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Researchers Submit Patent Application, "Anti-Angiogenic Therapy", for Approval

July 14, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors STASSEN, Jean-Marie (Lubbeek, BE); CARMELIET, Peter (Bladen, BE); COLLEN, Desire (Winksele, BE), filed on February 3, 2014, was made available online on July 3, 2014 (see also Patents).

No assignee for this patent application has been made.

News editors obtained the following quote from the background information supplied by the inventors: "Abnormal blood vessel formation contributes to the pathogenesis of numerous diseases with high morbidity and mortality. Elucidation of the mechanisms underlying vascular growth might allow the development of therapeutic strategies to stimulate vascular growth in ischemic tissues or to suppress their formation in tumours. Recent gene targeting studies in embryos have identified some of the mechanisms involved in the initial formation of endothelial channels (angiogenesis) and their subsequent maturation by coverage with smooth muscle cells (arteriogenesis). Evidence is emerging that distinct molecular mechanisms may mediate growth of blood vessels during pathological conditions, but the molecular players remain largely undetermined

"It has been established that Vascular Endothelial Growth Factor (VEGF) is implicated in development and pathological growth of the vasculature (Ferrara N. et al, 1999, Curr Top Microbiol Immunol 237, 1-30). Furthermore, it has also been shown that Placental growth factor (P1GF), a homologue of VEGF, is a specific modulator of VEGF during a variety of pathological conditions, such as ischemic retinopathy, tumourigenesis, inflammatory disorders, and oedema. It has been shown that P1GF.sup.-/- mice have an impaired angiogenesis and arteriogenesis in disease (Carmeliet P. et al., 2000, J. Pathol. 190, 387-405), while the physiological angiogenesis in normal health remains unaffected. Thus inhibitors of P1GF have a huge potential for the treatment of diseases in which angiogenesis or arteriogenesis contribute to the pathogenicity of the disease.

"Inhibitors for P1GF are known in the art, such as a goat polyclonal antibody against human P1GF (R&D pharmaceuticals, Abingdon, UK) and a chicken polyclonal antibody (Gassmann et al., 1990, Faseb J. 4, 2528). Those antibodies are used for Western blotting, histochemistry and immunoprecipitation studies. WO01/85796 describes the use of inhibitors of P1GF, including monoclonal anti-P1GF antibodies, for the treatment or prevention of diseases, such as tumour formation. More specifically, the preparation of murine monoclonal antibodies which fully inhibit murine P1GF-2 binding to its receptor Flt-1, is described, whereby the antibody Mab-PL5D11, is selected as having the most efficient inhibitory activity. Use of the antibody in animal models of pathological angiogenesis is described.

"Antibodies generated in animals have characteristics which may severely limit their use in human therapy. As foreign proteins, they may elicit an anti-immunoglobulin response (which for mouse antibodies is referred to as human anti-mouse antibody or HAMA) that reduces or destroys their therapeutic efficacy and/or provokes allergic or hypersensitivity reactions in patients, as taught by Jaffers et al., 1986 (Transplantation 1986 41:572). While the use of human monoclonal antibodies would address this limitation, it has proven difficult to generate large amounts of human anti-human antibodies by conventional hybridoma technology. Recombinant technology has therefore been used in the art to construct 'humanized' antibodies that maintain the high binding affinity of animal, such as murine monoclonal antibodies but exhibit reduced immunogenicity in humans. In particular, chimeric antibodies have been suggested in which the variable region (V) of a non-human antibody is combined with the constant (C) region of a human antibody. Methods of obtaining such chimerical immunoglobulins are described in detail in U.S. Pat. No. 5,770,198. In other attempts to reduce the immunogenicity of murine antibodies, only the complementarity determining region (CDR), i.e. regions of hypervariability in the V regions, rather than the entire V domain, are transplanted to a human antibody. Such humanized antibodies are known as CDR-grafted antibodies. The construction of CDR-grafted antibodies recognizing more complex antigens has resulted in antibodies having binding activity significantly lower than the native non-humanized antibodies. In numerous cases it was demonstrated that the mere introduction of non-human CDRs into a human antibody backbone is insufficient to maintain full binding activity. While a refined computer model of the murine antibody of interest is required in order to identify critical amino-acids to be considered in the design of a humanized antibody, and general theoretical guidelines were proposed for such design, in all cases the procedure must be tailored and optimized for the particular non-human antibody of interest.

