng or nonreducing ions using Coomassie blue or, ably, silver stain. Isolated naturally occurring antibody es the antibody in situ within recombinant cells since at least one component ofthe antibody's l environment will not be present. Ordinarily, however, isolated antibody will be ed by at least one purification step. ody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted 1g bound onto Fc gamma receptors (FcyRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell e. g. with cytotoxins. To assess ADCC activity of an antibody of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 or US.
Patent No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells.
"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first ent of the complement system (Clq) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess ment activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods _ 22 _ 202: 163 (1996), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased Clq binding are described, e.g., in US Patent No. 6,194,551 Bl and W0 1999/51642.
The term immunoconjugate (interchangeably referred to as "antibody-drug conjugate," or ”ADC") refers to an dy ated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, a enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Immunoconjugates have been used for the local ry of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (e.g. Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623)). Immunoconjugates allow for the targeted delivery Ofa drug moiety to a tumor, and intracellular accumulation n, where systemic administration of ugated drugs may result in unacceptable levels oftoxicity to normal cells and/or s. Toxins used, in antibody-toxin conjugates e bacterial toxins such as eria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin. The toxins may exert their cytotoxic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.
"Percent (%) sequence identity" with respect to a reference cleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after ng the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not ered as part of the sequence identity. Preferred are un-gapped alignments. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using ly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
Those skilled in the art can ine appropriate parameters for aligning sequences, _ 23 _ including any algorithms needed to achieve maximal alignment over the full length of the sequences being ed.
The term ‘maturated antibodies’ or ‘maturated antigen-binding fragments’ such as maturated Fab ts includes derivatives of an antibody or antibody fragment exhibiting stronger binding - i. e. binding with increased affinity - to a given antigen such as the extracellular domain of the FGFRZ. tion is the process of identifying a small number of mutations within the six CDRs of an antibody or antibody fragment leading to this affinity increase. The tion process is the combination of molecular y methods for introduction of mutations into the dy and screening for identifying the improved binders.
Antibodies of the invention The present invention relates to methods to inhibit growth of FGFRZ-positive cancer cells and the progression of neoplastic disease by providing anti-FGFRZ antibodies.
Provided are binding proteins, antibodies, antigen-binding dy fragments thereof, and variants of the antibodies and fragments that reduce the surface expression of FGFRZ after binding to FGFR2 in both, a cell overexpressing FGFR2 and a cell expressing mutated FGFRZ. It is another embodiment of the ion to provide antibodies, or antigen-binding antibody fragments thereof, or variants thereof, which bind to a broad range of different FGFRZ expressing cell lines both in cells overexpressing FGFR2 as well as in cells expressing mutated FGFRZ including, but not limited to SNU16 CRL—5974) and MFM223 (ECACC-98050130) which overexpress FGFR2 and AN3—CA ACC 267) and 6 (ECACC-9803l 10]) which express mutated FGFRZ.
Toward, these ends, it is an embodiment of the invention to e isolated human, humanized or chimeric antibodies, or antigen binding antibody fragments thereof, that specifically bind to a FGFR2 epitope which is present in different forms of the mature human FGFRZ polypeptide (for example see SEQ ID NO:61 for FGFR2 alpha IIlb, and SEQ ID NO:62 for FGFR2 beta HIb), which is presented by FGFRZ expressing cancer cell cancer cells, and/or which is bound by these antibodies with high affinities. As used _ 24 _ herein, different ‘forms’ of FGFR2 include, but are not cted to, different isoforms, ent splice variants, different glycoforms or FGFR2 polypeptides which undergo different translational and posttranslational modifications. The FGFR2 polypeptide is named ‘FGFRZ’ herein.
It is another embodiment of the invention to provide antibodies, or antigen-binding antibody nts thereof, or variants thereof that are safe for human administration.
It is another embodiment of the invention to provide antibodies, or antigen-binding antibody fragments thereof, or variants thereof, which bind to human FGFR2 and are cross— reactive to FGFR2 of another species including, but not limited to , rat, macaca mulatta, rabbit, pig and dog FGFRZ. ably, said other species is a rodent, such as for example mouse or rat. Most preferably, the antibodies, or antigen-binding antibody fragments thereof, or variants thereof bind to human FGFRZ and are cross-reactive to murine FGFRZ.
It is another embodiment of the invention to provide antibodies, or antigen-binding antibody nts f, or ts thereof, which are internalized efficiently ing binding to a FGFR2 expressing cell. An antibody of the invention might be co-administered with known medicaments, and in some instances the antibody might itself be d. For e, an antibody could be conjugated to a cytotoxic agent, immunotoxin, toxophore or radioisotope to potentially r increase efficacy.
It is another embodiment of the invention to provide antibodies, or antigen-binding antibody fragments thereof, or variants thereof, which activate FGFR2 on the short term and after internalization lead to FGFR2 degradation thus resulting in a desensitization of ent FGFR2—expressing cancer cells or tumor cells for FGF stimulus and finally inhibit tumor growth in vivo.
It is another embodiment of the invention to provide antibodies which constitute a tool for diagnosis of malignant or dysplastic conditions in which FGFR2 expression is elevated compared to normal tissue or where FGFR2 is shed from the cell surface and becoming able in serum. Provided are anti-FGFR2 antibodies conjugated to a _ 25 _ able marker. Preferred markers are a radiolabel, an , a chromophore or a fluorescer.
In one aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that contains an antigen-binding region that binds to cell surface expressed FGFR2 and reduce after g to FGFR2 the cell surface expression of FGFR2 in both a cell overexpressing FGFRZ and a cell expressing mutated FGFRZ. In one embodiment, the invention provides an isolated antibody or n-binding fragment thereof that contains an n-binding region that specifically binds to native, cell surface expressed FGFR2 and reduces after binding to FGFRZ the cell surface expression of FGFR2 in both a cell overexpressing FGFR2 and a cell expressing mutated FGFRZ. In one embodiment, the isolated antibody or antigen-binding fragment that binds specifically to native, cell surface expressed FGFR2 and reduces after binding to FGFR2 the cell e expression of FGFR2 in both at least two different cells overexpressing FGFR2 and at least two different cells expressing mutated FGFRZ.
In a further embodiment the antibody or antigen-binding fragment thereof specifically binds to native, cell surface sed FGFR2 and (i) reduces after binding to FGFR2 the cell e expression of FGFR2 in both, a cell overexpressing FGFR2 and a cell expressing mutated FGFR2 and (ii) induces FGFR2 phosphorylation, In a further embodiment the antibody or antigen-binding fragment f specifically binds to native, cell surface expressed FGFR2 and (i) reduces after binding to FGFRZ the cell surface expression of FGFRZ in both, a cell overexpressing FGFRZ and a cell expressing mutated FGFRZ and (ii) induces FGFRZ phosphorylation, wherein the antibody desensitizes a FGFRZ expressing cell for stimulation with FGF7. In a further embodiment the desensitization is the desensitization of a FGFR2 overexpressing cell.
In a further embodiment the antibody or n-binding nt thereof cally binds to native, cell surface sed FGFRZ and, (i) reduces after binding to FGFR2 the cell surface expression of FGFRZ in both, a cell overexpressing FGFR2 and a cell expressing mutated FGFRZ and (ii) induces internalization of FGFR2 resulting in FGFR2 degradation.
In a further embodiment the antibody or antigen-binding fragment thereof specifically binds to native, cell surface expressed FGFR2 and (i) reduces after binding to FGFR2 the cell surface expression of FGFR2 in both a cell overexpressing FGFR2 and a cell expressing mutated FGFR2 and (ii) reduces tumor-growth in xenograft tumor experiments.
In a further embodiment the antibody or antigen-binding fragment f is capable to reduce the FGFR2 cell surface expression in different cell lines including, but not limited to SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130) which overexpress FGFR2 and in cell lines AN3-CA ACC 267) and MFE-296 (ECACC— 98031 101) which express mutated FGFRZ.
In a r embodiment the antibody or antigen-binding fragment thereof is capable to reduce after binding to FGFR2 the FGFR2 cell e expression in SNU16 (ATCC-CRL-5974) and MFM223 (ECACC-98050130) cells which overexpress FGFR2 and in the cell lines AN3-CA (DSMZ-ACC 267) and MFE-296 (ECACC-98031101) which express mutated FGFR2.
In a preferred embodiment the cell surface reduction is at least 10%, 15%, 20%, % or 30% compared to the FGFR2 cell surface expression of the non-treated or the control treated cell.
In a further preferred embodiment the cell surface ion after 96 hours is at least 10%, 15%, 20%, 25% or 30% compared to the FGFR2 cell surface sion of the eated or the control treated cell.
In a further ment the antibody or antigen-binding fragment thereof binds ically to the ellular N—terminal epitope (IRPSFSLVEDTTLEPEIS) of FGFRZ (SEQ ID N0263). Critical residues for binding of the antibody or antigen-binding fragment f within the N-terminal epitope (IRPSFSLVEDTTLEPE‘S) of FGFR2 include, but are not limited to, Arg 1, Pro 2 Phe 4, Ser 5, Leu 6 and Glu 8. 2012/073325 _ 27 _ In a further embodiment the binding of the antibody or n—binding nt thereof of the invention to the extracellular N—terminal epitope (SEQ ID N0263) is mediated by at least one e residue selected from the group of residues consisting of Arg 1, Pro 2, Phe 4, Ser 5, Leu 6, and Glu 8.
In a further embodiment the binding of the antibody or antigen-binding fragment thereof of the invention to the extracellular N—terminal epitope (SEQ ID NO:63) is reduced by substitution of at least one e residue selected from the group of residues consisting of Arg 1, Pro 2, Phe 4, Ser 5, Leu 6, and Glu 8 by the amino acid Alanine.
In a further embodiment the binding of the antibody or antigen-binding fragment thereof of the invention to the extracellular N-terminal epitope (SEQ ID N0263) is mediated by at least one epitope residue selected from the group of residues consisting of Pro 2, Leu 6 and Glu 8.
In a r embodiment the binding of the antibody or antigen-binding fragment f of the invention to the extracellular N—terminal epitope (SEQ ID N0263) is reduced by substitution of at least one e residue selected from the group of residues consisting of Pro 2, Leu 6 and Glu 8 by the amino acid Alanine.
In another embodiment the binding of the antibody or antigen-binding fragment thereof of the invention to the extracellular N—terminal epitope (SEQ ID N0263) is mediated by at least one epitope residue selected from the group of residues consisting of Pro 2, Leu 6 and Glu 8 and the binding to the epitope is invariant to sequence alterations of position 5 of the e.
In a further embodiment the binding of the antibody or antigen-binding fragment thereof of the invention to the extracellular N—terminal epitope (SEQ ID N0263) is reduced by substitution of at least one epitope residue selected from the group of residues consisting of Pro 2, Leu 6 and Glu 8 by the amino acid e and the binding to the epitope is invariant to sequence tions of position 5 of the epitope.
In a further embodiment the antibody or n-binding nt thereof loses more than 50% of its ELISA signal by changing of at least one of the amino acid residues —28— in the N—terminal epitope (IRPSFSLVEDTTLEPEIS) of FGFR2 into an Alanine, (i) said residue selected from the group Pro 2, Leu, 6 and Glu 8, or (ii) said residue selected from the group Arg 1, Pro 2, Phe 4 and Ser 5.
In a further red embodiment, the isolated antibodies or antigen-binding fragments thereof lose more than 50% of their ELISA signal by changing of at least one of the amino acid residues within the N—terminal e (IRPSFSLVEDTTLEPEIS) of FGFR2 into an Alanine wherein said residue is selected from the groups including, but not limited to a) Pro 2, Leu 6 and Glu 8 or b) Arg 1, Pro 2, Phe 4 and Ser 5, as depicted in Table 7.
In a further embodiment the antibodies or antigen-binding fragments compete in binding to FGFR2 with at least one antibody selected from the group “M048-D01”, “M047- D08”, “M017-B02”, “M021-H02”, “M054-A05”, “M054-D03”, “TPP-1397”, “TPP-1398”, 399”, “TPP-1400”, “TPP-1401”, “TPP-1402”, “TPP-1403”, “TPP-1406”, “TPP- 1407”, “TPP-1408”, “TPP-1409”, “TPP-1410”, “TPP-1411”, 412”, and “TPP-1415” Throughout this nt, reference is made to the following preferred antibodies of the invention as depicted in Table 9 and Table 10: “M017—B02”, “M021-H02”, “M047- D08”, “M048-D01”, “M054-A05”, “M054-D03”, “TPP-1397”, “TPP-1398”, “TPP-1399”, “TPP-1400”, “TPP-1401”, “TPP-1402”, “TPP-1403”, 406”, 407”, “TPP- 1408”, “TPP-1409”, “TPP-1410”, “TPP-1411”, “TPP-1412”, and 415”.
M017-802 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 3 SEQ ID NO: 1 (protein) and a variable light chain region corresponding to SEQ ID NO: 4 (DNA)/SEQ ID NO: 2 (protein).
M021-HO2 ents an antibody sing a le heavy chain region corresponding to SEQ ID NO: 13 (DNA)/SEQ ID NO: 11 (protein) and a variable light chain region corresponding to SEQ ID NO: 14 (DNA)/SEQ ID NO: 12 (protein).
M047-D08 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 23 (DNA)/SEQ ID NO: 21 (protein) and a variable light chain region corresponding to SEQ ID NO: 24 (DNA)/SEQ ID NO: 22 (protein). _ 29 _ M048-D01 represents an antibody sing a variable heavy chain region corresponding to SEQ ID NO: 33 (DNA)/SEQ ID NO: 31 (protein) and a le light chain region corresponding to SEQ ID NO: 34 (DNA)/SEQ ID NO: 32 (protein).
M054-D03 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 43 (DNA)/SEQ ID NO: 41 (protein) and a variable light chain region corresponding to SEQ ID NO: 44 (DNA)/SEQ ID NO: 42 (protein).
M054-A05 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 53 (DNA)./SEQ ID NO: 51 (protein) and a variable light chain region corresponding to SEQ ID NO: 54 (DNA)/SEQ ID NO: 52 (protein).
TPP-1397 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 83 (protein) and a variable light chain region corresponding to SEQ ID NO: 84 (protein).
TPP-1398 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 93 in) and a variable light chain region corresponding to SEQ ID NO: 94 (protein).
TPP-1399 represents an antibody comprising a le heavy chain region ponding to SEQ ID NO: 103 (protein) and a variable light chain region corresponding to SEQ ID NO: 104 (protein).
TPP-1400 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 113 (protein) and, a variable light chain region corresponding to SEQ ID NO: 1 14 in).
TPP-1401 ents an dy comprising a variable heavy chain region corresponding to SEQ ID NO: 123 (protein) and a variable light chain region corresponding to SEQ ID NO: 124 (protein).
TPP-1402 represents an dy comprising a variable heavy chain region corresponding to SEQ ID NO: 133 (protein) and a variable light chain region corresponding to SEQ ID NO: 134 (protein). _ 30 _ 03 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 73 (protein) and a le light chain region corresponding to SEQ ID NO: 74 (protein).
TPP-1406 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 153 (protein) and a variable light chain region corresponding to SEQ ID NO: 154 (protein).
TPP-1407 represents an antibody comprising a variable heavy chain region ponding to SEQ ID NO: 163 (protein) and a le light chain region corresponding to SEQ ID NO: 164 (protein).
TPP-1408 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 173 (protein) and a variable light chain region corresponding to SEQ ID NO: 174 in). 09 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 183 (protein) and a variable light chain region corresponding to SEQ ID NO: 184 (protein).
TPP-1410 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 193 (protein) and a variable light chain region corresponding to SEQ ID NO: 194 (protein).
TPP-141 1 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 203 (protein) and a variable light chain region corresponding to SEQ ID NO: 204 in).
TPP-1412 represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 213 (protein) and a variable light chain region corresponding to SEQ ID NO: 214 (protein). represents an antibody comprising a variable heavy chain region corresponding to SEQ ID NO: 143 (protein) and a le light chain region corresponding to SEQ ID NO: 144 (protein).In a further preferred embodiment the antibodies or n- binding fragments comprise heavy or light chain CDR sequences which are at least 50%, _ 31 _ 55%, 60% 70%, 80%, 90, or 95% identical to at least one, preferably corresponding, CDR sequence ofthe antibodies “M048-D01”, “M047-D08”, “M017-B02”, “M021-HOZ”, “M054-A05”, D03”, “TPP-1397”, “TPP-1398”, “TPP-1399”, “TPP-1400”, “TPP- 1401”, “TPP—1402”, “TPP-1403”, “TPP-1406”, “TPP-1407”, “TPP-1408”, “TPP-1409”, “TPP-1410”, “TPP-1411”, “TPP-1412” or “TPP-1415” or at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical to the VH or VL sequence of “M048—D01”, “M047-D08”, “M017-B02”, “M021-HO2”, “M054-A05”, “M054-D03”, “TIT-1397”, “TPP-1398”, “TPP- 1399”, 400”, “TPP-1401”, “TPP-1402”, “TPP-1403”, “TPP-1406”, “TPP-1407”, “TPP-1408”, 409”, “TPP-1410”, “TPP-1411”, “TPP-1412” or “TPP-1415”, respectively.
In a further preferred embodiment the antibody or antigen-binding fragment of the invention comprises at least one CDR sequence or at least one variable heavy chain or light chain sequence as depicted in Table 9 and Table 10.
In a more red embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N025 (H-CDRl), SEQ ID N026 (H—CDRZ) and SEQ ID N027 R3) and comprises a light chain antigen-binding region that comprises SEQ ID N028 (L-CDRl), SEQ ID N029 (L-CDRZ) and SEQ ID NO:10 (L-CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain n-binding region that ses SEQ ID N0215 (H-CDRl), SEQ ID N0216 (H-CDR2) and SEQ ID NO:17 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N0218 (L-CDRl), SEQ ID NO:19 Z) and SEQ ID N0220 (L-CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment f comprises a heavy chain n-binding region that comprises SEQ ID N0225 (H-CDRl), SEQ ID N0226 (H-CDRZ) and SEQ ID N0227 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N0228 (L-CDRl), SEQ ID N0229 Z) and SEQ ID N0230 (L-CDR3). _ 32 _ In a more preferred embodiment the antibody of the invention or antigen—binding fragment thereof comprises a heavy chain n-binding region that comprises SEQ ID N035 (H-CDRI), SEQ ID N036 (H-CDRZ) and SEQ ID N037 (H-CDR3) and ses a light chain antigen-binding region that comprises SEQ ID N038 (L-CDRI), SEQ ID N039 (L-CDRZ) and SEQ ID N0240 3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen—binding region that ses SEQ ID N0245 (H-CDRI), SEQ ID N0246 (II-CDRZ) and SEQ ID N0247 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N0248 (L-CDRI), SEQ ID NO:49 (L-CDRZ) and SEQ ID N0250 3).
In a more preferred embodiment the antibody of the invention or antigen—binding fragment thereof comprises a heavy chain n-binding region that comprises SEQ ID N0255 (II-CDRI), SEQ ID N0256 RZ) and SEQ ID N0257 (H-CDR3) and comprises a light chain n-binding region that comprises SEQ ID N025 8 (L-CDRI), SEQ ID NO:59 (L—CDR2) and SEQ ID NO:60 (L-CDRS).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N0275 (H-CDRI), SEQ ID N0276 RZ) and SEQ ID N0277 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N0278 (L-CDRI), SEQ ID N0279 (L-CDRZ) and, SEQ ID N0280 (L—CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N0285 (II-CDRI), SEQ ID N0286 (H-CDRZ) and SEQ ID N0287 (II-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N0288 (L-CDRI), SEQ ID N0289 (L-CDRZ) and SEQ ID N0290 (L-CDR3).
