OA16258A - Novel anti-cMet Antibody. - Google Patents

Novel anti-cMet Antibody. Download PDF

Info

Publication number
OA16258A
OA16258A OA1201200490 OA16258A OA 16258 A OA16258 A OA 16258A OA 1201200490 OA1201200490 OA 1201200490 OA 16258 A OA16258 A OA 16258A
Authority
OA
OAPI
Prior art keywords
antibody
amino acid
met
acid sequence
sequence seq
Prior art date
Application number
OA1201200490
Inventor
Liliane Goetsch
Thierry Wurch
Cédric Bes
Original Assignee
Pierre Fabre Medicament
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pierre Fabre Medicament filed Critical Pierre Fabre Medicament
Publication of OA16258A publication Critical patent/OA16258A/en

Links

Abstract

The present invention relates to a novel divalent antibody capable of binding specifically to the human c-Met receptor and/or capable of specifically inhibiting the tyrosine kinase activity of said receptor, preferably both in a liganddependent and in a ligand-independent manner as well as the amino acid and nucleic acid sequences coding for said antibody. More preferably said antibody comprises a modified hinge region and exhibits an improved antagonistic activity. More particularly, the antibody according to the invention is capable of inhibiting the c-Met dimerization. The invention likewise comprises the use of said antibody as a medicament for the prophylactic and/or therapeutic treatment of cancers, preferably for cancer characterized by a ligand- independent activation of c-Met,or any pathology connected with the over expression of said receptor as well as in processes or kits for diagnosis of illnesses connected with the over-expression of c-Met. The invention finally comprises products and/or compositions comprising such an antibody in combination with other antibodies and/or chemical compounds directed against other growth factors involved in tumor progression or metastasis and/or compounds and/or anti-cancer agents or agents conjugated with toxins and their use for the prevention and/or the treatment of certain cancers.

Description

NOVEL ANTI-cMET ANTIBODY
The présent invention relates to a novel divalent antibody capable of binding specifically to the human c-Met receptor and/or capable of specifically inhibiting the tyrosine kinase activîty of said receptor, preferably both in a ligand-dependent and in a ligand-independent manner as well as the amino acid and nucleic acid sequences coding for said antibody. More preferably said antibody comprises a modified hinge région and exhibits an improved antagoniste activîty. More particularly, the antibody according to the invention is capable of inhibiting the c-Met dimerization. The invention lîkewîse comprises the use of said antibody as a médicament for the prophylactic and/or therapeutic treatment of cancers, preferably for cancer characterized by a ligandindependent activation of c-Met, or any pathology connected with the overexpression of said receptor as well as in processes or kits for diagnosis of illncsses connected with the over-expression of c-Met. The invention final ly comprises products and/or compositions comprising such an antibody in combination with other antibodies and/or chemical compounds directed against other growth factors involved in tumor progression or metastasis and/or compounds and/or anti-cancer agents or agents conjugated with toxîns and their use for the prévention and/or the treatment of certain cancers.
Receptor tyrosine kinase (RTK) targeted agents such as trastuzumab, cetuxîmab, bevacizumab, imatinib and gefitinib inhibitors hâve illustrated the interest of targeting this protein class for treatment of selected cancers.
c-Met, is the prototypic member of a sub-family of RTKs which also includes RON and SEA. The c-Met RTK family îs structurally different from other RTK families and is the only known high-afïïnity receptor for hépatocyte growth factor (HGF), also called scatter factor (SF) [D.P. Bottaro et al., Science 1991, 251:802-804; L. Naldini et al., Eur. Mol. Biol. Org. J. 1991, 10:2867-2878]. c-Met and HGF are widely expressed in a varicty of tissuc and their expression is normally restricted to cells of épithélial and mesenchymal origin respectively [M.F. Di Renzo et al., Oncogene 1991, 6:1997-2003; E. Sonnenbcrg et al., J. Cell. Biol. 1993, 123:223-235]. They are both required for normal mammalian development and hâve been shown to be particularly important in cell migration, morphogcnic différentiation, and organîzation of the thrcc-dimensional tubular structures as well as growth and angiogenesis [F. Baldt et al., Nature 1995, 376:768-771; C. Schmidt et al., Nature. 1995:373:699-702; Tsarfaty et al., Science 1994, 263:98-101]. While the controlled régulation of c-Met and HGF hâve been shown to be important in mammalian development, tissue maintenance and repair [Nagayama T., Nagayama M., Kohara S., Kamiguchi H., Shibuya M., Katoh Y., Itoh J., Shinohara Y., Brain Res. 2004, 5;999(2): 155-66; Tahara Y., Ido A., Yamamoto S., Miyata Y., Uto H., Hori T., Hayashi K., Tsubouchi H., J Pharmacol Exp Thcr. 2003, 307(1): 146-51], their dysrégulation is implicated in the progression of cancers.