"Subsequently, there remains a need for (monoclonal) antibodies which optimally inhibit human P1GF binding to its receptor. Furthermore, such antibodies also need to be non-immunogenic, in that they can not elicit HAMA (or have a low tendency to do so)."

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "The present invention relates to novel monoclonal antibodies and derivatives thereof directed to P1GF and capable of inhibiting the binding of P1GF to its receptor. The present ligands are the first to demonstrate inhibition of human P1GF in a pathological condition in vivo. More specifically, the antibodies and derivatives thereof according to the present invention are capable of reducing tumor size and vascularization of human tumor tissue in vivo. The antibodies of the present invention provide an alternative to anti-angiogenic therapies targeting VEGF currently used, with the important advantage that the side-effects caused by inhibition of physiological angiogenesis associated with these therapies is significantly reduced.

"The present invention relates to antigen-binding molecules, particularly monoclonal antibodies, fragments or derivatives thereof, including humanized antibodies and antibody fragments, which bind to the same epitope of P1GF as the antibody referred to herein as 16D3.

"A first object of the present invention is the provision of novel monoclonal antibodies capable of binding to P1GF and having the capacity to inhibit the functioning of P1GF, more specifically antibodies characterized in that their heavy chain variable region comprises the sequence of SEQ ID NO: 2 or a sequence having at least 80%, more particularly at least 90%, most particularly 95% sequence identity therewith within the CDR regions and/or in that their light chain variable region comprises the sequence of SEQ ID NO: 4 or a sequence having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity therewith within the CDR regions, such as antibody 16D3 or derivatives thereof. Additionally or alternatively, in a further embodiment, the antigen-binding molecules of the present invention have a sequence identity within the heavy and/or light chain variable regions outside the CDR regions which is at least 70%, particularly at least 80%, more particularly at least 90%, most particularly at least 95% identical with the sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

"According to a particular embodiment of the invention, the antibody is a humanized antibody, more particularly a hybrid antibody, most particularly a mouse/human hybrid antibody, more particularly a hybrid mouse 16D3/human IgG1.kappa. or IgG4.kappa.. Alternatively the humanized antibody is one which comprises the CDR regions of the mouse 16D3 antibody of the present invention capable of binding to P1GF, grafted onto the backbone of a human antibody.

"A further embodiment of the present invention relates to antigen-binding fragments of the mouse 16D3 antibody or a derivative thereof, such as a humanized antibody thereof, such as but not limited to an Fab, Fab' or F(ab').sub.2, a combination of at least two complementarity determining regions (CDRs), a soluble or membrane-anchored single-chain variable region, or single variable domain. Particular embodiments of such antigen-binding fragments include fragments which comprise at least two CDRs of 16D3 or derivatives thereof or more particularly at least two CDRs selected from the group consisting of SEQ ID NO: 17 (GYTFTDYY), SEQ ID NO: 18 (IYPGSGNT); SEQ ID NO:19 (VRDSPFFDY), SEQ ID NO: 20 (QSLLNSGMRKSF), SEQ ID NO: 21 (WAS) and SEQ ID NO: 22 (KQSYHLFT), or which comprise at least two sequences having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity therewith. A particular embodiment of the invention relates to the provision of single-chain variable fragments (scFvs) of the mouse 16D3 antibody and humanized scFvs which are capable of inhibiting P1GF activity. Most particularly the present invention provides scFvs comprising the amino acid sequence of SEQ ID NO:24 or SEQ ID NO: 26 or a sequence having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity thereto within the CDRs capable of binding P1GF.

"Yet a further object of the present invention is the provision of cell lines producing the monoclonal antibodies of the present invention, more particularly cell line 16D3 which produces the 16D3 antibody, but also other cell lines which are capable of producing the antigen-binding molecules derived from 16D3 or fragments thereof, for instance as a result of recombinant technology.