In a more red embodiment the antibody of the invention or antigen—binding fragment thereof ses a heavy chain antigen-binding region that comprises SEQ ID N0295 (H-CDRI), SEQ ID N0296 (H—CDRZ) and SEQ ID N0297 (II-CDR3) and _ 33 _ comprises a light chain antigen—binding region that comprises SEQ ID N0298 (L—CDRl), SEQ ID NOz99 (L-CDR2) and SEQ ID NO:100 (L-CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID NO:105 (H—CDRI), SEQ ID N02106 (H-CDRZ) and SEQ ID NO:107 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:]08 (L-CDRl), SEQ ID No;109 (L-CDRZ) and SEQ ID NO:110 (L-CDR3).
In a more red embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02115 (H-CDRl), SEQ ID NO:1]6 (H-CDRZ) and SEQ ID NO:117 (H-CDR3) and comprises a light chain antigen-binding region that ses SEQ ID N02] 18 (L-CDR] ), SEQ ID N02] 19 (L-CDRZ) and SEQ ID NO: 120 (L-CDR3).
In a more preferred ment the antibody of the invention or antigen—binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02125 (H-CDRl), SEQ ID NO:126 (H-CDRZ) and SEQ ID N02127 (H—CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N02128 (L-CDRI), SEQ ID N02129 (L-CDRZ) and SEQ ID NO: 130 (L-CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02135 (H—CDRI), SEQ ID N02136 (H-CDRZ) and SEQ ID N02137 (H-CDR3) and comprises a light chain n-binding region that comprises SEQ ID N02138 (L-CDRl), SEQ ID No;139 (L-CDRZ) and SEQ ID NO: 140 3).
In a more preferred embodiment the dy of the invention or antigen-binding nt thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02145 (H-CDRl), SEQ ID NO:146 Z) and SEQ ID NO:147 (H—CDR3) and comprises a light chain antigen—binding region that ses SEQ ID N02148 (L-CDRl), SEQ ID N02149 (L-CDR2) and SEQ ID NO: 150 (L-CDR3). _ 34 _ In a more preferred embodiment the antibody of the invention or antigen—binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID NO:155 (H-CDRl), SEQ ID NO:156 (H-CDRZ) and SEQ ID NO:]57 (H—CDR3) and comprises a light chain antigen—binding region that comprises SEQ ID NO:158 (L-CDRI), SEQ ID NQ:159 (L-CDRZ) and SEQ ID NO: 160 (L-CDR3).
In a more preferred embodiment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID NO:165 (H-CDRl), SEQ ID N02166 (H-CDRZ) and SEQ ID NO:167 (H-CDR3) and ses a light chain antigen-binding region that comprises SEQ ID NO:168 l), SEQ ID N02169 (L-CDRZ) and SEQ ID NO:170 (L-CDR3).
In a more preferred embodiment the antibody of the invention or n—binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID NO:175 (II-CDRI), SEQ ID N02176 (H-CDR2) and SEQ ID NO:177 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID NO:]78 (L-CDRI), SEQ ID NO:179 (L-CDRZ) and SEQ ID N02180 (L-CDR3).
In a more preferred embodiment the antibody of the ion or antigen-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID NO:]85 (H-CDRI), SEQ ID N02186 (H-CDRZ) and SEQ ID NO:187 (H-CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N02] 88 (L-CDRl), SEQ ID N02189 2) and SEQ ID NO:190 (L-CDR3).
In a more preferred ment the antibody of the invention or n-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02195 (H-CDRI), SEQ ID N02196 (H-CDR2) and SEQ ID NO:197 (II-CDR3) and ses a light chain antigen-binding region that comprises SEQ ID N02198 (L-CDRl), SEQ ID N02199 (L-CDRZ) and SEQ ID NQ;200 (L-CDR3).
In a more preferred, ment the antibody of the invention or antigen-binding fragment thereof comprises a heavy chain n-binding region that comprises SEQ ID NO:205 (II-CDRI), SEQ ID N02206 (H-CDR2) and SEQ ID N02207 (H-CDR3) and, _ 35 _ comprises a light chain antigen—binding region that ses SEQ ID N02208 (L—CDRl), SEQ ID NO:209 (L-CDRZ) and SEQ ID No;210 (L-CDR3).
In a more preferred embodiment the dy of the invention or n-binding fragment thereof comprises a heavy chain antigen-binding region that comprises SEQ ID N02215 (H-CDRl), SEQ ID NO:2]6 (H-CDRZ) and SEQ ID NO:217 (H—CDR3) and comprises a light chain antigen-binding region that comprises SEQ ID N022] 8 (L-CDRl), SEQ ID NOz2l9 (L-CDRZ) and SEQ ID NO:220 (L—CDR3).
An antibody ofthe invention may be an IgG (e.g., IgGl IgGZ, IgG3, IgG4), while an antibody fragment may be a Fab, Fab’, F(ab’)2 or scFv, for example. An inventive dy fragment, accordingly, may be, or may contain, an antigen-binding region that behaves in one or more ways as described herein.
For example the antibody Fab fragment M048-D01 (SEQ ID NO:31 for VH chain, and SEQ ID NOz32 for VL chain) was expressed as human IgG1 M048-DOl-hIgGl (SEQ ID NO:67 for heavy chain, and SEQ ID N0268 for light chain) and Fab nt M047- D08 (SEQ ID N0221 for VH chain, and SEQ ID NO:22 for VL chain) was expressed as human IgG1 M047-D08-hIgGl (SEQ ID NO:69 for heavy chain, and SEQ ID NO:70 for light chain). For efficient cloning the first 3 amino acids of the inus of the heavy chains [EVQ] (SEQ ID NO:67 and SEQ ID NO:69) can also atively be expressed as [QVE], for example as a variant of the heavy chain ofhuman IgG1 M048-D01-hIgGl (SEQ ID N02222). For efficient g the N—terminus of light chains can be extended by amino acid residues e. g. Alanin.
In a preferred embodiment the antibodies or antigen-binding antibody fragments of the invention are monoclonal. In a further red embodiment the antibodies or antigen- binding antibody fragments of the invention are human, humanized or chimeric.
In another aspect, the invention provides antibodies or antigen-binding nts having an antigen-binding region that bind specifically to and/or has a high affinity for FGFRZ independent of alpha and beta isoforms as well as IIIb and IIIc splice forms (for example see SEQ ID NOz6l for FGFR2 alpha IIIb and SEQ ID N0262 for FGFR2 beta _ 36 _ lllb). An antibody or antigen—binding fragment is said to have a “high ty” for an antigen if the affinity measurement is less than 250 nM (monovalent affinity of the antibody or antigen-binding fragment). An inventive antibody or antigen-binding region preferably can bind to human FGFR2 with an affinity of less than 250 nM, preferably less than 150 nM, determined as monovalent affinity to human FGFRZ. For instance, the affinity of an dy of the invention against FGFRZ from different species may be around 100 nM (monovalent affinity of the antibody or antigen-binding fragment) as shown in Table 8 exemplarily for M048-D1 and M047-D08.
The IgGl format was used for the cell-based affinity determination by fluorescence- activated cell sorting (FAC S). Table 6 provides a summary of the binding th (ECso) of representative anti-FGFRZ-IgG antibodies on cancer cell lines of human (SNU16, MFM223), murine (4T1) and rat (RUCA) origin.
An IgGl is said to have a “high y” for an antigen if the affinity ement measured by FACS is less than 100 nM (apparent affinity of IgG). An inventive bivalent antibody or antigen-binding fragment preferably can bind to FGFR2 with an y of less than 100 nM, more preferably less than 50 nM, and still more preferably less than 10 nM.
Further preferred are bivalent dies that bind to FGFRZ with an affinity of less than 5 nM, and more preferably less than 1 nM ined as apparent affinity of an IgG to FGFR2. For instance, the apparent affinity of an antibody of the invention against FGFR2 may be about 89.5 nM or less than 0.] nM on different tumor cell lines of human, murine and rat origin as determined by FACS analysis as depicted in Table 6.
An antibody or antigen-binding fragment of the invention internalizes “efficiently” when its time of half maximal internalization (t 1/2) into FGFR2 expressing tumor cells is r than 180 min or more preferably shorter than 120 min and still more ably shorter than 90 min. r preferred are antibodies or antigen-binding fragments with half maximal internalization times (t 1/2) of 60 minutes or less as determined by the protocol described in example 12. _ 37 _ Co—staining of small G—proteins can be used for a more detailed evaluation of the king pathway of antibodies after internalization. For instance Rab GTPases which regulate many steps of membrane traffic, including vesicle formation, vesicle movement along actin and tubulin ks, and membrane fusion can be used to distinguish between different pathways. Thereby, co-staining of d dies with Rab7, which is expressed in late mes and mes, indicates that after internalization of FGFRZ the complex enters the mal , lysosomal pathway, whereas co-staining with Rabll, which is expressed in early and recycling endosomes, indicates that these antibodies internalize afier binding to FGFRZ and favor the recycling pathway. Entering the endosomal - lysosomal pathway enables the antibodies to induce degradation of FGFR2 after internalization which finally results in desensitization of this pathway. Figure 7 shows the co-staining ns of representative antibodies of the invention with Rab7 and Rabll as described in example 12. lnternalizable antibodies or antigen-binding fragments of the invention are suitable as ing moiety of an antibody-drug ate (ADC). An antibody or antigen-binding fragment is suitable in an in vitro or in vivo method to deliver a compound, preferably a cytotoxic agent, into a FGFR2 expressing cell.
In some ments, the antibody, antigen-binding fragment thereof, or derivative thereof or nucleic acid encoding the same is isolated. An isolated biological ent (such as a nucleic acid molecule or protein such as an antibody) is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, e.g., other chromosomal and extra- chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" e nucleic acids and proteins purified by rd purification methods as described for example in Sambrook et al., 1989 (Sambrook, 1., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA) and Robert K. Scopes et al. 1994 (Protein Purification, - Principles and Practice, Springer Science and Business Media LLC). The _ 38 _ term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
An antibody of the invention may be derived from a recombinant antibody library that is based on amino acid sequences that have been isolated from the antibodies of a large number of healthy volunteers. Using the n-CoDeR® logy the fully human CDRs are recombined into new antibody molecules. The unique recombination process allows the library to contain a wider variety of antibodies than could have been created lly by the human immune . dy Generation A fully human N-CoDeR antibody phage display library was used to isolate FGFRZ— specific, human monoclonal antibodies of the present ion by a combination of whole cell and protein panning and through the development of specific methods. These methods include the development of g procedures and screening assays capable of identifying antibodies that entially bind to FGFR2 displayed on the cell surface and that are cross-reactive to murine FGFR2 and FGFRZ from other species and have a g and functional activity which is independent of FGFR2 over-expression and common mutations of FGFR2 found in FGFRZ-related diseases such as, cancer.
Antibodies to the cell-surface FGFR2 were developed by a combination of three non- conventional approaches in phage-display technology (PDT). First, ions were performed with inant, soluble, human and murine FGFR2 Fc-fusion proteins of several splice variants (alpha, beta, IIIb and Illc) to select for a very broad splice variant reactivity. Second, in addition urface selections were performed with KATO 111 cells expressing FGFRZ on their cell-surface. Third, screening methods were developed which allowed for successive screening of the phage outputs obtained in panning on whole KATOHI cells and recombinant, soluble, human and murine FGFRl, FGFR2, FGFR3, and FGFR4 Fc fusion proteins of several splice variants (alpha, beta, HIb and IIIc) to select for FGFR2 specific binders (no binding to FGFRl, FGFR3, and FGFR4) with a very broad splice variant cross-reactivity.
WO 76186 _ 39 _ After identification of preferred Fab nts these were expressed as full length IgGs. For example the antibody Fab fragment M048-D01 (SEQ ID NO:31 for VH chain, and SEQ ID N032 for VL chain) was expressed as human IgG] M048-D01-hIgG1 (SEQ ID NO:67 for heavy chain, and SEQ ID NO:68 for light chain) and Fab nt M047- D08 (SEQ ID N0221 for VH chain, and SEQ ID N0222 for VL chain) was expressed as human IgG1 M047-D08-hIgG1 (SEQ ID N0269 for heavy chain, and SEQ ID NO:70 for light . For efficient g the first 3 amino acids of the N—terminus of the heavy chains [EVQ] (SEQ ID NO:67 and SEQ ID N0269) can also alternatively be expressed as [QVE], for example as a variant of the heavy chain ofhuman IgG1 M048-D01-hIgG] (SEQ ID N02222). For efficient cloning the N—terminus of light chains can be extended by amino acid residues eg. Alanin Theses constructs were for e transiently expressed in ian cells as described in Tom et al., Chapter 12 in Methods Express: Expression s edited by Micheal R. Dyson and Yves Durocher, Scion Publishing Ltd, 2007. uriefly, a CMV-Promoter based expression plasmid was transfected into HEK293-6E cells and incubated in Fernbach iFlasks or Wave-Bags. Expression was at 37°C for 5 to 6 days in F17 Medium (Invitrogen). 5 g/l Tryptone TNl (Organotechnie), 1% Ultra-Low IgG FCS (Invitrogen) and, 0.5 mM Valproic acid (Sigma) were supplemented 24 h post-transfection.
These antibodies were further characterized by their binding affinity in ELISA’s, and by BIAcore binding to soluble FGFRZ. FACS binding with cells from different species was performed to select for cell binding antibodies which have a high affinity on mouse, rat and human cancer cell lines.
The combination of these c methods allowed the isolation of the unique antibodies “M017-B02”, “M021-H02”, “M047-D08”, “M048-D01”, “M054-A05” and, “M054-D03”.
Further characterization revealed, that the ed antibodies bind to a unique e at the N—terminus of FGFRZ resulting in their special features. These unique antibodies were further characterized in in vitro phosphorylation assays, internalization assays, and in vivo tumor xenograft experiments. The selected antibodies show a strong and significant anti-tumor activity in tumor xenograft experiments with SNU16 cells.
Peptide Variants Antibodies or antigen—binding fragments of the invention are not limited to the specific peptide sequences provided herein. Rather, the invention also embodies variants of these polypeptides. With reference to the instant disclosure and conventionally available logies and references, the d worker will be able to prepare, test and utilize functional variants of the antibodies disclosed herein, while appreciating these variants having the y to bind to FGFR2 fall within the scope of the present invention.
A variant can include, for example, an antibody that has at least one altered complementary determining region (CDR) (hyper-variable) and/or framework (FR) (variable) /position, vis-a-vis a e sequence disclosed herein. To better rate this concept, a brief description of antibody structure follows.
An antibody is composed of two peptide chains, each containing one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), the latter of which is in each case made up of four FR regions and three interspaced CDRs. The antigen-binding site is formed by one or more CDRs, yet the FR regions provide the structural framework for the CDRs and, hence, play an ant role in antigen binding. By altering one or more amino acid residues in a CDR or FR region, the skilled worker routinely can generate mutated or diversified antibody sequences, which can be screened t the antigen, for new or improved properties, for example.
A further preferred embodiment of the invention is an antibody or antigen-binding fragment in which the VH and VL sequences are selected as shown in Table 9. The skilled, worker can use the data in Table 9 to design peptide ts that are within the scope of the present invention. It is preferred that variants are constructed by changing amino acids within one or more CDR regions; a variant might also have one or more altered framework regions. Alterations also may be made in the framework regions. For e, a peptide FR domain might be altered where there is a deviation in a residue compared to a germline sequence.
WO 76186 _ 41 _ Alternatively, the skilled worker could make the same analysis by comparing the amino acid sequences disclosed herein to known sequences ofthe same class of such antibodies, using, for example, the procedure described by Knappik A., et al., JMB 2000, 296257-86.
Furthermore, variants may be Obtained by using one dy as starting point for optimization by diversifying one or more amino acid residues in the dy, preferably amino acid es in one or more CDRs, and by screening the resulting collection of antibody variants for variants with improved properties. Particularly preferred is diversification of one or more amino acid residues in CDR3 of VL and/or VH.
Diversification can be done by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekas B. et al., Nucl. Acids Res. 1994, 22: 5600.). Antibodies or antigen-binding fragments thereofinclude molecules with modifications/variations including but not limited to e.g. modifications leading to altered half-life (e. g. modification of the Fc part or attachment of further molecules such as PEG), altered binding affinity or altered ADCC or CDC activity.
Examples of variants of antibodies are given for M048—D01 (TPP-l397, TPP-1398, TPP-l399, TPP—l400, TPP-l401, TPP-1402 and TPP—1403) and 08 (TPP-1406, TPP-1407, 08, TPP-l409, TPP-1410, TPP-1411, TPP-l412, and TPP-l415) as depicted in Table 10. The improved properties of these variant antibodies are shown in Table 11.
Conservative Amino Acid Variants Polypeptide variants may be made that conserve the overall molecular structure of an antibody peptide ce described herein. Given the properties of the dual amino acids, some rational substitutions will be recognized by the d worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of rity in polarity, charge, solubility, hobicity, hydrophilicity, and/or the athic nature of the residues involved. _ 42 _ For example, (a) ar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, and nine; (b) polar neutral amino acids include glycine, serine, threonine, ne, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and, (d) negatively charged, (acidic) amino acids include aspartic acid, and glutamic acid. tutions typically may be made within groups (a)—(d). In addition, e and proline may be substituted for one another based, on their ability to disrupt u-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, ine and lysine are more commonly found in Ct-helices, while valine, isoleucine, alanine, ne, tryptophan and threonine are more ly found in B-pleated, sheets. e, serine, aspartic acid, asparagine, and, proline are commonly found, in turns. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and, G; and (iii) A, V, L and 1. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can uct DNAs encoding the conservative amino acid, variants.
As used herein, "sequence identity" between two polypeptide sequences, indicates the percentage of amino acids that are cal between the sequences. ”Sequence gy" indicates the percentage of amino acids that either is identical or that represent conservative amino acid, substitutions.
DNA molecules of the ion The present invention also relates to the DNA molecules that encode an antibody of the invention or antigen-binding fragment thereof. These sequences include, but are not limited to, those DNA molecules set forth in SEQ IDs 3, 4, 13, 14, 23, 24, 33, 34, 43, 44, 53 and, 54.
DNA molecules of the invention are not limited to the sequences disclosed, herein, but also include variants thereof. DNA variants within the invention may be described by reference to their physical properties in hybridization. The skilled worker will recognize that DNA can be used to identify its complement and, since DNA is double stranded, its _ 43 _ equivalent or homolog, using nucleic acid ization techniques. It also will be recognized that ization can occur with less than 100% complementarity. However, given appropriate choice of conditions, hybridization techniques can be used to differentiate among DNA sequences based on their structural relatedness to a particular probe. For guidance ing such ions see, Sambrook et al., 1989 supra and Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and, Sons).
Structural similarity between two polynucleotide sequences can be expressed as a function of "stringency" of the conditions under which the two sequences will hybridize with one another. As used herein, the term "stringency" refers to the extent that the conditions disfavor hybridization. Stringent conditions strongly disfavor hybridization, and only the most structurally related molecules will hybridize to one another under such conditions. Conversely, non-stringent conditions favor hybridization of molecules displaying a lesser degree of structural relatedness. Hybridization stringency, ore, directly correlates with the structural relationships of two nucleic acid sequences. The following relationships are useful in correlating hybridization and relatedness (where Tm is the g temperature of a nucleic acid duplex): a. Tm = 69.3 + 0.41(G+C)% b. The Tm of a duplex DNA decreases by 10C with every increase of 1% in the number of mismatched base pairs.