Aberrant signalling driven by inappropriate activation of c-Met is one of the most frequent alteration observed in human cancers and plays a crucial rôle in tumorigenesis and metastasis [Birchmeier et al., Nat. Rev. Mol. Cell Biol. 2003, 4:915925; L. Trusolino and Comoglio P. M., Nat Rev. Cancer. 2002, 2(4):289-300].
Inappropriate c-Met activation can arise by ligand-dependent and independent mechanisms, which include overexpression of c-Met, and/or paracrine or autocrine activation, or through gain in fonction mutation [J.G. Christensen, Burrows J. and Salgia R., Cancer Latters. 2005, 226:1-26]. However an oligomérization of c-Met receptor, in presence or in absence of the ligand, is required to regulate the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates [Hays JL, Watowich SJ, Biochemistry, 2004 Aug 17, 43:10570-8]. Activated c-Met recruîts signalling effectors to its multidocking site located in the cytoplasm domain, resulting in the activation of scvcral kcy signalling pathways, including RasMAPK, PI3K, Src and Stat3 [Gao CF, Vande Woude GF, Cell Res. 2005, 15( 1):49-51 ; Furge KA, Zhang YW, Vande Woude GF, Oncogene. 2000, 19(49):5582-9]. These pathways are essential for tumour cell prolifération, invasion and angiogenesis and for evadîng apoptosis [Furge KA, Zhang YW, Vande Woude GF, Oncogene, 2000, 19(49):5582-9; Gu H., Ncel BG, Trends Cell Biol. 2003 Mar, 13(3): 122-30; Fan S., Ma YX, Wang JA, Yuan RQ, Meng Q., Cao Y., Laterra JJ, Goldberg ID, Rosen EM, Oncogene. 2000 Apr 27, 19(18):2212-23]. In addition, a unique facet of the c-Met signalling relative to other RTK is its reported interaction with focal adhesion complexes and non kinase binding partners such as α6β4 integrins [Trusolino L., Bertotti A., Comoglio PM, Cell. 2001, 107:643-54], CD44v6 [Van der Voort R., Taher TE, Wielenga VJ, Spaargarcn M., Prevo R., Smit L., David G., Hartmann G., Gherardi
Ai
E., Pals ST, J, Biol. Chem. 1999, 274(10):6499-506], Plexin BI or semaphorins [Giordano S., Corso S., Conrotto P., Artigiani S., Gilestro G., Barberis D., Tamagnone L., Comoglio PM, Nat Cell Biol. 2002, 4(9):720-4; Conrotto P., Valdembri D., Corso S., Serini G., Tamagnone L., Comoglio PM, Bussolino F., Giordano S., Blood. 2005, 105(11):4321-9; Conrotto P., Corso S., Gambcrini S., Comoglio PM, Giordano S., Oncogène. 2004, 23:5131-7] which may further add to the complcxity of régulation of cell function by this receptor. Fînally récent data demonstrate that c-Met could be involved in tumor résistance to gefitinib or erlotinib suggesting that combination of compound targetîng both EGFR and c-Met might be of significant interest [Engelman JA et al., Science, 2007, 316:1039-43].