"Yet a further object of the present invention is the provision of a pharmaceutical composition for the prevention or treatment of (undesired) angiogenesis in pathological conditions or disorders in mammals or for the prevention or treatment of bone resorption, which comprises an antibody against P1GF which is 16D3 or a fragment or derivative, more particularly a humanized version of 16D3 or an antigen-binding fragment thereof in admixture with a pharmaceutically acceptable carrier. A specific embodiment of the invention is a pharmaceutical composition which comprises an antigen-binding fragment of 16D3 or a derivative thereof, which is selected from the group consisting of an Fab, Fab' or F(ab')2, a soluble or membrane-anchored single-chain variable part or a single variable domain. Most particular embodiments of the present invention relate to pharmaceutical compositions comprising an antigen-binding fragment which comprises at least two CDRs selected from the group consisting of SEQ ID NO: 17 (GYTFTDYY), SEQ ID NO: 18 (IYPGSGNT); SEQ ID NO:19 (VRDSPFFDY), SEQ ID NO: 20 (QSLLNSGMRKSF), SEQ ID NO: 21 (WAS) and SEQ ID NO: 22 (KQSYHLFT), or at least two having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity with two different sequences selected from the group consisting of SEQ ID NO: 17 to 22. A particular embodiment of thereof relates to pharmaceutical compositions comprising a scFv of the 16D3 antibody of the invention, more particularly comprising a scFV comprising at least two CDRs selected from the group consisting of SEQ ID NO: 17 (GYTFTDYY), SEQ ID NO: 18 (IYPGSGNT); SEQ ID NO:19 (VRDSPFFDY), SEQ ID NO: 20 (QSLLNSGMRKSF), SEQ ID NO: 21 (WAS) and SEQ ID NO: 22 (KQSYHLFT) or at least two sequences having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity with two different sequences selected from the group consisting of SEQ ID NO: 17 to 22, such as a scFv comprising SEQ ID NO:24. Most particularly the pharmaceutical composition comprises a humanized scFv of 16D3, such as, but not limited to the humanized scFv comprising SEQ ID NO:26 or a sequence having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity therewith within the CDRs capable of binding P1GF.

"In yet another particular embodiment of the pharmaceutical composition according to the invention, a therapeutically effective amount of another anti-angiogenic agent is included in addition to the antigen-binding molecule capable of binding to P1GF of the invention. Most particularly in this respect anti-angiogenic agents such as VEGF- and bFGF-inhibitors are envisaged, most particularly anti-VEGF antibodies.

"Another object of the present invention is to provide nucleotide sequences encoding the antigen-binding fragments of the antibodies binding to P1GF disclosed herein, more particularly nucleotide sequences encoding the heavy and light chain variable regions of 16D3 produced by cell line 16D3. Most specifically the nucleotide sequence encoding the variable regions of SEQ ID NO: 2 and SEQ ID NO: 4 are envisaged. Additionally polynucleotide sequences encoding antigen-binding fragments comprising at least two CDRs of 16D3, more specifically, polynucleotides encoding at least two of the CDRs selected from the group consisting of SEQ ID NO: 17 (GYTFTDYY), SEQ ID NO: 18 (IYPGSGNT); SEQ ID NO:19 (VRDSPFFDY), SEQ ID NO: 20 (QSLLNSGMRKSF), SEQ ID NO: 21 (WAS) and SEQ ID NO: 22 (KQSYHLFT), or encoding sequences comprising at least two CDRs having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity with SEQ ID NO: 17 to 22. Specific embodiments of the nucleotide sequences of the present invention are provided in SEQ ID Nos 1, 3, 5 and 6. Further specific embodiments include the nucleotide sequences encoding the scFv of 16D3 and humanized versions thereof, most particularly the sequence of SEQ ID NO: 23 and SEQ ID NO: 25 and sequences having at least 80%, particularly at least 85%, more particularly at least 90%, most particularly at least 95% sequence identity therewith, most particularly within the regions encoding the CDR regions of the scFv. It will be appreciated however that a multitude of nucleotide sequences exist which fall under the scope of the present invention as a result of the redundancy in the genetic code.