C. (Tm)H2' (T110111 2 18.5 lOglo‘LLZ/jyll where ul and u2 are the ionic ths of two solutions.
Hybridization stringency is a function of many factors, including overall DNA tration, ionic strength, temperature, probe size and the ce of agents which disrupt hydrogen bonding. Factors promoting hybridization e high DNA concentrations, high ionic strengths, low temperatures, longer probe size and the absence of agents that disrupt hydrogen g. ization typically is performed in two phases: the “binding” phase and the “washing” phase. _ 44 _ Functionally Equivalent Variants Yet r class of DNA variants within the scope of the invention may be described with reference to the product they encode. These functionally equivalent polynucleotides are characterized by the fact that they encode the same e ces found in SEQ ID NOS: 1, 2, 5-12, 15-22, 25-32, 35-42, 45-52, 55-60 due to the degeneracy ofthe genetic code.
It is recognized, that variants of DNA les provided herein can be constructed in several different ways. For example, they may be ucted as completely synthetic DNAs. s of efficiently synthesizing oligonucleotides in the range of 20 to about 150 nucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993).
Overlapping ucleotides may be synthesized and assembled in a fashion first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2. Synthetic DNAs preferably are designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.
As indicated, a method of generating variants is to start with one of the DNAs disclosed herein and then to conduct site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, a target DNA is cloned into a single-stranded DNA bacteriophage vehicle. Single-stranded DNA is ed and hybridized with an ucleotide containing the desired nucleotide alteration(s). The complementary strand, is synthesized and the double stranded phage is introduced into a host. Some of the resulting progeny will contain the d mutant, which can be confirmed, using DNA sequencing. In addition, various methods are available that increase the probability that the progeny phage will be the desired . These methods are well known to those in the field and kits are commercially available for generating such mutants.
Recombinant DNA ucts and expression The present invention further provides recombinant DNA constructs comprising one or more ofthe nucleotide ces of the present invention. The recombinant constructs of the present invention are used in connection with a vector, such as a plasmid, WO 76186 _ 45 _ phagemid, phage or viral vector, into which a DNA le encoding an antibody of the invention or antigen-binding fragment thereof is inserted.
An antibody, n binding portion, or derivative f provided herein can be ed by recombinant expression of nucleic acid sequences encoding light and heavy chains or portions thereof in a host cell. To express an antibody, antigen binding portion, or derivative thereof recombinantly, a host cell can be transfected with one or more recombinant sion vectors carrying DNA nts ng the light and/or heavy chains or portions thereof such that the light and heavy chains are expressed in the host cell.
Standard inant DNA methodologies are used prepare and/or obtain c acids encoding the heavy and light chains, incorporate these nucleic acids into recombinant expression vectors and, introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds) t Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in US. Pat. No. 4,816,397 by Boss et al..
In addition, the nucleic acid sequences ng variable regions ofthe heavy and/or light chains can be converted, for example, to nucleic acid sequences encoding full- length antibody chains, Fab fragments, or to scFv. The VL- or VH-encoding DNA fragment can be operatively linked, (such that the amino acid sequences encoded by the two DNA fragments are me) to another DNA fragment encoding, for example, an antibody constant region or a flexible linker. The sequences of human heavy chain and light chain nt regions are known in the art (see e.g., Kabat, E. A., el al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
In certain assays an expression of the antibodies of this invention as murine IgG is preferred, e.g. immunohistochemistry with human samples can be analyzed more easily by using murine antibodies. Therefore, for example the antibody Fab fragment M048—D01 (SEQ ID NO:31 for VH chain, and, SEQ ID N032 for VL chain) was expressed as murine —46— IgG2a called M048—D01—mIgG2a (SEQ ID NO:221 for heavy . This antibody was also used in Example 17 as control.
To create a polynucleotide sequence that encodes a scFv, the VH— and, VL-encoding nucleic acids can be operatively linked to another fragment encoding a flexible linker such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) e 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348 :552-554).
To express the antibodies, antigen binding portions or derivatives thereof standard inant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif.
). For example, DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. Suitable host cells are prokaryotic and otic cells. Examples for prokaryotic host cells are e. g. bacteria, es for eukaryotic host cells are yeast, insect or ian cells. In some embodiments, the DNAs encoding the heavy and light chains are inserted into separate vectors. In other embodiments, the DNA encoding the heavy and light chains is ed into the same vector. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.
Bacterial Expression Useful expression vectors for ial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a onal promoter. The vector will se one or more phenotypic selectable s and an origin of replication to ensure maintenance ofthe vector and, if desirable, to provide amplification within the host.
Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella WO 76186 _ 47 _ Uphimurium and s species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid- based. These vectors can contain a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well— known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an riate cell y, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically ted by centrifugation, disrupted by physical or al means, and the resulting crude extract retained for further purification.
In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the protein being expressed. For e, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide ies, for example, vectors which direct the expression of high levels of fusion n products that are readily purified may be desirable.
Therefore an embodiment ofthe present invention is an expression vector comprising a nucleic acid sequence encoding for the novel antibodies ofthe present invention. See Example 2 for an exemplary description.
Antibodies of the present invention or antigen-binding fragment thereof include lly purified, products, products of chemical synthetic procedures, and, products produced by recombinant ques from a prokaryotic host, including, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and s species within the genera Pseudomonas, Streptomyces, and lococcus, preferably, from E. coli cells.
Mammalian Expression & Purification Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV —48— promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., US. 5,168,062 by i, US. 4,510,245 by Bell et al. and US. 615 by Schaffner et al.. The inant expression vectors can also include origins of replication and selectable markers (see e.g., US. 4,399,216, 4,634,665 and US. 5,179,017, by Axel et al.). le selectable markers include genes that confer resistance to drugs such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. For example, the dihydrofolate reductase (DHFR) gene confers resistance to methotrexate and the neo gene s resistance to G418. For nt cloning the first 3 amino acids of the inus of the heavy chains [EVQ] (SEQ ID NO:67 and SEQ ID NO:69) can also alternatively be expressed as [QVE], for example as a variant of the heavy chain of human IgG1 M048- D01-hIgG1 (SEQ ID NO:222). For efficient cloning the N—terminus of light chains can be extended by amino acid residues e. g. Alanin.
Transfection of the expression vector into a host cell can be carried out using standard techniques such as electroporation, m-phosphate precipitation, and DEAE— dextran transfection.
Suitable mammalian host cells for expressing the antibodies, antigen binding portions, or derivatives thereof provided herein include e Hamster Ovary (CHO cells) [including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.
Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J.
Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621J], NSO myeloma cells, COS cells and SP2 cells. In some embodiments, the expression vector is ed such that the expressed protein is secreted, into the culture medium in which the host cells are grown. The antibodies, antigen binding portions, or derivatives thereof can be recovered from the culture medium using standard protein purification s. dies of the invention or an antigen-binding fragment thereof can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to ammonium sulfate or l precipitation, acid extraction, Protein A 2012/073325 _ 49 _ chromatography, Protein G chromatography, anion or cation ge chromatography, phospho-cellulose chromatography, hydrophobic ction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
Antibodies of the present invention or antigen-binding fragment thereof include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated. Such methods are described, in many rd laboratory manuals, such as ok, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20.
Therefore an embodiment of the present invention are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.
Another embodiment of the present invention is a method of using the host cell to produce an antibody and antigen binding fragments, sing culturing the host cell under suitable ions and recovering said antibody. ore another embodiment of the t invention is the production of the antibodies according to this invention (for e antibody M048—D01-hIgG1) with the host cells ofthe present invention and purification ofthese dies to at least 95% neity by weight. 2012/073325 _ 50 _ Therapeutic Methods Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of an antibody or antigen-binding nt thereof contemplated by the invention. A "therapeutically effective" amount hereby is defined as the amount of an antibody or antigen-binding fragment that is of sufficient quantity to deplete FGFRZ-positive cells in a treated area of a subject - either as a single dose or according to a le dose regimen, alone or in combination with other agents, which leads to the alleviation of an e condition, yet which amount is toxicologically tolerable. The subject may be a human or non—human animal (e.g., rabbit, rat, mouse, dog, monkey or other lower-order e).
An antibody of the invention or antigen-binding nt thereof might be co- stered with known medicaments, and in some instances the antibody might itself be modified. For example, an antibody could be conjugated to a cytotoxic agent or radioisotope to potentially further increase efficacy.
Antibodies of the present invention may be administered as the sole pharmaceutical agent or in combination with one or more additional eutic agents where the combination causes no ptable adverse effects. This combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the invention and one or more additional therapeutic agents, as well as administration of an antibody ofthe invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation. For e, an antibody of the invention and a therapeutic agent may be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent may be administered in separate dosage formulations.
Where separate dosage formulations are used, an antibody of the invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially). _ 51 _ In particular, antibodies of the present invention may be used in fixed or separate combination with other anti-tumor agents such as alkylating agents, anti-metabolites, plant- derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, ed drugs, antibodies, interferons and/or biological response modifiers, anti-angiogenic compounds, and other anti-tumor drugs. In this regard, the following is a non-limiting list of examples of secondary agents that may be used in combination with the antibodies of the present invention: Alkylating agents include, but are not limited to, nitrogen mustard N—oxide, cyclophosphamide, ifosfamide, thiotepa, stine, ine, temozolomide, altretamine, apaziquone, brostallicin, ustine, carmustine, estramustine, fotemustine, glufosfamide, mafosfamide, bendamustin, and ctol; platinum-coordinated alkylating compounds include, but are not limited to, cisplatin, latin, eptaplatin, lobaplatin, nedaplatin, oxaliplatin, and latin; Anti-metabolites include, but are not limited to, rexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or in combination with leucovorin, tegafur, doxifluridine, carmofur, bine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabin, S-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethynylcytidine, cytosine oside, yurea, lan, nelarabine, nolatrexed, ocfosfite, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine; Hormonal therapy agents include, but are not limited to, exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, 11-beta ysteroid dehydrogenase 1 inhibitors, 17-alpha hydroxylase/17,20 lyase inhibitors such as abiraterone acetate, 5- alpha reductase tors such as finasteride and epristeride, anti-estrogens such as tamoxifen citrate and fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, letrozole, anti-androgens such as tamide, flutamide, mifepristone, nilutamide, Casodex, and anti-progesterones and combinations thereof; 2012/073325 _ 52 _ Plant-derived anti—tumor substances include, e. g., those selected from mitotic inhibitors, for example epothilones such as sagopilone, ilone and epothilone B, vinblastine, vinflunine, docetaxel, and paclitaxel; Cytotoxic topoisomerase inhibiting agents include, but are not d to, bicin, doxorubicin, amonafide, belotecan, camptothecin, 10—hydroxycamptothecin, 9- aminocamptothecin, diflomotecan, irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, and combinations thereof; lmmunologicals include interferons such as interferon alpha, interferon alpha—2a, interferon 2b, interferon beta, interferon gamma-1a and interferon gamma-n1, and other immune enhancing agents such as 2 and other 1L2 derivatives, filgrastim, lentinan, sizofilan, TheraCys, ex, aldesleukin, alemtuzumab, BAM—002, dacarbazine, daclizumab, denileukin, gemtuzumab, ozogamicin, ibritumomab, imiquimod, lenograstim, lentinan, melanoma vaccine a), molgramostim, mostim, tasonermin, tecleukin, thymalasin, tositumomab, Vimlizin, epratuzumab, mitumomab, oregovomab, pemtumomab, and Provenge; Biological response modifiers are agents that modify defense mechanisms of living organisms or biological responses such as survival, growth or differentiation of tissue cells to direct them to have umor activity; such agents include, e.g., krestin, lentinan, sizofiran, picibanil, ProMune, and ubenimex; Anti-angiogenic compounds include, but are not limited to, acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, bevacizumab, brivanib at, cilengtide, tastatin, endostatin, inide, halofuginone, pazopanib, zumab, rebimastat, recentin, regorafenib, removab, revlimid, sorafenib, squalamine, sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin; Antibodies include, but are not limited to, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, lumiliximab, catumaxomab, atacicept, oregovomab, and alemtuzumab; WO 76186 _ 53 _ VEGF inhibitors such as, e.g., sorafenib, regorafenib, bevacizumab, nib, recentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib, pazopanib, and ranibizurnab; EGFR (HERl) inhibitors such as, e.g., cetuximab, paniturnurnab, vectibix, gefitinib, erlotinib, and Zactima; HER2 inhibitors such as, e. g., lapatinib, tratuzurnab, and pertuzurnab; niTOR inhibitors such as, e.g., ternsirolimus, sirolirnus/Raparnycin, and everolirnus; c-Met inhibitors; PI3K and AKT inhibitors; CDK inhibitors such as roscovitine and flavopiridol; Spindle assembly oints inhibitors and, ed anti-mitotic agents such as PLK tors, Aurora inhibitors (e. g. Hesperadin), checkpoint kinase inhibitors, and KSP inhibitors; HDAC inhibitors such as, e.g., panobinostat, vorinostat, M8275, belinostat, and LBH589; HSP90 and HSP70 inhibitors; Proteasorne inhibitors such as ornib and carfilzomib; Serine/threonine kinase tors including MEK inhibitors and Raf inhibitors such as sorafenib; Farnesyl transferase inhibitors such as, e.g., tipifarnib; Tyrosine kinase inhibitors including, e.g., dasatinib, nilotibib, regorafenib, bosutinib, nib, bevacizumab, sunitinib, cediranib, axitinib, aflibercept, telatinib, imatinib rnesylate, brivanib alaninate, pazopanib, ranibizumab, vatalanib, cetuxirnab, paniturnurnab, vectibix, gefitinib, erlotinib, lapatinib, tratuzumab, pertuzurnab, and c—Kit tors; Vitamin D receptor agonists; _ 54 _ Bel-2 protein tors such as lax, oblimersen , and gossypol; Cluster of entiation 20 receptor antagonists such as, e. g., rituximab; Ribonucleotide reductase inhibitors such as, e.g., gemcitabine; Tumor necrosis apoptosis inducing ligand receptor 1 agonists such as, e.g., mapatumumab ; -Hydroxytryptamine receptor antagonists such as, e.g., rEV598, xaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-lOOl ; Integrin inhibitors including alphaS-betal integrin inhibitors such as, e.g., E7820, JSM 6425, volociximab, and atin; Androgen receptor antagonists including, e.g., nandrolone ate, fluoxymesterone, Android, Prost-aid, andromustine, bicalutamide, flutamide, apo- cyproterone, apo-flutamide, adinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide; Aromatase inhibitors such as, e.g., anastrozole, letrozole, actone, exemestane, aminoglutethimide, and formestane; Matrix metalloproteinase inhibitors; Other anti-cancer agents including, e.g., alitretinoin, ampligen, atrasentan bexarotene, bortezomib, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, raginase, bazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazaroten, velcade, gallium nitrate, canfosfamide, darinaparsin, and tretinoin.
In a preferred embodiment, the antibodies of the present invention may be used in combination with chemotherapy (i.e. cytotoxic agents), anti-hormones and/or ed therapies such as other kinase inhibitors (for example, EGFR inhibitors), mTOR inhibitors and angiogenesis inhibitors. 2012/073325 _ 55 _ The compounds of the present invention may also be employed in cancer treatment in conjunction with radiation therapy and/or surgical intervention.
An dy of the invention or antigen-binding fragment thereof might in some instances itself be d. For example, an antibody could be conjugated to any of but not limited to the compounds mentioned above or any radioisotope to potentially further se efficacy. Furthermore, the antibodies of the invention may be utilized, as such or in compositions, in research and diagnostics, or as analytical reference standards, and the like, which are well known in the art.
The inventive antibodies or antigen-binding fragments thereof can be used as a therapeutic or a diagnostic tool in a variety of situations with aberrant FGFRZ-signaling, e. g. cell proliferative disorders such as cancer or fibrotic diseases. ers and ions particularly suitable for ent with an antibody of the inventions are solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, d, yroid, and their distant metastases.
Those disorders also include lymphomas, sarcomas and leukemias.
Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers .
Examples of esophageal cancer include, but are not limited to esophageal cell carcinomas and adenocarcinomas, as well as squamous cell carcinomas, leiomyosarcoma, malignant melanoma, rhabdomyosarcoma and lymphoma.
Examples of gastric cancer include, but are not d to intestinal type and diffuse type gastric adenocarcinoma.
Examples of pancreatic cancer e, but are not limited to ductal adenocarcinoma, adenosquamous carcinomas and pancreatic endocrine tumors. —56— Examples of breast cancer include, but are not d to triple negative breast cancer, invasive ductal carcinoma, invasive lobular carcinoma, ductal oma in Sim, and lobular carcinoma in situ. es of cancers of the respiratory tract include, but are not limited to small- cell and, non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, astoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor.
Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, l and vulvar cancer, as well as sarcoma of the uterus.
Examples of ovarian cancer include, but are not limited to serous tumour, endometrioid tumor, mucinous cystadenocarcinoma, granulosa cell tumor, Sertoli-Leydig cell tumor and arrhenoblastoma Examples of cervical cancer include, but are not limited to squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, small cell carcinoma, neuroendocrine tumour, glassy cell carcinoma and villoglandular arcinoma.
Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, urethral, and hereditary and sporadic papillary renal cancers.
Examples of kidney cancer include, but are not limited to renal cell carcinoma, lial cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal toma, Bellini duct carcinoma, cell sarcoma of the kidney, mesoblastic nephroma and Wilms‘ tumor.
Examples of bladder cancer include, but are not limited to transitional cell carcinoma, squamous cell oma, adenocarcinoma, a and small cell carcinoma. _ 57 _ Eye s include, but are not limited to intraocular melanoma and retinoblastoma.
Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck s include, but are not limited to squamous cell cancer of the head and neck, laryngeal, hypopharyngeal, nasopharyngeal, oropharyngeal cancer, lip and oral cavity cancer, and squamous cell cancer.
Lymphomas e, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, ous T-cell lymphoma, t lymphoma, Hodgkin's disease, and ma of the central nervous system.
Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous cytoma, lymphosarcoma, and rhabdomyosarcoma.
Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. In a preferred embodiment, the antibodies or antigen-binding nts thereof ofthe invention are suitable for a therapeutic or diagnostic method for the treatment or diagnosis of a cancer disease comprised in a group consisting of gastric cancer, breast cancer, pancreatic cancer, ctal , kidney cancer, prostate cancer, ovarian cancer, cervical cancers, lung cancer, endometrial cancer, esophageal cancer, head and neck cancer, hepatocellular carcinoma, melanoma and bladder cancer. In addition, the ive antibodies or antigen-binding nts thereof can also be used as a therapeutic or a stic tool in a variety of other disorders n FGFRZ is involved such as, but not limited to fibrotic diseases such as intraalveolar fibrosis, silica-induced pulmonary fibrosis, experimental lung fibrosis, idiopathic lung fibrosis, renal fibrosis, as well as lymphangioleiomyomatosis, stic ovary syndrome, acne, psoriasis, cholesteatoma, —58— cholesteatomatous chronic otitis media, periodontitis, solar lentigines, bowel e, atherosclerosis or endometriosis.
The disorders mentioned above have been well characterized in humans, but also exist with a similar etiology in other animals, including mammals, and can be treated by administering pharmaceutical compositions of the present invention.