In the past few years, many different strategies hâve been developed to attenuate c-Met signalling in cancer cell lincs. These strategies include i) neutralizing antibodies against c-Met or HGF/SF [Cao B., Su Y., Oskarsson M., Zhao P., Kort EJ, Fisher RJ, Wang LM, Vande Woude GF, Proc Natl Acad Sci USA. 2001, 98( 13):7443-8; Martens T., Schmidt NO, Eckerich C., Fillbrandt R., Merchant M., Schwall R., Westphal M., Lamszus K.., Clin Cancer Res. 2006, 12(20):6144-52] or the use of HGF/SF antagonist NK4 to prevent ligand binding to c-Met [Kuba K., Matsumoto K.., Date K., Shimura H., Tanaka M., Nakamura T., Cancer Res., 2000, 60:6737-43], ii) small ATP binding site inhibitors to c-Met that block kinase activity [Christensen JG, Schreck R., Burrows J., Kuruganti P., Chan E, Le P., Chen J., Wang X., Ruslim L., Blake R., Lipson ΚΈ, Ramphal J., Do S., Cui JJ, Cherrington JM, Mendel DB, Cancer Res. 2003, 63:7345-55], ni) engîneered SH2 domain polypeptide that interfères with access to the multidocking site and RNAi or ribozyme that reduce receptor or ligand expression. Most of these approaches display a sélective inhibition of c-Met resulting in tumor inhibition and showing that c-Met could be of interest for therapeutic intervention in cancer.
Within the molécules generated for c-Met targetîng, some are antibodies. The most extensively described is the anti-c-Met 5D5 antibody generated by Genentech [WO 96/38557] which behaves as a potent agonist when added alone in various models and as an antagonist when used as a Fab fragment. A monovalent engineered form of this antibody described as one armed 5D5 (OA5D5) and produced as a recombinant protein in E, Coli is also the subjcct of a patent application [WO 2006/015371] by ÜC
Genentech. However, this molécule that could not be considered as an antibody because of its particular scaffold, displays also mutations that could bc immunogenic in humans. ïn terms of activity, this unglycosylated molécule is devoided of effector fonctions and finally, no clear data demonstratc that OA5D5 inhibits dimerization of c-Met. Moreover, when tested in the G55 in vivo model, a glioblastoma cell line that expresses c-Met but not HGF mRNA and protein and that grows independently of the ligand, the one armed antî-c-Met had no significant effect on G55 tumor growth suggesting that OA5D5 acts primarily by b locking HGF binding and is not able to target tumors activated independently of HGF [Martens T. et al, Clin. Cancer Res., 2006, 12(20):6144-6152].
Another antibody targeting c-Met is described by Pfizer as an antibody aetîng “prédominant ly as c-Met antagonist, and in some instance as a c-Met agonist” [WO 2005/016382]. No data showing any effect of Pfizer antibodies on c-Met dimerization is described in this application.
One of the innovative aspects of the présent invention is to generate a chimeric and/or humanized monoclonal antibody wîthout intrinsic agonist activity and inhibiting c-Met dimerization. More particularly, an innovative aspect of the présent invention is to generate a chimeric and/or humanized monoclonal antibody with antagonist activity and inhibiting c-Met dimerization.
In addition of targeting ligand-dependent tumors, this approach will also impair ligand-independent activations of c-Met due to its overexpression or mutations of the întra cellular domains which remained dépendent to oligomérization for signalling. Another aspect of the activity of this antibody could be a steric hindrance for c-Met interaction with its partners that will resuit in impairment of c-Met fonctions. This antibody is humanized and engincered preferentially, but not limited, as human IgG! to get effector fonctions such as ADCC and CDC in addition to fonctions linked to the spécifie blockade of the c-Met receptor.
Surprisingly, for the first time, inventors hâve managed to generate a chimeric and/or humanized monoclonal antagonist antibody capable of binding to c-Met but also capable of inhibiting the c-Met dimerization, said monoclonal antibody being divalent contrary to existing antagonist antibodies directed against c-Met. If it is true that, in the prior art, it is sometimes suggested that an antibody capable of inhibiting the dimerization of c-Met with its partners could be an interestîng one, it has never been disclosed, or clearly suggested, an antibody capable of doîng so. Moreover, regarding antibody specificity, it was not évident at ail to succeed in the génération of such an active divalent antibody.