"Another object of the present invention is to provide a method of treatment and/or prevention of undesired (or pathological) angiogenesis in pathological condition in a mammal, which method comprises administering to a mammal in need of such treatment or prevention a therapeutically effective amount of an active ingredient which is antibody 16D3 of the present invention or an antigen-binding fragment or derivative thereof, most particularly a scFv as described herein. Particularly suited for the methods of the present invention are the humanized antibodies and antibody fragments, such as scFvs of 16D3 or derivatives thereof. A particular embodiment of the method of the invention relates to the treatment and/or prevention of pathological conditions such as, but not limited to cancer, inflammation, diseases of the eye, pulmonary hypertension and vascular leakage. A particular aim of the present invention is to provide an effective and safe therapy (i.e. without side-effects) for pathological angiogenesis, more in particular for tumor growth, inflammation, diseases of the eye or vascular leakage, in mammals, more particularly in humans. Most particularly the method of the present invention is suitable for the treatment and/or prevention of solid tumors, more particularly for the treatment and/or prevention of colon cancer, breast cancer, pancreatic cancer and melanomas.

"Further uses of the antibodies and antigen-binding fragments of the present invention relate to the immunological detection of P1GF in human samples, as labeled targeting moieties in diagnostic methods and for the screening of compounds with an additive effect to P1GF inhibition in the treatment of cancer.

"The present invention is based on the surprising determination of new ligands, namely new murine and humanized monoclonal antibodies and fragments, derivatives and homologs thereof which inhibit P1GF very efficiently. Most particularly the present invention demonstrates ligands capable of reducing tumor growth, most particularly capable of reducing the size of a tumor between 20% and 50%.

BRIEF DESCRIPTION OF THE FIGURES

"The following description, not intended to limit the invention to specific embodiments described therein, may be understood in conjunction with the accompanying Figures, incorporated herein by reference, in which:

"FIG. 1: Binding of antibody 16D3 (A) or humanized antibody 16D3 (hu16D3)(B) to human P1GF-2 (produced in Pichia) with an ELISA test in accordance with an embodiment of the present invention.

"FIG. 2: Inhibition of P1GF-2 binding to human Flt-1 receptor by the antibody 16D3 (squares) or the humanized antibody 16D3 (triangles) with an ELISA test in accordance with an embodiment of the present invention.

"FIG. 3: Results of the Biacore experiments wherein rhuP1GF-2 is immobilized on a CM5 chip in order to investigate the inhibition potential of the antibody 16D3 and its Fab fragment test in accordance with an embodiment of the present invention.

"FIG. 4: Monoclonal antibody 16D3 inhibits tumor growth in a subcutaneous human pancreatic DanG xenograft tumor model. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-tPA ('control IgG') (1C8; 50 mg/kg body weight; n=10), anti-hP1GF 16D3 (' anti-hP1GF')(50 mg/kg body weight; n=10), anti-mP1GF (PL5D11D4 (obtained as described in WO01/85796; 50 mg/kg body weigth; n=10), a combination of anti-hP1GF 16D3 and an anti-mP1GF PL5D11D4 ('anti-hP1GF/anti-mP1GF') (each 25 mg/kg body weigth; n=10) and 'vehicle' (n=10) in accordance with an embodiment of the present invention. (A) Tumor size (B) tumor weight 18 days after tumor inoculation.

"FIG. 5: Monoclonal antibody 16D3 inhibits tumor growth in a subcutaneous human pancreatic DanG xenograft tumor model in a dose dependent manner. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (50 mg/kg='1000 .mu.g'/'C', 37.5 mg/kg='750 .mu.g'/'D', 25 mg/kg='500 .mu.g'/'E', 12.5 mg/kg='250 .mu.g'/'F'; n=10 for each concentration), control IgG ('1C8'/'B'; 50 mg/kg) and 'vehicle'/'A' (n=10) in accordance with an embodiment of the present invention. A: evolution of mean tumor size after tumor cell inoculation; B: average mean tumor size at day 20.

"FIG. 6: Monoclonal antibody 16D3 inhibits tumor growth in a subcutaneous human breast MDA-MB xenograft tumor model. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (50 mg/kg body weight; n=10) or vehicle three times a week in accordance with an embodiment of the present invention. A: tumor weight and B: tumor volume as determined 32 days post inoculation.

"FIG. 7: Monoclonal antibody 16D3 inhibits tumor growth in a subcutaneous human colon LOVO xenograft tumor model. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (50 mg/kg body weight; n=10) or vehicle three times a week in accordance with an embodiment of the present invention. A: tumor weight and B: tumor volume as determined 30 days post inoculation.