To treat any ofthe ing disorders, pharmaceutical compositions for use in ance with the present invention may be formulated, in a conventional manner using one or more physiologically acceptable carriers or excipients. An antibody of the ion or antigen-binding fragment thereof can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., uscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if d for local immunosuppressive treatment, intralesional administration. In addition, an antibody of the invention might be stered by pulse infusion, with, e.g., declining doses of the dy. Preferably, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. The amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered. The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated.
Determining a therapeutically effective amount of the novel polypeptide, according to this invention, largely will depend on particular patient characteristics, route of stration, and the nature of the disorder being treated. General guidance can be found, for example, in the publications of the International Conference on Harmonization and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and, 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed, Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining a eutically effective amount will depend on such s as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the _ 59 _ art and, found in the ing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods bed below in the Examples. stic Methods FGFR2 dies or n-binding fragments thereof can be used for detecting the presence of FGFRZ-expressing tumors. The presence of FGFRZ-containing cells or shed FGFR2 within s biological samples, including serum, and tissue biopsy specimens, may be detected with FGFR2 antibodies. In addition, FGFRZ antibodies may be used in various imaging methodologies such as immunoscintigraphy with a 99To (or other isotope) conjugated antibody. For example, an imaging protocol similar to the one ly described using a 111In conjugated anti-PSMA antibody may be used to detect atic or ovarian carcinomas (Sodee et al., Clin. Nuc. Med. 21: 759-766, 1997). Another method of ion that can be used is positron emitting tomography by conjugating the antibodies of the invention with a suitable isotope (see Herzog et al., J. Nucl. Med. 342222-2226, 1993).
Pharmaceutical Compositions and Administration An embodiment of the present invention are pharmaceutical compositions which comprise FGFR2 antibodies or antigen-binding fragment thereof, alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A further embodiment are pharmaceutical itions comprising a FGFR2 binding dy or antigen-binding fragment thereof and a further pharmaceutically active compound that is suitable to treat FGFR2 related diseases such as cancer. Any of these molecules can be administered to a t alone, or in ation with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.
The present invention also relates to the administration of pharmaceutical compositions. Such administration is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, _ 60 _ subcutaneous, intramedullary, intrathecal, entricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these pharmaceutical itions may contain suitable pharmaceutically acceptable carriers comprising excipients and aries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in s suitable for oral stration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, symps, slurries, suspensions and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid ent, optionally grinding a resulting e, and processing the mixture of granules, after adding suitable auxiliaries, if d, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or n fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl, cellulose, hydroxypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums ing arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, ol gel, polyethylene glycol and/or titanium e, r solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the ty of active compound, i.e. dosage. 2012/073325 _ 61 _ Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit es can contain active ingredients mixed with a filler or binders such as lactose or es, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active nds may be dissolved, or suspended in le liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, 's solution, or physiologically buffered saline. Aqueous ion suspensions may n substances that increase viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular r to be ted are used in the formulation. Such penetrants are generally known in the art.
Kits The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more ofthe ingredients ofthe aforementioned compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a mental agency ting the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration. _ 62 _ In another embodiment, the kits may contain DNA sequences encoding the antibodies of the invention. Preferably the DNA sequences encoding these antibodies are provided in a plasmid suitable for transfection into and expression by a host cell. The plasmid may contain a promoter (often an inducible promoter) to regulate expression of the DNA in the host cell. The plasmid may also contain appropriate restriction sites to facilitate the ion of other DNA sequences into the plasmid to produce various antibodies. The plasmids may also contain numerous other elements to facilitate cloning and expression of the encoded proteins. Such ts are well known to those of skill in the art and include, for e, able markers, tion codons, termination codons, and the like.
Manufacture and Storage.
The pharmaceutical compositions of the present invention may be ctured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, ting, emulsifying, encapsulating, entrapping or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with acids, including by not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more e in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lized powder in l mM-SO mM histidine, O.l%-2% sucrose, 2%-7% mannitol at a pH range of4.5 to 5.5 that is combined with buffer prior to use.
After pharmaceutical compositions comprising a compound ofthe invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate ner and labeled for treatment of an indicated ion. For administration of FGFRZ antibodies or antigen-binding nt thereof, such labeling would include amount, frequency and method of administration.
Therapeutically Effective Dose.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve _ 63 _ the intended purpose, i.e. treatment of a particular disease state characterized by FGFR2 expression. The determination of an ive dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., neoplastic cells, or in animal models, usually mice, rabbits, dogs, pigs or monkeys. The animal model is also used to achieve a desirable concentration range and route of administration. Such ation can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of antibody or antigen-binding nt thereof, that ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by rd pharmaceutical procedures in cell cultures or mental s, e.g., ED50 (the dose eutically ive in 50% of the population) and LD50 (the dose lethal to 50% ofthe population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, EDso/LDso. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage of such compounds lies ably within a range of circulating concentrations what include the 131350 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional s that may be taken into account include the severity of the disease state, e.g., tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction ivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. _ 64 _ Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 2 g, depending upon the route of administration. Guidance as to particular dosages and methods of ry is provided in the literature. See US. Pat. No. 4,657,760; ,206,344; or 5,225,212. Those skilled in the art will employ different formulations for polynucleotides than for proteins or their tors. Similarly, ry of polynucleotides or ptides will be specific to particular cells, conditions, locations, etc. Preferred specific activities for a radiolabelled antibody may range from 0.1 to 10 mCi/mg of protein (Riva et al., Clin. Cancer Res. 523275—3280, 1999; Ulaner et al., 2008 Radiology 246(3):895-902) The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. e molecular biology techniques of the following examples can be carried out as described in standard tory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold, Spring , N.Y., 1989.
A preferred embodiment of the invention is: A. An isolated antibody or antigen-binding fragment thereof which reduces the cell surface expression of FGFR2 after binding to FGFR2 in cell lines SNU16 (ATCC- 74) and MFM223 (ECACC-98050130) which overexpress FGFR2 and in cell lines AN3-CA (DSMZ-ACC 267) and, MFE-296 -98031101) which express mutated FGFR2.
B. An isolated antibody or antigen-binding fragment thereof according to claim A wherein the dy or antigen-binding fragment thereof specifically binds to the _ 65 _ extracellular inal epitope (lRPSFSLVEDTTLEPEIS) of FGFRZ as presented by (SEQ ID N0263).
C. An isolated antibody or antigen-binding fragment f according to claim B wherein binding of the antibody to the extracellular N—terminal epitope (SEQ ID NO:63) is mediated by at least one epitope residue selected from the group of residues consisting of Arg 1, Pro 2, Phe 4, Ser 5, Leu 6, and Glu 8.
D. An ed antibody or antigen-binding fragment f according to any one of claims B-C wherein the antibody or antigen-binding fragment thereof loses more than 50% of its ELISA signal by changing of at least one of the amino acid es in the N-terminal epitope (IRPSFSLVEDTTLEPEIS) of FGFR2 into an Alanine a) said residue selected from the group Pro 2, Leu 6 and Glu 8, or b) said, residue selected from the group Arg 1, Pro 2, Phe 4 and Ser 5.
E. The antibody or antigen-binding nt according to any one of claims A to D, wherein the antibody or n-binding fragment competes in binding to FGFR2 with at least one antibody selected from the group “M048-D01”, “M047-D08”, “M017-B02”, “M021—H02”, “M054-A05” “M054-D03”, “TPP-1397”, “TPP- 1398”, “TPP-1399”, “TPP-1400”, “TPP—1401”, “TPP—1402”, “TPP-1403”, “TPP- 1406”, “TPP-1407”, “TPP-1408”, “TPP-1409”, “TPP-1410”, “TPP-1411”, “TPP- 1412”, and 415”.
F. The antibody or antigen-binding fragment according to claim E, wherein the amino acid sequence of the antibody or antigen-binding fragment is at least 50%, 55%, 60% 70%, 80%, 90, or 95% identical to at least one CDR sequence of “MO48-D01”, D08”, B02”, “M021-H02”, “M054-A05”, “M054- D03”, “TPP-1397”, “TPP-1398”, “TPP-1399”, “TPP-1400”, “TPP-1401”, “TPP— 1402”, “TPP-1403”, “TPP-1406”, “TPP-1407”, “TPP-1408”, “TPP-1409”, “TPP— 1410”, “TPP-1411”, “TPP-1412”, or “TPP-1415”, or at least 50%, 60%, 70%, 80%, 90%, 92% or 95% identical to the VH or VL sequence of “M048-D01”, _ 66 _ “M047-D08”, “M017—B02”, “M021-H02”, “M054-A05”, “M054—D03”, “TPP- 1397”, 398”, “TPP-1399”, “TPP-1400”, “TPP-1401”, “TIT-1402”, “TPP- 1403”, “TPP-1406”, “TPP-l407”, “TPP-1408”, 409”, 410”, “TPP- 1411”, “TPP-l 412”, or “TPP-l415”.
The antibody or antigen-binding fragment according to any one of claims E to F, wherein the antibody or antigen-binding fragment comprises at least one CDR sequence or at least one le heavy chain or light chain sequence as depicted in Table 9 and Table 10.
H. The antibody or antigen-binding fragment according to claim A to G comprising a) the variable heavy chain CDR sequences as presented by SEQ ID NO: -7 and the variable light chain CDR sequences ted by SEQ ID NO: 8-10, or b) the le heavy chain CDR sequences as presented by SEQ ID NO: -17 and the variable light chain CDR sequences presented by SEQ ID NO: 18-20, or c) the variable heavy chain CDR sequences as presented by SEQ ID NO: -27 and the variable light chain CDR sequences presented, by SEQ ID NO: 28-30, or d) the variable heavy chain CDR sequences as presented by SEQ ID NO: -37 and the le light chain CDR sequences presented by SEQ ID NO: 38-40, or e) the variable heavy chain CDR sequences as presented by SEQ ID NO: 45-47 and the variable light chain CDR sequences presented by SEQ ID NO: 48-50, or f) the variable heavy chain CDR sequences as presented by SEQ ID NO: 55-57 and, the variable light chain CDR sequences presented by SEQ ID NO: 58-60, or _ 67 _ g) the variable heavy chain CDR sequences as presented by SEQ ID NO: 75-77 and the variable light chain CDR sequences ted by SEQ ID NO: 78-80, or b) the variable heavy chain CDR sequences as presented by SEQ ID NO: 85-87 and the variable light chain CDR ces presented by SEQ ID NO: 88-90, or i) the variable heavy chain CDR sequences as ted by SEQ ID NO: 95-97 and, the variable light chain CDR sequences presented by SEQ ID NO: 98-100, 0r j) the variable heavy chain CDR sequences as presented by SEQ ID NO: 7 and the variable light chain CDR sequences presented by SEQ ID -110, or k) the variable heavy chain CDR sequences as presented by SEQ ID NO: 115 -1 l7 and the variable light chain CDR sequences presented by SEQ ID N02118-120, 0r 1) the variable heavy chain CDR sequences as presented by SEQ ID NO: 125-127 and the variable light chain CDR sequences presented by SEQ ID NO: 128-130, or m) the variable heavy chain CDR sequences as presented by SEQ ID NO: 135-137 and the variable light chain CDR sequences presented by SEQ ID NO: 138-140, or n) the variable heavy chain CDR sequences as presented by SEQ ID NO: 145-l47 and the variable light chain CDR sequences presented by SEQ ID NO: 148-150, or 0) the variable heavy chain CDR sequences as presented by SEQ ID NO: 155-157 and the variable light chain CDR sequences ted by SEQ ID NO: 158-160, or _ 6g _ p) the variable heavy chain CDR sequences as presented by SEQ ID NO: 165-167 and the variable light chain CDR sequences presented by SEQ ID NO: 0, or q) the variable heavy chain CDR ces as presented by SEQ ID NO: 175-177 and the variable light chain CDR sequences presented, by SEQ ID NO: 178-180, or r) the le heavy chain CDR sequences as presented by SEQ ID NO: 185 -1 87 and the variable light chain CDR sequences presented by SEQ ID NO: 188-190, or s) the variable heavy chain CDR sequences as ted by SEQ ID NO: 195-197 and the variable light chain CDR sequences presented by SEQ ID NO: 198-200, or t) the variable heavy chain CDR ces as presented by SEQ ID NO: 205-207 and the le light chain CDR sequences presented by SEQ ID NO: 208-210, or u) the variable heavy chain CDR sequences as presented by SEQ ID NO: 215-2] 7 and the variable light chain CDR sequences presented by SEQ ID NO: 0.
I. The antibody or antigen-binding fragment according to claims A - H comprising a) a variable heavy chain sequence as presented by SEQ ID N021 and a variable light chain sequences as presented by SEQ ID N022, or b) a variable heavy chain sequence as presented by SEQ ID N0:ll and a variable light chain sequences as presented by SEQ ID N0212, or c) a variable heavy chain sequence as presented by SEQ ID N022] and a variable light chain sequences as presented by SEQ ID N0222, or d) a variable heavy chain sequence as presented by SEQ ID N031 and a variable light chain sequences as presented by SEQ ID N032, or 2012/073325 _ 69 _ e) a variable heavy chain sequence as presented by SEQ ID NO:41 and a variable light chain sequences as ted by SEQ ID NO:42, or f) a variable heavy chain sequence as presented by SEQ ID NO:51 and a variable light chain sequences as presented by SEQ ID N0252, or g) a le heavy chain sequence as presented by SEQ ID NO:73 and a variable light chain sequences as presented by SEQ ID NO:74, or h) a variable heavy chain sequence as presented by SEQ ID NO:83 and a variable light chain sequences as presented by SEQ ID N0284, or i) a variable heavy chain ce as presented by SEQ ID N0293 and a variable light chain sequences as presented by SEQ ID N0294, or j) a variable heavy chain sequence as ted by SEQ ID NO: 103 and a variable light chain sequences as presented by SEQ ID , or k) a variable heavy chain ce as presented by SEQ ID NO:113 and a variable light chain sequences as presented by SEQ ID NO:114, 0r 1) a variable heavy chain sequence as presented by SEQ ID NO: 123 and a variable light chain sequences as presented by SEQ ID N02124, 0r rn) a variable heavy chain sequence as presented by SEQ ID NO:133 and a variable light chain sequences as presented by SEQ ID N02134, or n) a variable heavy chain sequence as presented by SEQ ID NO: 143 and a variable light chain sequences as presented by SEQ ID NO:144, 0r 0) a variable heavy chain sequence as presented by SEQ ID NO:153 and a variable light chain sequences as presented by SEQ ID NO:154, or p) a variable heavy chain sequence as presented by SEQ ID NO:163 and a variable light chain sequences as presented by SEQ ID N02164, or q) a variable heavy chain sequence as presented by SEQ ID NO:173 and a variable light chain ces as presented by SEQ ID NO:174, 0r _ ’70 _ r) a variable heavy chain sequence as presented by SEQ ID N02183 and a variable light chain sequences as presented by SEQ ID NO:184, or s) a variable heavy chain ce as presented by SEQ ID N02193 and a variable light chain sequences as ted by SEQ ID NO:194, or t) a variable heavy chain sequence as ted by SEQ ID N02203 and a variable light chain sequences as presented by SEQ ID N02204, or u) a variable heavy chain sequence as presented by SEQ ID NO:213 and a variable light chain sequences as presented by SEQ ID N02214.
J. The dy according to any one of the preceding claims, which is an IgG antibody.
K. The antigen-binding fragment according to any one of the preceding claims, which is an scFv, Fab, Fab’ fragment or a F(ab’)2 fragment.
L. The antibody or antigen-binding fragment according to any one of the preceding claims, which is a monoclonal antibody or antigen-binding fragment.
M. The antibody or antigen-binding fragment according to any one of the preceding claims, which is human, humanized or chimeric antibody or antigen-binding fragment.
N. An antibody-drug conjugate, comprising an antibody or antigen g fragment thereof according to claims A to M. 0. An isolated c acid sequence that encodes the dy or antigen-binding nt according to claims A to M.
P. A vector comprising a nucleic acid sequence according to claim 0.
Q. An isolated cell expressing an antibody or antigen-binding fragment according to any one of the claims A , M and for comprising a nucleic acid according to claim 0 or a vector according to claim P.
R. An isolated cell according to claim Q, wherein said cell is a prokaryotic or an eukaryotic cell. _ ’71 _ S. A method of producing an antibody or n—binding fragment according to any one of the claims A , M comprising culturing of a cell according to claim R and purification of the antibody or antigen-binding fragment.
T. An antibody or antigen-binding fragment according to claims A , M or an antibody-drug conjugate according to claim N as a medicament.
U. An antibody or antigen antigen-binding fragment according to claims A7 M as a diagnostic agent.
V. An dy or antigen-binding fragment according to claims A , M or an antibody-drug conjugate according to claim N as a medicament for the treatment of cancer.
W. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to claims A 7 M or an dy-drug conjugate according to claim N.
X. A combination of a pharmaceutical ition according to claim W and one or more therapeutically active compounds.
Y. A method for ng a disorder or condition associated with the undesired presence of FGFRZ, comprising administering to a subject in need thereof an effective amount of the ceutical composition ing to claim W or a combination according to claim X. _ 72 _ EXAMPLES EXAMPLE 1: Antibody tion from n-CoDeR Libraries Tools usedfor Phage selections: Recombinant proteins used for the isolation of human antibodies ofthe t invention were obtained from R&D Systems and are listed in Table 1. All variants used were present as Fc-fusion proteins in carrier free ations. hTRAIL-Fc served as depletion agent to avoid Fc binder. Proteins were biotinylated according to manufacturer's instructions using an approximately 2-fold molar excess of biotin-LC-NHS (Pierce; Cat.
No. 21347) and desalted using Zeba desalting columns (Pierce; Cat. No. 89889).
Table 1: List of recombinant proteins used in phage selections and screening Protein Origin Cat. No. (R&D Systems) hFGFR2B—Fc (IHb) Human 665—FR mFGFR2B—FC (Hlb) Murine 708-MF OL—FC (lllb) Human 663 —FR hFGFR2B—FC (THC) Human 684—FR -Fc Human 63O—TR For phage selections on cells the human gastric carcinoma cell line KATO Ill (ATCC HTB-103) was employed, displaying native FGFRZ on its cell surface.
Phage Selections: The isolation of human antibodies ofthe present invention or antigen binding fragments thereof was performed by phage display technology ing the naive Fab antibody library n-CoDeR of Biolnvent International AB (Lund, ; described in 2012/073325 _ 73 _ Soderling et al., Nat. Biotech. 2000, -856), which is a Fab library in which all six CDRs are diversified. As summarized in Table 2, three different strategies for the selection of inventive antibodies were employed.
Table 2: Summary of selection strategies Round of gy I Strategy 11 Strategy [[1 selection: 200 nM biotinylated hFGFR2B-Fc (IIIb) 200 nM biotinylated 200 nM biotinylated KATO 111 cells mFGFRZB-Fc (Illb) hFGFRZB-Fc (ch) 100 11M biotinylated 100 nM biotinylated 200 nM biotinylated hFGFRZB—Fc (IIIb) hFGFRZoc—Fc (HIb) hFGFR20c—Fc (HIb) 100 nM ylated 200 nM biotinylated KATO 111 cells mFGFR2B—Fc (IIIb) hFGFR2B—Fc (IIIc) Standard buffers used, in this example are: 1X PBS: from Sigma (D5652—501) PBST: 1X PBS supplemented with 0.05% Tween20 (Sigma, P7949) ng buffer: PBST supplemented with 3% BSA (Sigma A4503) Precipitation buffer: 20% PEG (Calbiochem, 528877) in 2.5 M NaCl FACS-buffer: PBS supplemented with 3% FBS (GIBCO, 10082) and 0.01% NaN3 (Sigma, 71289) _ 74 _ Briefly, an aliquot of the Fab dy library was centrifuged at r.t for 5 min, the ing pellet was resuspended in 40 ml PBS and precipitated by addition of precipitation buffer followed by an incubation on ice for l h and a centrifugation step (1h at 4000 rpm).