As it was explained before, the inhibition of the c-Met dimerization is a capital aspect of the invention as such antibodies will présent a real interest for a larger population of patients. Not only ligand-dependent activated c-Met cancer, as it was the case until the présent invention, but also ligand-îndependent activated c-Met cancer could be traited with antibodies generated by the process of the présent invention.
Antibodies were evaluated by BRET analysis on cells expressing both c-MetRLuc/c-Met-YFP and selected on their ability to inhibit at least 40 %, preferably 45 %, 50 %, 55 % and most preferably 60 % of the BRET signal.
The BRET technology is known as being représentative of the protein dimerization [Angers et al., PNAS, 2000, 97:3684-89],
The BRET technology is well known by the man skill în the art and will bc detailed in the following examples. More particularly, BRET (Bioluminescence Résonance Energy Transfer) is a non-radiative energy transfer occurring between a bio luminescent donor (Renilla Luciferase (Rluc)) and a fluorescent acceptor, a mutant ofGFP (Green Fluorescent Protein) or YFP (Yellow fluorescent protein). In the présent case EYFP (Enhanced Yellow Fluorescent Protein) was used. The efficacy of transfer dépends on the orientation and the distance between the donor and the acceptor. Then, the energy transfer can occur only if the two molécules are in close proximîty (1-10 nm). This property is used to generate protein-protein interaction assays. Indeed, in order to study the interaction between two partners, the first one is genetically fiised to the Renilla Luciferase and the second one to the yellow mutant of the GFP. Fusion proteins are generally, but not obligatory, expressed in mammalian cells. In presence of its membrane permeable substrate (coelenterazine), Rluc emits blue light. If the GFP mutant is doser than 10 nm from the Rluc, an energy transfer can occur and an additional yellow signal can be detectcd. The BRET signal is measured as the ratio between the light emitted by the acceptor and the light emitted by the donor. So the BRET signal will increase as the two fusion protéine are brought into proximity or îf a conformational change brings Rluc and GFP mutant doser.
if the BRET analysis consists in a preferred embodiment, any method known by the man skilled in the art can be used to mcasure c-Met dimerization. Wîthout limitation, the following technologies can be mentioned: FRET (Fluorescence Résonance Energy Transfer), HTRF (Homogenous Time resolved Fluorescence), FLIM (Fluorescence Lifetime Imaging Microscopy) or SW-FCCS single wavelength fluorescence cross-correlation spectroscopy).
Other classical technologies could also bc used, such as Coimmunoprecipitation, Alpha screen, Chemical cross-linking, Double-Hybrid, AfFmity Chromatography, ELISA or Far western blot.
The terms “antibodÿ”, “antibodies” or “immunoglobulin” are used interchangeably in the broadest sense and include monocional antibodies (e.g., fiill length or intact monocional antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecifîc antibodies so long as they exhibit the desired biological activity).
More particularly, such molécule consists in a glycoprotcin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable région (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant région. The heavy chain constant région is comprised of three domains, CHl, CH2 and CH3. Each light chain is comprised of a light chain variable région (abbreviated herein as LCVR or VL) and a light chain constant région. The light chain constant région is comprised of one domain, CL. The VH and VL régions can be further subdivîded into régions of hypervariability, termed complementarity determining régions (CDR), înterspersed with régions that are more conserved, termed framework régions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable régions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant régions of the antibodies may médiate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complément system.
The heavy chains of immunoglobulins can be divided into three functional régions: the Fd région, the hinge région, and the Fc région (fragment crystallizablc). The «C
Fd région comprises the VH and CHl domains and, in combination with the light chain, forms Fab - the antigen-binding fragment. The Fc fragment is responsible for the immunoglobulin cf'fector fonctions, which includes, for example, complément fixation and binding to cognate Fc receptors of cffcctor cells. The hinge région, found in IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer that allows the Fab portion to move freely in space relative to the Fc région. The hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.