"FIG. 8: Monoclonal antibody 16D3 inhibits tumor growth in a subcutaneous human melanoma Mel2a xenograft tumor model. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (50 mg/kg body weight; n=10) or vehicle three times a week in accordance with an embodiment of the present invention. Tumor weight as determined 53 days post inoculation.

"FIG. 9: Monoclonal antibody 16D3 prevents body weight loss in a subcutaneous human pancreatic DanG xenograft tumor model. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (50 mg/kg body weight; n=10), control IgG, a combination of anti-hP1GF and anti-mP1GF (each 25 mg/kg body weigth; n=10) and vehicle (n=10) in accordance with an embodiment of the present invention. Tumor weight was subtracted from body weight prior to calculation of the percentage of body weight loss. Open bars: body weight on first day of treatment; filled bars: body weight on last day of treatment

"FIG. 10: Monoclonal antibody 16D3 and Avastin exert additional effect on inhibition of tumor growth in a subcutaneous human pancreatic DanG. Treatment of nu/nu mice with tumors of approximately 60 mm.sup.3 with anti-hP1GF 16D3 (37,5 mg/kg, 25 mg/kg, 12.5 mg/kg body weight; n=10 for each concentration), control IgG (1C8; 50 mg/kg), Avastin (15 mg/kg and 5 mg/kg body weight, i.p. twice weekly, n=10 each) and a combination of anti-hP1GF (12.5 mg/kg body weight) and Avastin (5 mg/kg body weigth; n=10) in accordance with an embodiment of the present invention. Mice were sacrificed after 20 days of tumor inoculation and tumor volume was determined

"FIG. 11: Nucleotide and amino acid sequences of variable parts of murine antibody 16D3 in accordance with an embodiment of the present invention. A. Nucleotide sequence encoding variable part of heavy chain; B. Nucleotide sequence encoding variable part of light chain; C. Amino acid sequence of variable part of heavy chain; D Amino acid sequence of variable part of light chain. Nucleotides or amino acids which can be modified for humanization purposes according to one embodiment of the invention are underlined.

"FIG. 12: Nucleotide and amino acid sequences of humanized variable parts of 16D3 in accordance with an embodiment of the present invention. A. Nucleotide sequence encoding humanized variable part of heavy chain; B. Nucleotide sequence encoding humanized variable part of light chain; C Amino acid sequence of humanized variable part of heavy chain; D. Amino acid sequence of humanized variable part of light chain. Nucleotides or amino acids modified for humanization purposes are underlined.

"FIG. 13: Illustration of the vector for expression of humanized 16D3 scFv in 293 cells

"FIG. 14: A: Binding of scFv16D3 and humanized scFv16D3 to P1GF; B: Inhibition of huP1GF-2 binding to its receptor huFlt-1 by scFv16D3 and humanized scFv16D3 (B).

"FIG. 15: A: Amino acid sequence of scFv of murine antibody 16D3; B: Amino acid sequence of humanized scFv of antibody 16D3. Regions outside heavy and light chain variable regions are underlined.

"FIG. 16: Illustration of the vector for expression of humanized 16D3 Fab in 293 cells in accordance with an embodiment of the invention.

"FIG. 17: A: Binding of humanized Fab 16D3 to huP1GF with an ELISA test in accordance with an embodiment of the present invention; B: Inhibition of huP1GF-2 binding to its receptor huFlt-1 by humanized Fab16D3"

For additional information on this patent application, see: STASSEN, Jean-Marie; CARMELIET, Peter; COLLEN, Desire. Anti-Angiogenic Therapy. Filed February 3, 2014 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3641&p=73&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: VEGF, Biotechnology, Biomedical Engineering, Patents, Genetics, Oncology, Pancreas, Peptides, Xenograft, Immunology, Amino Acids, Angiogenesis, Inflammation, Arteriogenesis, Blood Proteins, Immunoglobulins, Protein Kinases, Serum Globulins, Gastroenterology, Membrane Proteins, Xenotransplantion, Angiogenic Proteins.

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Source: Cancer Gene Therapy Week


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