The precipitated library was subsequently resuspended in 1 ml blocking buffer and incubated at r.t. for 30 min.
Meanwhile, aliquots of streptaVidin-coated Dynabeads M280 (Invitrogen, 11206D) were prepared by washing 3 times with PBS for 30 min on an end-to-end rotator. After that some ts were mixed with 200 nM biotinylated TRAIL-Fc protein while the remaining were mixed with the biotinylated target n as indicated in Table 2. The mixtures were incubated at r.t. on an -end rotator for 30 min and subsequently washed 3 times in 1 ml PBS. Coated beads were finally blocked by resuspension in ] ml ng buffer followed by collection of the beads and removal of the supernatant.
For depletion of unwanted Fc binders the blocked library (described above) was added to blocked Dynabeads coated with TRAIL-Fc and ted at r.t. for 30 min while rotating. After collection of the beads on a magnetic rack, the atant was mixed with d Dynabeads coated with target protein. After 60 min incubation on an end-to-end rotator the samples were washed 3 times with blocking buffer followed by 5 times washing with PB ST. Bound phages were eluted by adding 100 ul triethanolamine solution (TEA, 100 mM). After 10 min incubation at r.t., samples were neutralized by adding 400 pl 1M Tris-Cl, pH 7.5.
Panning strategy I included 2 rounds of panning on whole cells as a source of target protein (see Table 2). For this purpose, KATO 111 cells were resuspended in ice cold FACS buffer at a density of 107 cells per ml. An aliquot of rescued phages were added to 1 ml cell suspension and incubated at 40C by end-over-end rotation. Subsequently, cells were washed times with 2.5 ml FACS buffer followed by an elution of bound phages with 300 pl 76 nM citric acid (pH 2.5). After 5 min incubation, cells were centrifuged for 5 min at 400 g and 4°C and the supernatant was neutralized by adding 300 ml 1 M Tris—Cl, pH 7.5 WO 76186 _ 75 _ Eluted phages were propagated and phage titers determined essentially as previously bed (Cicortas Gunnarsson et al., Protein Eng Des Sel 2004; 17 (3): 213— 21). Briefly, aliquots of the eluate solution were saved for titration experiments while the rest was used to transform exponentially growing E. coli TGl (from Stratagene) for preparation of new phage stocks used in a second, third and fourth ion round according to the strategies depicted in Table 2. For each selection round, both input and output phages were titrated, on exponentially growing E. coli TG1 and clones were picked from round 2 to 4 for analysis in Phage ELISA.
Enzyme-linked immunosorbent assay (ELISA): Phage ELISA: Selected phages from different selection rounds were analyzed for specificity using phage ELISA. Briefly, phage expression was med, by adding 10 pl of over night culture (in LB-medium supplemented with 100 ug/ml ampicillin (Sigma, A5354), 1% glucose) to 100 pl fresh medium (LB-medium supplemented with 100 ug/ml ampicillin and 0.1% e (Sigma, G8769) and shaking at 250 rpm and 37°C in 96-well MTP until an OD600 of 0.5 was reached. Subsequently 40 ul helper phage M13KO7 (lnvitrogen, 420311) was added and samples were incubated for another 15 min at 37°C without shaking. After addition of IPTG (f.c. of 0.5 mM; final volume 200 pl) cells were incubated over night at 30°C while shaking at 200 rpm. 96-well ELISA-plates pre-coated with streptavidin (Pierce, 15500) were coated over night at 4°C with 1 ptle ylated 2B Fc (IIIb) or biotinylated TRAIL—Fc.
The next day plates were washed 3 times with PBST, treated with blocking reagent, and washed again 3 times with PBST. Meanwhile, phage es were briefly centrifuged, than 125 pl of the supernatant was removed and mixed with 125 pl blocking buffer. After that 100 ul of the blocked phages were transferred per well and incubated for 1 h at r.t.. After washing 3 times with PBST, anti M13 antibody coupled to HRP (GE Healthcare, 27-9421— 01; 122500 diluted in PBST) was added and incubated for 1 h at r.t.. Color reaction was _ 76 _ developed by addition of 50 ul TMB (lnvitrogen, 2023) and stopped after 5-15 min by adding 50 ul 112SO4 (Merck, 1 120801000). Colorimetric reaction was recorded at 450 nM in a plate reader (Tecan). ing astabs by ELISA: For the generation of soluble Fab nts (sFabs) phagemid DNA from the selection rounds 3 and 4 was isolated and digested with restriction s Eagl (Fermentas, FD0334) and EcoRI (NEB, R0101L ) according to the providers instructions in order to remove the gene III sequence. The ing fragment was re-ligated and, constructs were transformed into chemically competent E. coli Top10 using standard methods. Single clones were picked, transferred to 96-well plates containing LB-media (100 ug/ml ampicillin (Sigma, A5354), 1% glucose) and shaken ON at 250 rpm and 37°C. The next g 10 ml of pre—culture was transferred to 150 pl fresh LB-media (100 ug/ml ampicillin (Sigma, A5354), 0.1% glucose) until an OD600 of 0.5 was reached. After that sFab production was induced by the addition of IPTG (f.c. 0.5 mM) and incubation was continued over night at 30°C while shaking at 200 rpm. Next morning 50 ul ffer (24.7 g/l boric acid; 18.7 g/l NaCl; 1.49 g/l EDTA pH 8.0; 2.5 mg/ml lysozyme )) was added to each well, the e was incubated 1 h at r.t. Subsequently, 1/3 volume of blocking buffer with 9% BSA was added and alter an additional 30 min incubation step at r.t., 50 pl of each well was analyzed for binding of sFabs to the target in an ELISA essentially as described for phages, except that detection was performed with an IgG (Fab-specific) coupled to HRP (122500 diluted; Sigma; A 0293).
E 2: Small-scale production of soluble Fab screening hits Unique screening hits were produced in small scale for the initial analysis of their binding to different variants of FGFR-proteins (see example 3). 20-50 ml of LB-medium (supplemented with 0.1 mg/ml ampicillin and 0.1% glucose) were inoculated with a pre- _ 77 _ culture of the tive E. coli Top 10 clone, ning a unique Fab sequence cloned into the intial pBIF-vector but lacking the gene 111 sequence. Production of sFabs was induced by the addition of 0.5 mM IPTG (final concentration) and incubation was continued over night at 30°C at 250 rpm shaking.
Subsequently, cells were harvested by centrifugation and gently lysed by 1 h incubation at 4°C in a lysis buffer, containing 20 % sucrose (w/v), 30 mM TRIS, 1 mM EDTA, pH 8.0, 1 mg/ml lysozyme (Sigma L-6876) and 2.5 U/ml benzonase (Sigma , followed by the addition of an equal volume of PBS. After that, the cleared supernatant was applied to Dynabeads for His-tag isolation (lnvitrogen, 101—03D) and incubated for 2 h at 4°C on an end-over-end rotator. Subsequently, the matrix was washed 3 times with buffer 1 (50 mM Na-phosphate buffer, pH 7.4, 300 mM NaCl, 5 mM imidazol, 0.01 % Tween-20) followed by a single wash step in buffer 2 (PBS containing 0.005% Tween-20). Finally, Fabs were eluted with buffer E (10 mM Na—phosphate buffer, pH 7.4, 300 mM NaCl, 300 mM imidazol) and concentrated in Vivaspin 500 (cut-off 10000; from GE; 2-25) using ffer. Fabs were analysed for protein content and for purity by SDS-PAGE.
EXAMPLE 3: Cross-reactivity profile of antibodies Unique ing hits were ed in small scale as described in Example 2 and tested in an ELISA for binding to different FGFR-variants listed in Table 3. _ ’78 _ Table 3: List of recombinant proteins used in ELISA for cross—reactivity profiling of binder Protein Origin Cat. No. (RnD Systems) hFGFRZB—Fc (lllb) Human 665-FR mFGFR2B-Fc (Illb) Murine hFGFR2B-Fc (Illc) Human hFGFR] B-Fc (lllc) Human 661~FR hFGFRl B-Fc (lllb) Human 765-FR hFGFR3-Fc (lllc) Human 766—FR hFGFR3-Fc (11113) Human 1264—FR hFGFR4-Fc Human 685—MF mFGFR2B—Fc (l [lc) Murine 716-MF mFGFR3-Fc (Illc) Murine 710-MF hTRAlL—Fc Human 63 0-TR All variants used were present as Fc—fusion proteins in carrier free ations.
Proteins were biotinylated using an approximately 2-fold molar excess of biotin-LC-NHS e; Cat. No. 21347) according to cturer's instructions and desalted using Zeba desalting columns e; Cat. No. .
For the ELISA 96-well plates pre-coated with streptavidin (Pierce, 15500) were coated over night at 4°C with lug/ml biotinylated protein. Wells coated with biotinylated TRAIL-Fe served as a reference. The next day plates were washed 3 times with PBST, treated with blocking reagent, and washed again 3 times with PB ST. 100 ul of purified Fabs (1 ug/ml) were added and incubated for 1 h at r.t.. After washing 3 times with PB ST, an anti-hIgG (Fab-specific) d to HRP 0 diluted; Sigma; A 0293) was added and incubated for 1 h at r.t.. Color reaction was developed by addition of 50 ul TMB (lnvitrogen, 2023) and stopped after 5-15 min by adding 50 111 H2804 (Merck, 1120801000). Colorimetric reaction was recorded at 450 nM in a plate reader (Tecan). _ ’79 _ Wells containing TRAIL—Fe were used as background values and the signal to background ratios were calculated as summarized in Table 4.
Table 4: Summary of ELISA-data on cross-reactivity of antibodies a a s E} $3 $3. a Li? a; a. L? 22? meamams Ea 5,22 2 mg; La: La: LL: E: E: LL: Lu: LAMA‘EEM 0v 0v owns/Lave o 04: 0,2 mvo LL 0 LL 0 LL 0 o 0 LL 0 Lu 0 ELL ELY-1 L w LL >4 0 LL and an an am swab/4:: ELLE—1 M048— +++ +++ +++ +++ 0 0 0 0 0 0 M017— +++ +++ +++ +++ 0 0 0 M02 1— +++ +++ +++ +++ 0 0 0 M054- ++ ++ +++ ++ 0 0 0 M047— +++ +++ +++ ++ 0 0 0 M054- + + ++ + 0 0 0 Signal to background ratios: 0: <2; +: 2-3; ++z 3-5; +++z >5 As shown in Table 4 the dies of this ion bind to human and murine FGFR2 independent of alpha and beta as well as Illb and lllc splice form. The antibodies of this invention do not bind to FGFRl, FGFR3, and FGFR4 as shown in Table 4.
EXAMPLE 4: Binding of FGFRZ antibodies to cell surface of cancer cell lines To determine the binding teristics of the anti-FGFR2 antibodies on mouse, rat and human cancer cell lines, binding was tested by flow cytometry to a panel of cell _ 80 _ lines. Adherent cells were washed twice with PBS (without Ca and Mg) and detached by enzyme-free PBS based cell dissociation buffer (lnvitrogen). Cells were suspended at approximately 105 well in FACS buffer (PBS without Ca/Mg, Biochrom containing 3% FCS, Biochrom). Cells were centrifuged (250g, 5min, 4°C) and supernatant discarded.
Cell were resuspended in dilutions ofthe antibodies ofinterest (5 ug/ml in 80ul if not indicated otherwise) in FACS buffer, and incubated on ice for 1h. 1n the ing cells were washed once with 100ul cold FACS buffer and 80p] secondary antibody diluted at 1:150 (PE goat anti-human IgG, Dianova 15-098, or PE Goat Anti-Mouse IgG, Jackson Immuno Research #115-1 15-164) was added. After incubation for 1h on ice cells were again washed with cold FACS buffer, resuspended in 100ul FACS buffer and analyzed, by flow cytometry using a FACS-Array (BD ences). Results are calculated as Geo Mean of the detection by the antibody of interest subtracted by background fluorescence as measured by detection with the secondary antibody alone. Values are scored according to the following system: Geo Mean - Geo Mean of secondary antibody alone >10: +, >100: ++, >1000: +++, 10000: ++++, close to ry border in 0.
List of cell lines used, for cross—reactivity ng of antibodies: SNU16 ATCC—CRL—5974 KATOlII ATCC-HTB-103 NCI—N87 CRL—5822 _ HS746T NCI-60 Panel, Lot 507285 MFM223 ECACC-98050130 ATCC-CRL-2539 ATCC—CRL-2755 ATCC—HTB—l l3 ATCC-CRL—2923 MFEZSD ECACC-98050131 RUCA PE Asterand, Source Steven Ethier _ 81 _ As shown in Table 5, all anti FGFR2 antibodies of this ion used at a concentration of Sug/ml bind a broad range of tumor cells expressing FGFR2 of murine (4T1, EMT6), rat (RUCA) and human (all other cell lines included in the table) origin.
Table 5: Binding of anti FGFR2 antibodies Sag/ml to different cell lines by scoring of FACS analysis Gastric cancer cells Breast cancer cells NCISNU16 N87 HS746T MFMZZ3 _E—MT6 MOI7-BOZ—hIgGl +++ M021—H02-hIgG1 M048-D01—hIgGl E- Endometrial cancer cells ECCl MFE296 MFE280 AN3CA .l:_021—H02—hlgG1 + + + 1 M048—D01—hIgG1 —_ 1(1) 11 Mo4v-nos1M111 _-—_ (Geo Mean-Geo Mean of secondary antibody alone >10: +, >100: ++, >1000: +++, >10000: ++++, close to category border in 0) To determine the EC50 values for binding of dies to selected cancer cell lines, cells were stained with FGFR2 dies as described above, but with various concentrations of antibodies ranging from 01-100 nM. ECso values were determined using Graph Pad Prism Software and are presented in Table 6. Three antibodies with highest affinity (MOl7-B02-hIgGl, 01-hIgGl, M047-D08-hIgGl) show subnanomolar to low nanomolar EC50 values in human (SNU—l 6, MFM223), murine (4T1) and rat (Ruca) cell lines. M021-HOZ-hlgGl, 05-hIgGl and M054—D03-hIgGl show also low nM 2012/073325 _ 82 _ cellular EC50 values in murine and human cell lines. Thus, all tested antibodies are cross reactive in binding to human, murine and, rat cells expressing FGFR2.
Table 6: EC50 values of anti FGFR2 antibodies binding to cell lines of human (SNUI 6, MFM223), murine (4T1) and rat (RUCA) origin analyzed by FACS ECsu [HM] 0.1 0.2 ' ' .8 n.d. . . 0.1 0.9 M054-A05-hIgG1 . .
M054-D03-hIgG1 MO47-D08—hIgG1 (n.d. stands for not determined/measured) EXAMPLE 5: Epitope g by Pepscan’s Chemically Linked Peptides 0n Scaffolds (CLIPS) technology To determine the binding characteristics of the antibodies found, an intensive epitope mapping based on Pepscan’s proprietary Chemically Linked, Peptides on Scaffolds (CLIPS) technology (Timmerman et al., J. Mol. it. 2007, 20:283-99) was performed. In total 8653 different CLIPS peptides of 15AA and 30AA length covering linear, conformational and discontinuous epitopes on the native human FGFRZ were designed. The peptides were synthesized on peptide arrays. Antibodies of this invention were tested on the e arrays in human IgG1 format in an based assay. The es that gave the highest ELISA values were analyzed to fy shared similar amino acid ces.
To reconstruct discontinuous epitopes of the target molecule a library of structured peptides was synthesized. This was done using Pepscan’s proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology (Timmerman et al., J. Mol. Recognit. 2007, :283-99). CLIPS technology allows to structure peptides into single loops, -loops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates are _ g3 _ coupled to cysteine residues. The hains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the T2 CLIPS template 1,3-bis (bromomethyl) benzene was dissolved in um bicarbonate (20 mM, pH 7.9)facetonitrile (1 :1(v/v)). This solution was added onto the peptide arrays. The CLIPS template bound to side-chains of two cysteines as present in the solid-phase bound peptides of the peptide-arrays (455 wells plate with 3 ul wells). The e arrays were gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays were washed extensively with excess of HzO and ted in disrupt- buffer containing 1 percent SDS/0.1 percent beta-mercaptoethanol in PBS (pH 7.2) at 70°C for 30 minutes, ed by sonication in HgO for another 45 minutes. The T3 CLIPS carrying peptides were made in a similar way but now with three cysteines.
The binding of antibody to each peptide was tested in a PEPSCAN—based ELISA (Slootstra et al., Molecular Diversity 1996, 1: 87-96). The e arrays were pre- incubated with 5% to 100%-binding buffer (1 hr, 200C). The binding buffer was composed of 1% Tween-80, 4% horse-serum, 5% Ovalbumin (w/v) and was diluted with PBS. After washing the peptide arrays were incubated with primary dy on (1 to 5 ug/ml) in PBS containing 1% Tween-80 (overnight at 4°C). After washing, the peptide arrays were incubated with a 1/1000 on in 100% binding buffer of an antibody peroxidase conjugate for one hour at 25OC (anti-human). After washing, the peroxidase substrate 2,2’- azino-diethylbenzthiazoline sulfonate (ABTS) and 2 iter/milliliter of 3 percent H202 were added. After one hour, the color development was measured. The color development was quantified with a charge d device (CCD) - camera and an image processing system.
Data processing The raw data are optical values obtained by a CCD-camera. The values range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The binding values were ted for analysis. Occasionally, a well contains an air-bubble resulting in a false- _ g4 _ positive value, the cards were manually inspected and any values caused by an air—bubble were scored as 0.
All antibodies of this invention bind to the same epitope, which comprises of the N- terminal residues of FGFRZ (IRPSFSLVEDTTLEPEIS). Analysis of 1257 CLIPS and linear peptides showed tent high ELISA values for N—terminal peptides.
The N—terminal residues (1RPSFSLVEDTTLEPE15) are present in all splice variants of human FGFRZ independent of alternative splicing in D3 resulting in IIIb and, IIIc isoforms (see Figure 1). The epitope is also present if domain D1 is spliced out of the full length FGFR2 (SEQ ID NO:61; FGFRZ alpha) resulting in the shorter beta form of FGFR2 (SEQ ID N0262). In this case the e is directly in front of domain D2 (see Figure 1).
Of special interest is that the N—terminal sequence is conserved in human, mouse, rat and macaca inulatta. This enables broad inter species cross reactivity.
This new epitope is outside the well-known ligand binding site and the heparin g site (see Figure l) and results in novel features of the antibodies of this invention.
EXAMPLE 6: Epitope fine mapping by Alanine scanning of peptides To define the binding characteristics ofthe antibodies ofthe ion in more detail an e-scanning was performed. As described in example 5, peptides of lSAA and 30 AA lengths were synthesized and each amino acid of the human FGFR2 sequence was replaced for a n peptide by an Alanine residue. Binding of the antibodies was analyzed as described in Example 5. Ifthe exchange of an amino acid residue for an Alanine results in a icant reduction of the binding signal, this residue was accounted as critical for the binding.