According to crystallographic studies, the immunoglobulin hinge région can be further subdivided structurally and fonctionally into three régions; the upper hinge, the core, and the lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge includes amino acids from the carboxyl end of CHl to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge région correlates with the segmentai flexîbility of the antibody. The core hinge région contains the inter-heavy chain disulfide bridges. The lower hinge région joins the amino terminal end of, and includes residues in the CH2 domain. The core hinge région of human IgGl contains the sequence Cys-Pro-Pro-Cys that, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexîbility, Conformational changes permitted by the structure and flexîbility of the immunoglobulin hinge région polypeptide sequence may affect the effector fonctions of the Fc portion of the antibody.
The term « Monoclonal Antibody » is used in accordance with its ordinary mcaning to dénoté an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical cxcept for possible naturally occurring mutations that may be présent in minor amounts. In other words, a monoclonal antibody consists in a homogenous antibody resulting from the prolifération of a single clone of cells (e.g., hybridoma cells, eukaryotic host cells transfected with DNA encoding the homogenous antibody, prokaryotic host cells transformed with DNA encoding the homogenous antibody, etc.), and which is generally characterized by heavy chains of a single class and subclass, and light chains of a single type. Monoclonal antibodies arc highly spécifie, being dircctcd against a single antigen. Furthermore, in contras t to polyclonal antibodies préparations that typîcally include different antibodies directed against different déterminants, or epitope, each monoclonal antibody is directed against a single déterminant on the antigen.
In the présent description, the terms polypeptides, polypeptide sequences, amino acid sequences, peptides and proteins attached to antibody compounds or to their sequence are interchangeable.
The invention relates to a monoclonal antibody, or a divalent functional fragment or dérivâtive thereof, capable to inhibit the c-Met dimerization and comprising a heavy chain comprising CDR-H l, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3 or a sequence having at least 80 % identity after optimum alignment with sequences SEQ 1D Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80% identity after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody being further characterized in that it also comprises a hinge région comprising the amino acid sequence SEQ ID No. 56.
In a preferred embodiment, the présent invention is directed to a monoclonal antibody, or a divalent functional fragment or dérivative thereof, capable to inhibit the c-Met dimerization, said antibody comprising a heavy chain comprising CDR-H 1, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7, said antibody further comprising a hinge région comprising the amino acid sequence SEQ ID No. 56;
for use for the prévention or the treatment of a patient in nced thereof having a cancer characterized by ligand-independent activation of c-Met, preferably a cancer further characterized by overexpression of c-Met, said c-Met overexpression resulting more preferably from génie amplification of c-Met, and, also more preferably, resulting in ligand-independent activation of c-Met.
In this aspect, the présent invention comprises a method for the prévention or the treatment of a patient in nced thereof having a cancer characterized by ligand*1 L independent activation of c-Met, preferably a cancer further characterized by overexpression of c-Met, said c-Met overexpression resulting more preferably from génie amplification of c-Met, and, also more preferably, resulting in ligand-independent activation of c-Met, said method comprising the step of administering a composition comprising a monoclonal antibody, or a divalent functional fragment or dérivât ive thereof, capable to inhibit the c-Met dimerization, said antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7, said antibody further comprising a hinge région comprising the amino acid sequence SEQ IDNo. 56.
In this particular aspect and in a more preferred embodiment, said cancer characterized by ligand-independent activation of c-Met, and preferably further characterized by overexpression of c-Met, resulting more preferably from génie amplification of c-Met, and, also more preferably, resulting in ligand-independent activation of c-Met, is selected from the group consisting of rénal cell carcinoma and gastric cancer.
More partîcularly, the invention relates to a monoclonal antibody, or a divalent functional fragment or dérivative thereof, as above described characterized in that said hinge région comprises the amino acid séquence SEQ JD No. 57.
In other words, the invention relates to a monoclonal antibody, or a divalent functional fragment or dérivative thereof, capable to inhibit the c-Met dimerization and comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1,2 and 3 or a sequence having at least 80% identity after optimum alignaient with sequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80% identity after optimum alignaient with sequences SEQ ID Nos. 5, 6 or 7, said antibody being further characterized in that it also comprises a hinge région comprising the amino acid sequence SEQ ID No. 57.
ψνϋ
More particularly, the invention relates to a monoclonal antibody, or a divalent functional fragment or dérivative thereof, as above described characterized in that it also comprises a hinge région comprising the amino acid sequence SEQ ID No. 21.