Table 7 shows for the antibodies of this ion the critical residues in the N- terminal part (IRPSFSLV’EDTTLEPEIS) of FGFRZ. _ 85 _ Table 7: Critical residues in the inal part SLVEDTTLEPEIS) of FGFR2 for binding of antibodies of this invention position I L) W J; (A 0‘ J W \O >4 O >4 H H N >—a L2) #4 J‘— ,_. U1 M017- X X X X 1302 M021- X M047- X X X X M048— X X X M054- X X X X M054- X X (Residues being critical for binding are marked by an (X). By changing this residue into an Alanine more than 50% of the ELISA signal is lost) Antibodies M048-D01 and MOZI-HOZ are of special interest because they are binding independently of variations at position Ser-S. This enables them to bind in addition to human, mouse, rat and macaca mulatta FGFR2 (SEQ ID NO:63) to rabbit (SEQ ID , pig (SEQ ID N0265) and dog (SEQ ID NO:66) FGFRZ making it possible to use even more species for preclinical development.
EXAMPLE 7: Affinity of antibodies for the N-terminal epitope analyzed by Biacore To define the binding affinities for the N—terminal peptides characterized as epitopes Biacore e plasmon resonace experiments were performed.
Binding affinities of anti FGFR2 dies were determined by surface plasmon resonance analysis on a Biacore T100 instrument (GE Healthcare Biacore, Inc.). Antibodies as human IgG1 were immobilized onto a CMS sensor chip through an indirect capturing reagent, anti-human IgG(Fc). Reagents from the “Human Antibody Capture Kit” (BR 39, GE Healthcare Biacore, Inc.) were used as described by the manufacturer.
Approximately 5000 RU monoclonal mouse uman IgG (Fc) antibody were _ g6 _ immobilized per cell. Anti FGFR2 antibodies were injected at a concentration of 5 ug/ml at lOul/min for 10 sec. Various concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.12 nM) in HEPES-EP buffer (GE Healthcare Biacore, Inc.) of es derived from the first 15 amino acids of FGFR2 of ent species (human, mouse, rat, macaca mulatta FGFR2 (SEQ ID , rabbit (SEQ ID NO:64), pig (SEQ ID NO:65) and dog (SEQ ID NO:66)) were injected over immobilized anti FGFR2 antibodies at a flow rate of 60 uL/min for 3 minutes and the dissociation was allowed for 5 minutes.
Sensograms were generated after in—line reference cell correction followed by buffer sample subtraction. The dissociation equilibrium nt (K3) was calculated based on the ratio of association (km) and dissociation rated (koff) constants, ed by fitting sensograms with a first order 1:] binding model using Biayaluation re (version 4.0).
M048-D01-hIgG1 and M047-D08-hIgG1. bind with a KD value around 100 nM human, murine, rat and macaca mulatta FGFR2 (for details see Table 8). As supported by the Alanine-scanning M048-D01 showed nearly the same KD value for all peptides derived from several species (see Table 8).
Table 8: Monovalent KD values of antibodies M048-D01 and M047—D08 as measured by Biacore with 15 aminoacid long peptides.
N—terminal peptide of M048-D01—hIgG1 M047-D08—hIgG1 species human, mouse, rat, macaca 105 nM mulatta [SEQ ID NO:63] rabbit [SEQ ID N0264j 88 nM no binding pig [SEQ ID N0265j 70 nM no binding dog [SEQ ID N0266] 72 nM no binding _ g7 _ EXAMPLE 8: Stimulation of P-FGFRZ (phosphorylated FGFRZ) levels after short term incubation with anti FGFRZ dies on FGFRZ overexpressing cell lines To determine the effect of anti FGFRZ antibodies on cellular levels of phosphorylated FGFR2 (P-FGFR2) after short term incubation, P-FGFR2 ELISAS were performed. MFM223 cells were plated at 7000 cells per well in growth medium (MEM Earle (Biochrom; F0315) + 10% FCS + 2mM Glutamin) in 96well plates. 24h after plating cells were incubated with dies (lop/ml) for 15min, followed by two washing steps with PBS and lysis in 100ul of cold lysis buffer ting of 50mM Hepes pH 7,2, 150mM NaCl, 1mM MgClz, 10mM 7, 100mM NaF, 10% Glycerin, 1.5% Triton X-100 and freshly added te Protease Inhibitor cocktail (Roche No. 1873580001), 4mM Na3V04, pH adjusted to 7.4 with NaOH by shaking for 5 min. Samples were shock frozen and stored at -80°C until analysis. Measurement of P-FGFRZ levels was carried out using a P-FGFRZ ELISA kit from R&D s according to the cturer’s instructions. OD was measured at 450 nM (Tecan Spectra, Rainbow) with background correction. Levels of P-FGFRZ were ated as % of untreated control levels. To control for non-specific effects of the antibody , parallel samples were incubated with non-cell binding control IgGs of the same isotype.
Results are shown in Figure 2 and, indicate a pronounced induction of P-FGFR2 levels by anti FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1. In contrast neither the control IgG dy nor anti FGFR2 antibodies commercially available from R&D (MAB665, MAB684, 3) show any significant effect on P-FGFRZ levels after short—term incubation. These results reveal an agonistic effect of anti FGFR2 antibodies described within this invention on FGFRZ after short-term incubation.
EXAMPLE 9: Desensitizing of FGFR2 overexpressing cells against stimulation of P- FGFRZ by FGF7 after long-term incubation with anti FGFRZ antibodies To determine the effect of anti FGFR2 antibodies on cellular levels of phosphorylated FGFRZ (P—FGFRZ) after long term incubation and the effect of antibody _ 88 _ treatment on the power of FGF7 to induce FGFRZ phosphorylation, P—FGFR2 ELISAS were performed. MFM223 cells were plated at 7000 cells per well in growth medium (MEM Earle (Biochrom; F0315) + 10% FCS + 2mM Glutamin) in 96well plates. 24h after plating cells were incubated with antibodies (10u/ml) for 24min, followed by incubation in the presence or absence of FGF7 (R&D Systems, 25ng/ml) for 15 min. Cells were washed twice with PBS and lysed in lysis buffer consisting of (50mM Hepes pH 7,2, 150mM NaCl, 1mM MgClg, 10mM Na4P207, 100mM NaF, 10% Glycerin, 1.5% Triton X-100, freshly added te Protease Inhibitor il (Roche No. 1873580001), 4mM Na3V04, pH adjusted to 7.4 with NaOH) and shaking for 5min at room temperature. s were snap frozen and stored at -800C until is by the P-FGFR2 ELISA from R&D according to the manufacturer’s instructions. Optical density was measured at 450 nM (Tecan Spectra, Rainbow) with background tion. Levels of P-F GFR2 were calculated as % of untreated control levels. To control for non-specific effects of the antibody format, parallel s were incubated with non-cell g control IgGs 0f the same isotype.
Corresponding results are presented in Figure 3. In cells treated without antibody treatment as well as in cells treated with isotype control IgG stimulation with FGF7 lead to an about 4 fold se of P-FGFR2 . In contrast, in samples pretreated with anti FGFR2 antibodies for 24h, FGF7 only induced P-FGFR2 levels by 1.37-1.4 fold.
Taken together these results show that prolonged incubation of cells with anti FGFRZ antibodies of this invention leads to desensitization towards stimulation with FGF7.
EXAMPLE 10: Downregulation of FGFRZ surface expression after incubation of cell lines with anti FGFRZ dies To analyze FGFR2 surface expression after treatment with anti FGFR2 antibodies FACS analysis was carried out in different cell lines with FGFR2 overexpression 3, SNU16) or FGFR2 mutation (AN3-CA, MFE-296). Adherent cells were washed twice with PBS (without Ca and Mg) and detached by enzyme-free PBS based cell dissociation buffer (Invitrogen). Cells were suspended at 0.5 >"105 cells/well in 80p] growth _ 89 _ medium (MFM223, MFE—296: MEM Earle (Biochrom; F0315) + 10% FCS + 2mM Glutamin, SNU—16: RPMI 1640 (Biochrom, FG]215) + 10% FBS; AN3-CA2MEM Earle (Biochrom; ) + 10% FCS + 1mM pyruvate +1x NEA: non essential amino acids Biochrom K0293). 20ul of 5 fold concentrated antibody dilution was added (final concentration of 10ug/ml) and incubated for 4.511 at 37°C. After the end of the incubation time cells were washed once with 100ul FACS buffer, stained with detection dy (at Sug/ml, mouse GFRZ for hlgGs, human anti-FGFR2 for mIgGs) for 45min at 40C, followed by an additional wash with 100ul FACS buffer. PE-Stained secondary antibody (PE goat anti-human IgG, Dianova #109098, or PE Goat Anti-Mouse IgG, Jackson lmmuno Research #1 15-] 15-164, 1:150 diluted) was added in 80ul volume, incubated for 45min at 40C and after an additional wash with FACS buffer cells analyzed by flow cytometry using a FACS array (BD Biosciences). In control experiments antibody competition for overlapping epitopes was excluded by parallel incubation with the antibody of interest and the corresponding detection antibody. Geo-Means measured after staining with secondary dies alone were subtracted from Geo-Means from peaks detected, after staining with anti FGFR2 antibodies. Results are calculated as % of Control cells that were ted for 4.5h without the presence of antibodies. s are depicted in Figure 4. Incubation of cells with control IgG leads to no decrease of FGFRZ e expression, whereas anti FGFR2 antibodies M048-D01-hIgG1 and M047-D08-hIgG1 downregulated FGFR2 surface levels significantly by 39-60% in all 4 cell lines independent of FGFRZ overexpression or mutation. In contrast no other anti FGFR2 dy either commercially available from R&D (MAB665, MAB684, MAB6843) or described ere for e (GAL-FR21, GAL-FRZZ; W02010/054265 and Zhao et al. (Clin Cancer Res. 20] 0,16:5750-5758)) showed FGFR2 surface downregulation in all 4 cell lines independent of FGFR2 overexpression or ons.
GAL-FRZ] downregulated FGFR2 surface levels in cell lines with FGFR2 amplification (SNU16 and MFM223), but had no impact on cell lines with FGFRZ mutation. GAL-FR22 reached 73 and 21% downregulation of FGFR2 surface expression in FGFR2 mutated cell lines (AN3-CA and 6 respectively), but had no significant impact on surface 2012/073325 FGFR2 levels in SNU16 and MFM223 cells. MAB684 and MAB6843 again induced around 60% reduction of FGFRZ surface levels in FGFR2 mutated cell lines without major effects on FGFRZ overexpressing cell lines. Finally, MAB665 did not show any impact on FGFR2 surface levels at all.
To summarize, anti FGFR2 antibodies MO48-D01-hIgG1 and M047-D08-h1gG1 are the only anti FGFR2 antibodies ng FGFR2 surface downregulation in cancer cell lines independent of FGFR2 pression or on.
EXAMPLE 1]: Donwregulation of total FGFRZ levels after long-term incubation of cancer cells with anti FGFRZ antibodies To analyze whether FGFRZ surface downregulation induced by anti FGFR2 antibodies leads to long-term decrease in total FGFR2 levels, total n levels of FGFR2 were analyzed by FGFRZ ELISA. SNU16 cells were plated at 5000 cells/well in 96well plates in growth medium (RPMI 1640 (Biochrome, FG1215) + 10% FBS). 2h later cells were incubated with anti FGFRZ antibodies at various trations as indicated or corresponding isotype control IgG. 96h after start of incubation with the antibodies cells were centrifuged for 5min at 300g at room ature, washed twice in PBS and lysed by addition of 100ul lysis buffer (50mM Hepes pH 7,2, l50mM NaCl, lmM MgClz, 10mM Nangov, 100mM NaF, 10% Glycerin, 1.5% Triton X-100, freshly added Complete Protease Inhibitor cocktail (Roche No. 1873580001), 4mM Na3V04, pH adjusted to 7.4 with NaOH) and shaking for 5min at room temperature. Samples were snap frozen and stored at -80°C until analysis using the Total-FGFRZ-ELISA Ki (R&D Systems) ing to the manufacturer’s instructions. Optical density was measures at 450 nM (Tecan Spectra, Rainbow) together with background correction. To calculate absolute levels of total FGFR2 standard, curve using isolated FGFRZ n was applied according to the manufacturer’s recommendations (R&D Systems). Results are depicted as % of FGFR2 levels measured in control cells that were incubated for 96h in the absence of antibody. _ 91 _ Results are ted in Figure 5. Incubation with anti FGFR2 antibodies of this invention for 96h leads to a reduction of total FGFR2 levels by 41-55%. Half maximal reduction is reached at doses of 3ug/ml of the anti FGFR2 antibodies. In contrast, incubation with isotype control antibody has no effect on total FGFR2 levels.
Taken er, these results indicate that anti FGFR2 dies M048-D01-hIgGl and 08-hIgGl do not only lead to a short term decrease in surface FGFR2 levels but also a long term reduction of total FGFR2 levels.
E 12: alization of anti FGFR2 antibodies into cells Anti FGFR2 antibodies ofthis invention were analyzed, for their capability to alize after binding to the FGFR2 antigen.
To visualize this process the FGFR2 specific antibodies M048-D01-hIgGl and M047-D08-hIgGl and an isotype control antibody were selected. The antibodies were conjugated in the presence of a two molar excess of CypHer 5E mono NHS ester (batch 357392, GE Healthcare) at pH 8.3. After the conjugation the reaction mixture was dialyzed (slide-A—Lyser Dialysis Cassettes MWCD lOkD, Fa. Pierce) overnight at 4°C to eliminate excess dye and, adjusting the pH-value. Afterwards the protein solution was concentrated (VIVASPIN 500, Fa Sartorius stedim biotec). In addition to the pH-dependent fluorescent dye CypHerSE the ph-independent dye Alexa 488 was used. The dye load of the antibody was determined with a spectrophotometer (Fa. NanoDrop). The dye load of 01— hIgGl and M047-D08-hIgGl and the isotype control (M014) were in a similar range. The affinity of the labeled antibodies was tested in a cell binding-assay to ensure that labeling did not alter the binding to FGFR2. These d antibodies were used in the following internalization assays. Prior to treatment cells (2x104/well) were seeded in 100ul medium in a 96-MTP (fat, black, clear bottom No 4308776, Fa. d Biosystems). After 18 h incubation at 37°C/5%C02 medium was changed and labeled anti FGFR2 antibodies M048-D01-hIgGl and M047-D08-hIgGl were added in various concentrations (10, 5, 2.5, l, 0.3, 0.1 ug/ml). The identical treatment was carried out with the isotope control antibody _ 92 _ ive control). The incubation time was chosen to be 0, 5h, 1h, 2h, 3h, 6h and 24h. The fluorescence measurement was performed with the InCellAnalyzer 1000 (Fa. GE Healthcare). Granule counts and total fluorescence intensity were measured in a kinetic A highly specific and significant alization of M048-D01-hIgGl and M047- DOS-hIgGl was observed in endogenous FGFR2 expressing cancer cell lines SNU16 (gastric cancer) and SUMSZPE (breast cancer).
This alization was target dependent as uptake could only be demonstrated using the anti FGFRZ antibodies while no internalization was ed with the isotype controls. During the first 6h the anti FGFR2 antibodies showed a 20fold increase of antibody internalization compared, to isotype controls. Isotype control showed a minor internalization after a long exposure (>24h).
Internalization of anti FGFR2 antibodies labeled with Alexa 488 upon binding reveals that more than 50% ofinternalized antibodies seem to follow the endocytotic pathway.
In Figure 6 a copic evaluation of the time course of specific internalization of M048-D01-hIgGl and M047-D08-hIgGl upon g to endogenous FGFRZ expressing cells is shown. Internalization of antibodies (2.5 ug/ml) was investigated on breast cancer cell line SUM SZPE. Granule counts were measured in a kinetic fashion.
Rapid internalization could be observed for 01-hIgGl and M047-D08-hlgG1, whereas the isotype control hlgGl does not internalize.
A more detailed evaluation of the trafficking pathway was performed with co- staining of small G-proteins. Rab GTPases regulate many steps of membrane c, including vesicle formation, vesicle movement along actin and tubulins networks, and membrane fusion. To guish n different pathways two Rab proteins were selected for staining - Rab7, which is expressed in late endosomes and lysosomes and Rab 1], which is expressed in early and recycling endosomes. After a 6h internalization of _ 93 _ labeled dies the cells were fixed and perrneabilized with ol prior to staining with Rab 7- and Rab 11- antibodies. The results are shown in Figure 7. 01-hlgGl and M047-D08-hIgGl show a significant co-staining with Rab 7, whereas the co-staining with Rab 11 is only minor. These results indicate that after internalization of FGFR2 the complex enters the mal - lysosomal pathway.
The staining pattern for other described antibodies like GAL-FRZl and GAL-FR22 (W02010/054265 and Zhao et al. (Clin Cancer Res. 2010,16: 758)) looks completely different. Here almost no staining could be detected with Rab7, but a major co- staining was ed with Rabll. This indicates that these antibodies internalize after binding to the FGFR2 receptor and favor the recycling pathway EXAMPLE 13: Test of anti FGFRZ antibodies of this invention in experimental tumors in mouse model In vivo efficacy ofthe anti FGFR2 antibodies of this invention was for example tested via subcuteanous neic or allogeneic tumor models. The expert knows prior art methods in order to proof for efficacy of the innovative antibodies. For e, mice were therefore subcutaneously ated with tumor cells, which express the target FGFR2.
Afterwards, tumor-bearing mice were either treated with FGFRZ-targeting antibodies of this invention, non-binding isotype control or phosphate-buffered saline (PBS). Application of dies was carried out intraperitoneally or intravenously two times weekly. In order to test for additive anti-tumor efficacy, the FGFRZ Abs of this invention were combined with common standard of cares and compared to the single agent efficacies. Tumor growth was monitored by frequent measurement of tumor area via a caliper. After tumor growth and treatment for some weeks, tumors were harvested and tumor weights or tumor sizes (tumor area calculated by the formula length x width) of animals treated with the anti FGFRZ antibodies of this invention were compared to those treated with PBS or isotype control antibodies. Mice treated with the anti FGFRZ dies of this invention displayed significantly smaller tumors.
WO 76186 _ 94 _ Human or murine tumor cells that express FGFR2 were subcutaneously ated onto the flank of immunocompromised mice, for example Nude- or SClD-mice. Per mouse 0.25-10 n cells were detached from cell culture flasks, centrifuged and suspended, in 100 pl PBS, 50% medium/ 50% Matrigel, or 100% Matrigel, respectively. Cells were than inoculated subcutaneously beneath the skin onto the flank of mice. In case of patient- derived tumor models, tumors harvested from gastric cancer patients were subcutaneously passaged on immunocompromised mice. For g efficacy of the anti FGFR2 antibodies, tumor pieces ofa defined size (2 x 2 mm) were subcutaneously transplanted onto the flank of mice. Within a couple of days a tumor was established. Treatment started earliest if tumors reached a size of 20 mm2 (cell line-derived tumors) or 100 mm3 (patient-derived tumors), whereby tumor area (mml) was calculated by the formula length x width and, tumor volume (mm3) by the formula length x widch/ 2.. Treatment with the antibodies was performed either intraperitoneally or intravenously via tail vein injection. Antibodies were either solved in PBS or 50 mM Na—acetat, 150 mM NaCl. Antibodies were d in a volume of 10 ml/kg. Treatment schedule was based on the pharmacokinetic behavior of the antibody. As standard, antibodies were applied twice weekly (alternating every third and fourth day). As standard, treatment was performed until control group reaches the maximal possible tumor size. Alternatively, treatment was stopped earlier. As standard, 8 mice per treatment group were used. Number of mice per treatment group can be increased, if higher variations in tumor growth were expected. In parallel to the treatment , a l group was treated with PBS following the same treatment schedule. During the study, tumor area was frequently ed by measuring length and width of tumors using a caliper. At study end, tumors were harvested and weighed. Ratio of mean tumor weights of the antibody-treated groups (T) and, mean tumor s of the control (C) was stated as T/C. If treatment and, control groups were terminated at different time points or tumor weight could not been used as read-out since tumors became necrotic, T/C ratios were calculated based on tumor area of the last common ement time point. 2 Mio human gastric cancer SNU—16 cells in 50% medium / 50% Matrigel were aneously inoculated onto the flank of female nodSCID mice. Intraperitoneal _ 95 _ treatment with the anti—FGFR2 antibodies started when tumors reach a mean size of 20—30 mm2 and was continued twice weekly until study end. If tumors of control group reached the maximal acceptable size, study was terminated and tumors are harvested and weighed.