In other words, the invention also relates to a monoclonal antibody, or a divalent functional fragment or dérivative thereof, capable to inhîbit the c-Met dimerization and comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3 or a sequence having at least 80% identity after optimum alignment with sequences SEQ ID Nos. 1, 2 and 3; and a light chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7 or a sequence having at least 80% identity after optimum alignment with sequences SEQ ID Nos. 5, 6 or 7, said antibody being further characterized in that it also comprises a hinge région comprising the amino acid sequence SEQ ID No. 21.
As ît will be apparent for the man skilled in the art, the consensus sequences SEQ ID Nos. 57 and 21 are comprîsed in the consensus sequence SEQ ID No. 56.
Table 1
#01 #02 #03 #04 #05 #06 #07 #08 #09 #10 #11 #12 #13 #14
SEQID NO 56 XI X2 X3 C X5 X6 X7 Xfl X9 c Xll X12 C X14
SEQ ID NO 57 XI X2 X3 C X5 X6 X7 X8 X9 c P P C P
SEQ ID NO 21 XI X2 X3 C X5 - C X8 X9 c Xll X12 C XI4
For SEQ ID No. 56:
XI : P, R, C, - X5: D, C, G, - X8: H, V, K, - X12: P, -
X2: K, C, R, - X6: K, C, - X9: T, C, E, P, - X14 : P, T
X3: S, C, D, - X7: T, C, - Xll : P, I
The expression “functional fragments and dérivatives” will be defined in details later in the présent spécification.
By CDR régions or CDR(s), it is intended to indicate the hypervariable régions of the heavy and light chains of the immunoglobulins as defined by IMGT.
The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P.,
Immunology Today 18, 509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommié, C,, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT unique numbering, the conserved amîno acids always hâve the samc position, for instance cysteine 23 (lst-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobie amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or JTRP). The IMGT unique numbering provides a standardized délimitation of the framework régions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining régions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information. The IMGT unique numbering is used in 2D graphical représentations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M.-P,, Current Bioînformatics, 2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].
Three heavy chain CDRs and 3 light chain CDRs exist. The term CDR or CDRs is used hcre in order to indicate, according to the case, one of these régions or several, or even the whole, of these régions which contain the majority of the amino acid residues responsable for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.
By “percentage of îdentîty” between two nucleîc acid or amino acid séquences in the sense of the présent invention, it is intended to indicate a percentage of nucléotides or of îdentical amino acid residues between the two sequences to be compared, obtained after the best alignaient (optimum alignaient), this percentage being purely statistical and the différences between the two sequences being distributed randomly and over their entire length. The comparisons of sequences between two nucleîc acid or amino acid sequences are traditionally carried out by comparîng these sequences after having aligned them in an optimum manner, said comparison being able to be carried out by segment or by “comparison wîndow”. The optimum alignaient of the séquences for the comparison can bc carried out, in addition to manualiy, by means
Æ-V of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or else by BLAST N or BLAST P comparison software).
The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned in an optimum manner and in which the nucleic acid or amino acid sequence to be compared can comprise additions or délétions with respect to the reference sequence for an optimum alignment between these two sequences. The percentage of identity is calculated by determining the number of îdentical positions for which the nucléotide or the amino acid residue is identîcal between the two sequences, by dividing this number of îdentical positions by the total number of positions in the comparison window and by multiplying the resuit obtained by 100 in order to obtain the percentage of identity between these two sequences.
For example, it is possible to use the BLAST program, “BLAST 2 sequences” (Tatusova et al., “Blast 2 sequences - a new tool for comparing protein and nucléotide sequences”, FEMS Microbîol Lett. 174:247-250) avai labié on the site http://www.ncbi.nlm.nih.gov/ gori7bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penalty”; 5, and “extension gap penalty”: 2; the matrix chosen being, for example, the matrix “BLOSUM 62” proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.