All tested anti FGFR2 antibodies of this invention reduced significantly tumor growth as compared to l. Treatment with a dose of 2 mg/kg of M017-B02-hIgG1, M021-H02-hIgG1, M048-D01-hIgG1, M054-A05-hIgG1, M054-D03-hlgG1 and M047- D08-hIgG1 resulted in T/Cs of 0.19, 0.22, 0.17, 0.19, 0.21 and 0.22, respectively (see Figures 8 to 13). 2.5 x 105 murine 4T1 breast cancer cells were subcutaneously inoculated in 100% PBS onto the flank ofNMRI nu/nu mice. compromised instead of syngeneic mice were chosen in order to avoid the development of neutralizing dies against the human IgG protein. Treatment of tumors d at the time point at which tumors have reached a mean size of 24 mmZ. In order to test for possible additive anti-tumor efficacy of M048- DOl-hIgGl mice were either treated with M048-D01-hIgG1, Lapatinib or Taxol, respectively, alone and in combination with M048—D01-hIgGl and Taxol or Lapatinib. As control, mice were treated with PBS alone. Treatment with M048-D01-hIgG1 was carried out twice weekly intravenously (i.v.), nib once daily per os (p.o.) and Taxol once weekly intravenously. All treatments were performed until end of the study. Since tumors became necrotic at the end of the study, tumor area at day 13 after tumor cell inoculation was used to determine anti-tumor efficacy. This study revealed that combination of M048- D01-hIgG1 with either ninb or Taxol achieved additive anti-tumor cy: Monotherapy with Lapatinib and Taxol, respectively did not significantly changed growth of tumors as compared to the vehicle control, while M048-D01-hIgG1 alone resulted in significant reduction as compared to vehicle with a T/C of 0.73. Combination with Lapatinib and Taxol reduced this T/C down to 0.58 and 0.52, both statistically significant versus both monotherapies (see Figures 14 and 15). 2 x 2 mm pieces of originally patient—derived gastric tumors, 608 and GC12-0811 (Prof. Huynh Hung, al University of ore (NUS)), passaged on immunocompromised mice, were aneously transplant e d 0 nt 0 fe m ale _ 96 _ immunocompromised nai‘ve mice. Tumor size was assessed frequently using a caliper measuring the tumor in two ions and, tumor volume was calculated by the formula length x widthz/ 2. ent with different doses of M048-D01-hIgG1 was started at the time point at which tumors reached a mean size of approximately 100 mm3. Treatment was performed intravenously twice weekly with doses of 5, 2 and 1 mg/kg M048-D01-hIgG1. In a tumor model with high FGFR2 protein sion (GC10-0608), all three doses resulted in significant reduction of tumor growth resulting in T/C values based on final tumor weight of 0.55, 0,60 and 0.41 (see Figure 16). In a model with markedly lower FGFR2 protein expression (GC12-081 1), 5 and 2 mg/kg of M048-D01—hIgG1 resulted in significant reduction of tumor weight resulting in T/Cs 0.70 and 0.67 (see Figure 17). In accordance with the lower FGFR2 expression, 1 mg/kg of M048-D01-hIgG1 did not result in significant ion of final tumor weight. For the treatment of two other tumor models, the breast cancer model MFM223 and the ctal cancer model NCI—H716 (ATCC-CCL- 251) we have not found an appropriate application scheme to reduce tumor growth cantly.
EXAMPLE 14: Downregulation of P-FGFR and total FGFR2 levels in xenograft tumors after treatment with anti FGFR2 antibodies To analyze whether the observed downregulation oftotal FGFR2 levels and concurrent reduction in 2 is also seen in xenograft tumors in vivo, SNU—16 tumors after treatment with anti FGFR2 antibodies were analyzed by Western Blot. Tumors were collected at the end of a aft experiment in NOD/SCID mice, treated with anti FGFR2 antibodies 2mg/kg i.p. twice weekly (see Example 13 for details). Tumors were taken 24h after the last injection of the antibodies, snap frozen in liquid nitrogen and stored at -800C until analysis. Prior to Western Blot analysis frozen tumors were cut in slices of around 5mm diameter and each slice deposited in a 2m] Eppendorf tube together with a precooled 5mm steel bull (Qiagen) and 500111 lysis buffer (50mM Hepes pH 7.2, 150mM NaCl, lmM MgClg, IOmM N34P207, 100mM NaF, 10% Glycerin, 1.5% Triton X-100, freshly added Complete Protease Inhibitor cocktail (Roche No. 1873580001), 4mM NagVO4, pH adjusted _ 97 _ to 7.4 with NaOH). Samples were lysed for 3 min at 300Hz in a lyzer n) followed by incubation on ice for 30min. In the following, samples were centrifuged for 10min at 13000 rpm at 4°C in a Micro-centrifuge (Eppendorf) and supernatants from slices coming from one original tumor pooled back together. Protein levels in the tumor lysates were determined by using the BCA protein assay kit (Novagen, lysates 1:50 diluted in HgO).
Samples were diluted to a final concentration of 5mg/ml and 50ul of sample were mixed with 7.7 ul of (10*) Sample Reducing agend and 19.2111 (4*) NuPAGE Sample Buffer (Invitrogen). s corresponding to 115ug of n were applied to NuPage 4-12% SDS page gels from Invitrogen and run for 2h45min at 120V. Blotting was carried out by an iBlot system (Invitrogen) according to the cturer’s recommendations. Membranes were d for 2h at room temperature in 5% BLOT QuickBlocker in PBST (Invitrogen), followed by incubation with primary antibodies over night at 4°C. Primary dies were as follows: : #AF3285, R&D Systems, 0.5ug/m; total FGFRZ: M017-B02-hIgG1, 4ug/ml in in 3% BLOT QuickBlocker in PBST. On the next day membranes were washed three times in PBST, followed by incubation with secondary antibodies (Peroxidase- conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch #111 003 or Peroxidase-conjugated AffiniPure Goat Anti-Human IgG + IgM (H+L) (Jackson ImmunoResearch #109127, l : 10000 in 3% BLOT QuickBlocker/PBST) for 211 at room temperature. Subsequently, membranes were washed four times for 10 min with PBST and signals were detected by chemoluminescence after incubation with ECL reagent.
To detect the loading control, membranes were stripped with stripping solution strong (1:10 in Milipore-H20) for 15 min shaking at room temperature, followed by blocking and detection with Anti-Actin antibody #A2066 ) 1 21000 in 3% QuickBlocker/PB ST.
Representative results from 2 animals per group treated with anti FGFR2 antibodies are shown in Figure 18 side by side with samples from animals treated with l IgG.
Total FGFRZ as well as P-FGFR levels were strongly reduced after treatment with anti FGFR2 antibodies of this invention. Thus, the mode of action of downregulation of total FGFR2 bed in the in Vitro studies is also relevant in aft tumors after treatment with anti FGFR2 antibodies of this invention. _ 98 _ EXAMPLE 15: Subcutaneous Xenograft cancer Model with dy drug conjugates: Anti FGFR2 antibodies can be conjugated to cytotoxic small molecules using protocols that are known in the art (e.g. Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618— 8623). A431 cells are maintained as adherent cultures in DMEM supplemented with 10 % FBS. NOD SCID or other immunocompromised mice of 6-7 weeks age will be inoculated aneously in the right flank with 1-5 x 10e6 cells in 0.1 ml of medium. When tumor sizes reach ca. 25 mm2 antibody drug conjugates will be stered intraperitoneal 3X every 4, 7 or 10 days at a dose of 1-10 mg/kg. Control mice will be treated with PBS or an vant monoclonal antibody conjugated with the same toxophore, Tumor size will be measured twice weekly with a sliding caliper. Anti-tumor efficacy will be evaluated by comparing tumor size of anti FGFR2 antibody drug conjugate treatment versus control treatment.
EXAMPLE 16: Generation of matured variants of ed antibodies with improved affinities Anti FGFR2 antibodies of this invention discovered by phage display as depicted in Table 9 were further optimized by affinity maturation.
Table 9: Sequences of antibodies discovered by phage display a) a) O O O O O O O z: 05 0‘5 05 z_ zN 2m 2 ZN z 23:) 23 2;) 22 mm mm mm a; om m2 me me m3 m8 LL]: mm mm LUL) LuA mg m m> LUH-t Lug Antibody U1 U) (/2 mg m m m> (11 (11> (11> M017—B02 7 8 9 10 l 2 3 4 M021 —HO2 17 18 19 2O 11 12 13 __ 4;. 27 28 [\J \O 30 21 37 38 34 =. M054A05 56 57 60--- _ 99 _ Antibody affinity maturation is a two-step process where tion mutagenesis and well-based high throughput screening are combined to fy a small number of mutations resulting in affinity increases. In the first round of affinity maturation positional diversification of ype antibody was introduced by site-directed mutagenesis using NNK-trinucleotide cassettes (whereby N ents a 25% mix each of adenine, thymine, guanine, and cytosine nucleotides and K represents a 50% mix each of thymine and guanine nucleotides) according to BMC Biotechnology 7: 65, 2007. This way, all 20 amino acids are introduced at an individual amino acid position. This positional ization is cted to the six complementarity determining regions (CDRs). In the second round of affinity maturation beneficial substitutions were recombined and screened for further improvements. Examples of such variants are ed in Table 10.
Table 10: Sequences of variant antibodies derived from M048—D01 and M047-D08, respectively ‘3 <5 c5 :5 6 <5 <5 5 .5 C5 .9 C5 .5 C5 ,5 E ZEZEZQZEZQZQZL; 22 25-2 22 ,5 Antibody 959395999929 95 BE 95 SE a: or: 0:: on 05 0/3 03 on 0/4 0/15 0;) > LU m m m m m m > m > m E m ,4 DO U) m U) m U) U) U) m U) TPP-1403 75 76 77 78 79 80 73 74 71 72 1131341397 85 86 87 88 89 90 83 84 81 82 g TPP-1398 95 96 97 98 99 100 93 94 91 92 g! TPP-1399 105 106 107 108 109 110 103 104 101 102 E TPP—1400 115 116 117 118 119 120 113 114 111 112 01 125 126 127 128 129 130 123 124 121 122 TPP-1402 135 136 137 138 139 140 133 134 131 132 TPP-1415 145 146 147 148 149 150 143 144 141 142 TPP-l406 155 156 157 158 159 160 154 151 152 TPP~1407 165 166 167 168 169 170 164 161 162 E, TPP—1408 175 176 178 179 180 174 171 172 a; TPP-1409 185 186 188 189 190 184 181 182 TPP—1410 195 196 198 199 200 194 191 192 TPP-141] 205 206 207 208 209 210 204 201 202 TPP-1412 215 216 217 218 219 220 214 211 212 — 100 — Two different types of ELISA were used to determine the binding improvement of mutated variants: a) e Binding ELISA: a synthetic peptide comprising the amino acid sequence of the epitope linked C-terminally to a biotinylated lysine VEDTTLEPEG-Ttds- Lys(Biotin) (peptide sequence derived from SEQ ID N0263, synthesized by JPT Peptide logy GmbH, Berlin, Germany), and b) Recombinant Protein Binding ELISA: recombinant human FGFR2 (DNA sequence of human FGFR2 ( NP7000132.3 ) Met 1 - Glu 377, fused with a polyhistidine tag at the C— terminus; # 10824-IIO8H, Sino Biological Inc., Beijing, China).
Briefly, in both ELISA s MTP plates (3 84well Maxisorp, Nunc) were coated with 20 pl, anti-human IgG Fc specific (# I2136; sigma) at 2.2 11le for 2.5 h at 37°C in coating buffer (# 121125 Candor ence GmbH). After one washing step using 50 ul PBST (phosphat buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM NagHPO4, 2 mM KHgPO4, pH 7.4, 0.05 % Tween20), plates were blocked with 50 pl of 10 % Smart Block (# 1 13500, Candor Bioscience GmbH) for 1h at 20 — 22 °C and the washing step was repeated 3 times. Anti-FGFRZ variants were immobilized in concentrations of 0.035 ug/ml de based assay) or 0.2 ug/ml (recombinant human FGFR2 protein based assay) in 10 % Smart Block in PBST depending on the format and ts to be analyzed by incubation of 20 ul for one hour at 20 - 22 0C. After one washing step using 50 ul PBST, 20 ul quadruplets of the antigen dilution series in 10 % SmartBlock in PBST with a maximum concentration of 100 nM were added and incubated for 111 at 20 - 22 OC and the washing step was ed 3 times. For the detection of the biotinylated epitope e 20 pl of streptavidine / POD conjugate (# $5512, Sigma) in a 1:1000 dilution in 10 % SmartBlock in PBST were applied for one hour at 20 - 22 0C. For the ion of the recombinant FGFR2 protein 20 ul of anti-His / HRP conjugate (# 71840, novagen) in a 1210000 dilution in 10 % SmartBlock in PBST were applied for one hour at 20 ~ 22 0C. After 3 washing steps 20 ul of 10 uM amplex red substrate (# A12222, Invitrogen) in 50 mM Sodium hydrogen phosphate, pH 7.6, were added and the fluorescence signal was detected using a common fluorescence reader, e.g. Tecan M1000. ECSO values were evaluated by fitting the data (Sigmoidal dose- response, variable slope, bottom set to background; GraphPad Prism software).
Provided in Table 10 are l es of variants with amino acid substitutions generated in the heavy and light chains of M048-D01 (TPP-1403). All variants showed strong improvement in antigen binding evaluated in two ELISA formats with different forms of antigen compared to the non CDR changed variant (Table 11).
Provided in Table 10 are several examples of variants with amino acid substitutions ted in the heavy and light chains of M047-D08 (TPP-1415). All variants showed significant improvement in antigen binding compared to the non CDR changed variant (Table 11).
The ences between both formats regarding the numeric results, ECSO and the factor of improvement, were unexpected but can be likely explained by the use of antigen in peptide or protein form and differences in the formation of the finally detected enzyme conjugate on top of the ELISA sandwich: The K13 of the anti-His—HRP ate and the His-tagged FGFR2 is not known, however it is very likely, that it is magnitudes of orders higher than the KD of biotin and streptavidin (IO-15 M) utilized in the detection of the epitope e. Consequently the sensitivity for the bound e is significant higher than the sensitivity for the His-tagged protein g to the potential of determining smaller ECSO. In addition or alternatively the ences may be caused by ions in the interaction of the anti-FGFRZ antibody with both antigens despite their identical sequence over a h of 15 amino acids; firstly the chemistry of the C-terminal following part Ofthe molecules is very different, secondly the 15 amino acids might take a 3D conformation not identical to the corresponding region in the FGFRZ protein. Both explanations could refer to the differences between the peptide and protein based ELISA showing smaller ECSO values in the peptide ELISA format.
The data sets in Table 11 y indicate that M048-D01 (TPP-1403) binds FGFR2 at its N—tenninal sequence as represented in the epitope e, and that several variants 2012/073325 — 102 — with amino acid substitutions in the CDRs surprisingly do the same even with higher y. Notably, the substitution N102I is present in five of the six other variants of TPP— 1403 accompanied by several other substitutions in CDR-L] -H2 and/or -H3, but , -L2, -L3, not in TPP-l399 showing surprisingly a lysine (K) at position HC-102.
The data sets in Table 11 indicate that M047-D08 (TPP-1415) binds FGFR2 at its N—terminal sequence as represented in the epitope peptide, and that l variants with amino acid substitutions in the CDRs surprisingly do the same even with higher affinity.
Variants of M047-D08 (TPP-1415) with multiple amino acid substitutions showed approximately four- to forty-fold improved binding, TPP-1409 least (2.1 nM) and TPP- 1406 (0.22 nM) most. y three of them have a G102L (TPP-1406, —1407 and -1412) and one a G102V 408) substitution accompanied by several other substitutions in , -L2, —L3, -H1 and/or H3 Table 11: Peptide binding ELISA results, n binding ELISA results and internalization efficacy data of variant antibodies derived from M048-D01 and M047-D08, respectively of Intemalization Peptide Binding Protein Binding Variant 6fficacy fold ECSO fold ECSO fold reduction reduction ement TPP-l397 TPP-1398 M048—D01 TPP—l399 TPP-1400 0.009 TPP-1401 0.006 >l700 —==..>1500 022 24 III-TPP1406 022 09 TPP—1408 TPP-1409 TPP-1410 TPP—1411 In addition in Table 11 the improvements of internalization efficacy of ted anti FGFRZ antibodies are summarized. The improvement factor is calculated based on — 103 — comparison of total granule intensity/cell achieved by internalization and degradation of maturated antibodies to the corresponding value of the parental antibody. Equal findings are achieved by comparison of granule count/cell resulting in the identical ranking of dies. Experimental details are described in Example 12. Notably all matured variants of M048-D01 (TPP-1403) showed an improved internalization efficacy (1.9 to 2.4 fold). In case of M047-D08 (TPP-1415) variant TPP-1412 showed a 1.5 fold improved internalization efficacy. Internalization is an important feature of the antibodies of this ion.
With the variants provided for M047-D08 and M048-D01 it could clearly be trated that variants of these antibodies can have similar or improved properties if the e is maintained.
EXAMPLE 17: Determination of competition with other GFRZ antibodies To analyze the competition between anti-FGFR2 antibodies ing to the invention and anti-FGFR2 antibodies described in the art, different antibodies were evaluated in a competitive ELISA format: MTP plates ll Maxisorp, Nunc) were coated, with 20 ul of 2 itng anti- human IgG (Fc specific (# 12136; sigma) in coating buffer (# 121125 Candor Bioscience GmbH) at 4°C over night. After one washing step using 50 ul PB ST (phosphat buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 0.05 % Tween20), plates were d with 50 ul of 100 % Smart Block (# 113500, Candor Bioscience GmbH) for 111 at 20 - 22 OC and the washing step was repeated 3 times. M048— DOl-hIgGl was immobilized in a trations of 1 ptle in 10 % Smart Block in PBST by incubation of 20 ul for one hour at 20 - 22 OC (indicated in Table 12; row 2 with M048- gGl capture yes); control wells without M048-D01—hIgG1 were incubated with 10 % Smart Block in PBST only (indicated in Table 12; row 2 with M048-D01-hIgGl capture no). The immobilization step was followed by three washing steps using 50 ul PBST. 20 ul quadruplets of the pre-incubated (1h at 20 - 22 0C) antigen / antibody mix composed by — 104 — recombinant human FGFRZ, 10 nM (#10824-H08H, SinoBiological) and anti—FGFR2 IgG in a 5-fold dilution series (1000 to 0.064 nM) in 10% lock in PBST were added and incubated for 1h at 20 - 22 OC, followed by three washing steps.
For the detection of the recombinant human FGFR2 20 ul of is / HRP conjugate (# 71840, novagen) in a 1210000 on in 10 % SmartBlock in PBST were applied for one hour at 20 - 22 0C. After 3 washing steps 20 ul of 10 uM amplex red substrate 22, Invitrogen) in 50 mM Sodium hydrogen phosphate, pH 7.6, were added and the fluorescence signal was detected using a common fluorescence reader, e. g.
Tecan M1000.
Three different FGFR2 binding antibodies called Z] GAL-FR22 and GAL-FR23 (described in W02010/054265 and Zhao et a1. (Clin Cancer Res. 2010,1625750- 5758)) have been described to bind to different domain epitopes. For evaluation of ence with these antibodies competition assays were performed.
Due to the different es of the analyzed dies the competition ELISA format has to ensure an equally and, directly comparable detection of the ition situation without superposition of additional effects due to use of different detection antibodies or different affinities of a single detection dy to the different IgG-isotypes.
The ELISA format described above fulfills this criterion by detection of the FGFR2 antigen via its His-tag instead of the detection of bound mouse or human IgGl or IgG2a. The immobilization of M048-D01-hIgG1 is specific with respect to its human Fc portion, otherwise significant amounts of FGFR2 would have been detected in ELISA plate wells coated with uman IgG (Fc specific), but not ed with M048-D01-hIgG1; a potential binding of mouse anti-FGFRZ-IgG to anti-human IgG (Fc specific) and subsequent binding of FGFR2 was not detected (Table 12, columns 8 - 11). Additionally, no significant unspecific binding of FGFR2 to the immobilized anti-human IgG (Fc specific) was observed (column 2). The “self-competition” of M048-D01-hIgG1 worked very clearly (column 6), and the same is true for M048-D01-mIgG2a, (column 7). The observation, that neither GAL-FRZl, -FR22 nor -FR23 showed dose dependent reduction in detectable FGFR2 (column 3 i 5) as M048-D01-hIgG1 and M048-D01-mIgG2a did, with an WO 76186 — 105 — >50 % decreased signal at 1.25 and 0.63 nM and higher concentrations of competing antibody, respectively, demonstrates the differences between M048-D01 and the three GAL antibodies. In contrast, after pre-incubation the monomeric FGFR2 (10 nM) with GAL— FR22 and GAL-FR23, but not with GAL-FR21, the amount of detectable FGFR2 appeared to be significantly increased. Since the GAL antibodies are neither fused to a His—Tag, checked by Western analysis, nor captured by the anti-human IgG (Fc specific), the most likely explanation is, that monomeric FGFRZ can be dimerized by the pre—incubation with antibodies leading to an avidity effect in the subsequent binding of FGFR2 to the lized 01-hIgG]. The situation of immobilized M048-D01-hIgG] bound directly to FGFRZ and ed by this indirectly to the dimerizing antibodies GAL-FR22 and GAL-FR23 would further rate, that M048—D01-hIgG1 binds to a te different FGFR2 e than the GAL antibodies, otherwise a simultaneous binding event could not occur. Notably, GAL—FRZI did not increase the amount of detectable FGFRZ.
This difference can be plausibly interpreted by taking the more particular description of the GAL antibodies as described in WO2010/054265 into account: Gal-FR22 binds to an epitope in IIa, and GAL-FR23 binds to one all or partly located in D1; both regions represented in the used recombinant human FGFRZ-IIIc molecule. But for GAL-FRZI the epitope is described to be d in D3—Hlb, a sequence stretch not represented in this FGFRZ-ch m; consequently GAL-FR21 is not able to bind the antigen and mediate an avidity effect. As shown, in none of the assays competition between M048-D01-hIgG1 and one of the GAL antibodies was observed. - 106 — Table 12: Antibody Competition ELISA. The e signals are given in relative to the corresponding value for 10 nM FGFR2 determined in the calibration series (column 1) column 1 2 3 4 5 6 7 8 9 10 ll M048— DOl-hIgGl capture yes no yes yes yes yes yes no no no no E a. s e. g E c, g , e. s 8 RD E m m a: . - m . N 3:3, is g_ a: a: n: L: e L: L: L: L? : : : 148% 3% 1000 77% 198% 219% 3% 3% 3% 3% 3% 5% 1 0 100% 2% 200 77% 250% 265% 4% 3% 2% 2% 2% 3% 51% 2% 40 77% 284% 297% 20% 5% 1% 2% 2% 3% 2. 5 23% 2% 8 87% 287% 294% 30% 10% 2% 2% 2% 3% I . 25 12% 2% I i 6 91% 248% 222% 44% 18% 2% 2% 2% 3% 0.63 6% 2% 0.32 81% 167% 151% 63% 34% 1% 2% 1% 3% 0.31 18% 2% 0.06 76% 92% 106% 81% 60% 2% 2% 2% 3% 0.16 4% 3% 0 79% 84% 93% 93% 82% 3% 3% 3% 5% The results of ition experiments, as described above, are supported by the observation, that all three GAL antibodies including GAL-FR23 (epitope all or partly located in D1) show no g to the synthetic peptide of the extracellular N-terminal epitope of FGFR2 (SEQ ID N0263) comprising the amino acid sequence of the epitope C- terminally linked to a biotinylated lysine SLVEDTTLEPEI5G-Ttds-Lys(Biotin)) even in the highest concentration in the IgG titration series applied (600 nM), whereas the strong binding of M048-D01-hlgGl (detected, by anti-human IgG (Fc specific) POD conjugate; # A5175, sigma) and M048-D01-mIgG2a, resulted in EC50 in the range $1 nM (detailed data not shown). For the detection of mouse antibodies ouse IgG (Fc ic) POD conjugate (# 71515, jakson) was used, checked positively for its ability to detect GAL-FR21, -FR22, -FR23 and M048-D01-mIgG2a bound to FGFR2-Mb alpha.

Claims (32)

1. An isolated antibody or antigen-binding fragment thereof specifically g to the extracellular N-terminal epitope (1RPSFSLVEDTTLEPE15) of FGFR2 as presented by SEQ ID NO:63.
2. An isolated antibody or n-binding fragment thereof according to claim 1 wherein binding of the dy to the extracellular N-terminal epitope (SEQ ID NO:63) is mediated by at least one epitope residue selected from the group of residues consisting of Arg 1, Pro 2, Phe 4, Ser 5, Leu 6, and Glu 8.
3. An isolated antibody or n-binding fragment thereof according to any one of claims 1 - 2 wherein the antibody or antigen-binding nt thereof loses more than 50% of its ELISA signal by changing of at least one of the amino acid residues in the N-terminal e (1RPSFSLVEDTTLEPE15) of FGFR2 into an Alanine a. said residue selected from the group Pro 2, Leu 6 and Glu 8, or b. said residue selected from the group Arg 1, Pro 2, Phe 4 and Ser 5.
4. The antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment competes in binding to FGFR2 with at least one antibody selected from the group “M048-D01” comprising a variable heavy chain region corresponding to SEQ ID NO: 31 and a variable light chain region ponding SEQ ID NO: 32, “M047- D08” comprising a variable heavy chain region corresponding SEQ ID NO: 21 and a variable light chain region corresponding SEQ ID NO: 22, “M017-B02” comprising a variable heavy chain region corresponding to SEQ ID NO: 1 and a variable light chain region corresponding to SEQ ID NO: 2, “M021-H02” comprising a variable heavy chain region corresponding SEQ ID NO: 11 and a le light chain region corresponding to SEQ ID NO: 12, “M054-A05” sing a variable heavy chain region corresponding to SEQ ID NO: 51 and a variable light chain region corresponding to SEQ ID NO: 52, “M054-D03” comprising a variable heavy chain region corresponding to SEQ ID NO: 41 and a variable light chain region ponding to SEQ ID NO: 42, “TPP-1397” comprising a variable heavy chain region corresponding to SEQ ID NO: 83 and a variable light chain region corresponding to SEQ ID NO: 84,, “TPP-1398” comprising a variable heavy chain region corresponding to SEQ ID NO: 93 and a variable light chain region ponding to SEQ ID NO: 94, “TPP-1399” comprising a variable heavy chain region corresponding to SEQ ID NO: 103 and a variable light chain region corresponding to SEQ ID NO: 104, “TPP-1400” comprising a variable heavy chain region corresponding to SEQ ID NO: 113 and a variable light chain region corresponding to SEQ ID NO: 114, “TPP-1401” sing a variable heavy chain region ponding to SEQ ID NO: 123 and a variable light chain region ponding to SEQ ID NO: 124, “TPP-1402” comprising a variable heavy chain region corresponding to SEQ ID NO: 133 and a variable light chain region corresponding to SEQ ID NO: 134, “TPP-1403” comprising a variable heavy chain region corresponding to SEQ ID NO: 73 and a variable light chain region corresponding to SEQ ID NO: 74, “TPP- 1406” comprising a variable heavy chain region corresponding to SEQ ID NO: 153 and a variable light chain region corresponding to SEQ ID NO: 154, “TPP-1407” comprising a variable heavy chain region corresponding to SEQ ID NO: 163 and a variable light chain region corresponding to SEQ ID NO: 164, 408” comprising a variable heavy chain region corresponding to SEQ ID NO: 173 and a variable light chain region corresponding to SEQ ID NO: 174, “TPP-1409” sing a variable heavy chain region corresponding to SEQ ID NO: 183 and a variable light chain region corresponding to SEQ ID NO: 184, “TPP-1410” comprising a variable heavy chain region corresponding to SEQ ID NO: 193 and a variable light chain region corresponding to SEQ ID NO: 194, 411” comprising a variable heavy chain region corresponding to SEQ ID NO: 203 and a variable light chain region corresponding to SEQ ID NO: 204, 412” comprising a variable heavy chain region corresponding to SEQ ID NO: 213 and a variable light chain region corresponding to SEQ ID NO: 214, and “TPP-1415” comprising a variable heavy chain region corresponding to SEQ ID NO: 143 and a variable light chain region corresponding to SEQ ID NO: 144, as depicted in tables 9 and 10, respectively.
5. The antibody or antigen-binding fragment ing to claim 1 to 4 comprising a. the variable heavy chain CDR ces as presented by SEQ ID NO: 5-7 and the le light chain CDR sequences presented by SEQ ID NO: 8-10, or b. the variable heavy chain CDR sequences as presented by SEQ ID NO: 15-17 and the le light chain CDR sequences presented by SEQ ID NO: 18-20, or c. the variable heavy chain CDR sequences as presented by SEQ ID NO: 25-27 and the variable light chain CDR sequences presented by SEQ ID NO: 28-30, or d. the variable heavy chain CDR sequences as presented by SEQ ID NO: 35-37 and the variable light chain CDR sequences presented by SEQ ID NO: 38-40, or e. the variable heavy chain CDR sequences as presented by SEQ ID NO: 45-47 and the variable light chain CDR sequences presented by SEQ ID NO: 48-50, or f. the variable heavy chain CDR sequences as presented by SEQ ID NO: 55-57 and the variable light chain CDR sequences presented by SEQ ID NO: 58-60, or g. the variable heavy chain CDR sequences as presented by SEQ ID NO: 75-77 and the le light chain CDR sequences presented by SEQ ID NO: 78-80, or h. the variable heavy chain CDR sequences as presented by SEQ ID NO: 85-87 and the variable light chain CDR sequences presented by SEQ ID NO: 88-90, or i. the variable heavy chain CDR ces as presented by SEQ ID NO: 95-97 and the variable light chain CDR ces presented by SEQ ID NO: 98-100, or j. the variable heavy chain CDR sequences as presented by SEQ ID NO: 105-107 and the variable light chain CDR sequences presented by SEQ ID NO: 108-110, or k. the variable heavy chain CDR sequences as presented by SEQ ID NO: 115-117 and the variable light chain CDR sequences presented by SEQ ID NO: 118-120, or l. the variable heavy chain CDR sequences as presented by SEQ ID NO: 125-127 and the variable light chain CDR sequences presented by SEQ ID NO: 128-130, or m. the variable heavy chain CDR sequences as presented by SEQ ID NO: 135-137 and the variable light chain CDR ces presented by SEQ ID NO: 138-140, or n. the variable heavy chain CDR sequences as presented by SEQ ID NO: 145-147 and the le light chain CDR sequences presented by SEQ ID NO: 148-150, or o. the variable heavy chain CDR sequences as ted by SEQ ID NO: 155-157 and the le light chain CDR sequences presented by SEQ ID NO: 158-160, or p. the le heavy chain CDR sequences as presented by SEQ ID NO: 165-167 and the variable light chain CDR sequences presented by SEQ ID NO: 168-170, or q. the variable heavy chain CDR sequences as presented by SEQ ID NO: 175-177 and the variable light chain CDR sequences presented by SEQ ID NO: 178-180, or r. the variable heavy chain CDR sequences as ted by SEQ ID NO: 185-187 and the variable light chain CDR sequences presented by SEQ ID NO: 188-190, or s. the variable heavy chain CDR sequences as presented by SEQ ID NO: 195-197 and the variable light chain CDR sequences presented by SEQ ID NO: 198-200, or t. the variable heavy chain CDR sequences as presented by SEQ ID NO: 205-207 and the variable light chain CDR sequences presented by SEQ ID NO: 208-210, or u. the variable heavy chain CDR sequences as presented by SEQ ID NO: 215-217 and the variable light chain CDR sequences presented by SEQ ID NO: 218-220.
6. The antibody or antigen-binding fragment according to claims 1 - 5 comprising a. a variable heavy chain ce as ted by SEQ ID NO:1 and a variable light chain sequences as ted by SEQ ID NO:2, or b. a variable heavy chain sequence as presented by SEQ ID NO:11 and a variable light chain sequences as presented by SEQ ID NO:12, or c. a variable heavy chain sequence as presented by SEQ ID NO:21 and a variable light chain sequences as presented by SEQ ID NO:22, or d. a variable heavy chain ce as presented by SEQ ID NO:31 and a variable light chain sequences as presented by SEQ ID NO:32, or e. a le heavy chain sequence as presented by SEQ ID NO:41 and a variable light chain sequences as ted by SEQ ID NO:42, or f. a variable heavy chain sequence as presented by SEQ ID NO:51 and a variable light chain sequences as presented by SEQ ID NO:52, or g. a le heavy chain sequence as presented by SEQ ID NO:73 and a variable light chain sequences as presented by SEQ ID NO:74, or h. a variable heavy chain sequence as presented by SEQ ID NO:83 and a variable light chain sequences as presented by SEQ ID NO:84, or i. a variable heavy chain sequence as presented by SEQ ID NO:93 and a variable light chain ces as presented by SEQ ID NO:94, or j. a variable heavy chain sequence as presented by SEQ ID NO:103 and a variable light chain sequences as presented by SEQ ID NO:104, or k. a variable heavy chain sequence as presented by SEQ ID NO:113 and a variable light chain sequences as presented by SEQ ID NO:114, or l. a variable heavy chain sequence as presented by SEQ ID NO:123 and a variable light chain sequences as presented by SEQ ID NO:124, or m. a variable heavy chain sequence as presented by SEQ ID NO:133 and a variable light chain sequences as presented by SEQ ID NO:134, or n. a variable heavy chain sequence as presented by SEQ ID NO:143 and a variable light chain sequences as presented by SEQ ID NO:144, or o. a variable heavy chain sequence as presented by SEQ ID NO:153 and a variable light chain sequences as presented by SEQ ID NO:154, or p. a variable heavy chain sequence as presented by SEQ ID NO:163 and a le light chain ces as presented by SEQ ID NO:164, or q. a variable heavy chain sequence as ted by SEQ ID NO:173 and a variable light chain sequences as presented by SEQ ID NO:174, or r. a variable heavy chain sequence as presented by SEQ ID NO:183 and a variable light chain sequences as presented by SEQ ID NO:184, or s. a le heavy chain sequence as presented by SEQ ID NO:193 and a variable light chain sequences as ted by SEQ ID NO:194, or t. a variable heavy chain ce as presented by SEQ ID NO:203 and a variable light chain sequences as presented by SEQ ID NO:204, or u. a variable heavy chain ce as presented by SEQ ID NO:213 and a le light chain sequences as presented by SEQ ID NO:214.
7. The antibody according to any one of the preceding claims, which is an IgG antibody.
8. The n-binding fragment according to any one of the preceding claims, which is an scFv, Fab, Fab’ fragment or a 2 fragment.
9. The antibody or antigen-binding nt according to any one of the preceding , which is a monoclonal antibody or antigen-binding fragment.
10. The antibody or antigen-binding fragment according to any one of the preceding claims, which is human, humanized or chimeric antibody or antigen-binding fragment.
11. An antibody-drug conjugate, comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 10.
12. An isolated nucleic acid sequence that encodes the antibody or antigen-binding fragment according to any one of claims 1 to 10.
13. A vector comprising a nucleic acid sequence according to claim 12.
14. An isolated cell expressing an antibody or antigen-binding fragment according to any one of the claims 1 to 10 and /or comprising a nucleic acid according to claim 12 or a vector according to claim 13.
15. An isolated cell according to claim 14, wherein said cell is a prokaryotic or an eukaryotic cell.
16. A method of producing an antibody or antigen-binding nt according to any one of the claims 1 – 10 comprising culturing of a cell according to claim 14 and purification of the antibody or antigen-binding fragment.
17. An antibody or n-binding fragment according to claims 1 – 10 or an antibody-drug conjugate according to claim 11 as a medicament.
18. An antibody or antigen antigen-binding nt according to any one of claims 1 – 10 as a diagnostic agent.
19. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to claims 1 – 10 or an antibody-drug conjugate according to claim 11.
20. A combination of a pharmaceutical composition ing to claim 19 and one or more therapeutically active compounds.
21. The use of an antibody or antigen-binding nt according to any one of claims 1-10, an antibody-drug ate according to claim 11, a pharmaceutical composition according to claim 19 or a combination according to claim 20, in the manufacture of a medicament for treating a disorder or condition associated with the undesired presence of FGFR2.
22. The use of an antibody or antigen-binding fragment according to any one of claims 1 – 10, an antibody-drug ate according to claim 11, a pharmaceutical composition according to claim 19 or a combination ing to claim 20, in the manufacture of a medicament for the treatment of cancer.
23. An antibody or antigen-binding fragment according to claim 1, substantially as herein described or ified.
24. An antibody-drug conjugate according to claim 11, substantially as herein described or exemplified.
25. A nucleic acid sequence according to claim 12, ntially as herein described or exemplified.
26. A vector according to claim 13, substantially as herein described or exemplified.
27. An isolated cell according to claim 14, substantially as herein described or exemplified.
28. A method according to claim 16, substantially as herein described or exemplified.
29. A pharmaceutical composition according to claim 19, substantially as herein described or exemplified.
30. A combination according to claim 20, ntially as herein described or exemplified.
31. A use according to claim 21, ntially as herein described or exemplified.
32. A use according to claim 22, substantially as herein described or exemplified.
NZ624534A 2011-11-23 2012-11-22 Anti-fgfr2 antibodies and uses thereof NZ624534B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP11190227.6 2011-11-23
EP11190227 2011-11-23
PCT/EP2012/073325 WO2013076186A1 (en) 2011-11-23 2012-11-22 Anti-fgfr2 antibodies and uses thereof

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NZ624534A NZ624534A (en) 2016-06-24
NZ624534B2 true NZ624534B2 (en) 2016-09-27

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