By amino acid sequence having at Ieast 80 %, preferably 85 %, 90 %, 95 % and 98 % identity with a reference amino acid sequence, those having, with respect to the reference sequence, certain modifications, in particular a délétion, addition or substitution of at Ieast one amino acid, a troncation or an élongation are preferred. In the case of a substitution of one or more consecutive or nonconsecutîve amino acid(s), the substitutions are preferred in which the substituted amino acids are replaced by “équivalent” amino acids. The expression “équivalent amino acids” is aimed hcrc at % l îndicating any amino acid capable of being substituted with one of the amino acids of the base structure without, however, essentially modifying the biological activities of the corresponding antibodies and such as will be defined later, espccially in the examples. These équivalent amino acids can bc determined either by relying on their 5 structural homology with the amino acids which they replace, or on results of comparative trials of biological activity between the different antibodies capable of being carried out.
By way of example, mention is made of the possîbilities of substitution capable of being carried out without resulting in a profound modification of the biological 10 activity of the corresponding modifïed antibody.
As non limitative example, the following table 2 is giving substitution possîbilities conceivable with a conservation of the biological activity of the modifïed antibody. The reverse substitutions are also, of course, possible in the same conditions.
Table 2
Original residue Substitution(s)
Ala (A) Val, Gly, Pro
Arg (R) Lys, His
Asn (N) Gin
Asp(D) Glu
Cys(C) Ser
Gln(Q) Asn
Glu (G) Asp
Gly (G) Ala
His (H) Arg
Ile(I) Leu
Leu (L) Ile, Val, Met
Lys (K) Arg
Met (M) Leu
Phe (F) Tyr
Pro (P) Ala
Ser (S) Thr, Cys
Thr (T) Ser
Trp (W) Tyr .
Tyr (Y) Phe, Trp
Val (V) Leu, Ala
It must be understood here that the invention does not relate to the antibodies in natural form, that is to say they are not in their natural environment but that they hâve been able to be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they can then contain unnatural amino acids as will be described further on.
It must also be understood, as prcviously mentioned, that the invention concems more particularly a chimeric and/or a humanized divalent antibody, or any divalent functional fragment or dérivâtive, with an antagonistic activity. Divalent antibodies of the prior art are agonîsts or partial agonists. The monoclonal antibody of the invention, including a modified hinge as previously described, i.e. including a hinge région comprising the amino acid sequence SEQ ID No. 56, 57 or 21, is novel and présents the particularity to hâve a improved antagonistic activity comparcd to the chimeric or humanized antibody 224G11 without such a modified hinge as ît will appear from the following examples.
Contrary to the prior art, inventors hâve obtained an improved antagonistic activity without modifying the format of the antibody. Actually, in the closest prior art represented by the antibody 5D5, it has been necessary to develop a monovalent fragment of the antibody to generate an antagonistic activity. In the présent application, by the use of the hinge of the invention, it is possible for the first time to obtain a füll divalent antibody with increased antagonistic activity, and this contrary to the general knowledge.
In a preferred embodiment, the antibody of the invention comprises a hinge région comprising an amino acid sequence selected from the group consîsting of SEQ ID Nos. 22 to 28 and 58 to 72, or a sequence having at least 80% îdentity after optimum alignaient with sequences SEQ ID Nos. 22 to 28 and 58 to 72.
For more clarity, the following tables 3 and 4 regroup the amino acids and nucléotides sequences of the different preferred hinges of the invention.
Table 3
SEQ ID No. Amino acids SEQ ID No. Nucléotides
22 RKCCVECPPCP 29 AtKJAAGTGCTGTÜTGGAGTGCCCCCCCTGCCCA
23 PRDCGCKPCICT 30 CCCCGGGACTGTGGGTGCAAGCCTTGCATTTGTACC
24 PKSCGCKPC1CT 31 CCCAAGACiCTGTGGGTGCAAGCCTTGCATTTGTACC
25 PKSCGCKPCICP 32 CCAAAGAGCTGCGGCTGCAAGCCTTGTATCTGTCCC
26 PRDCGCKPCPPCP 33 CCACGGGACTGTGGCTGCAAGCCCTGCCCTCCGTGTCCA
27 PRDCGCHTCPPCP 34 CCCAGAGACTGTGGGTGTCACACCTGCCCTCCTTGTCCT
28 PKSCDCHCPPCP 35 CCCAAAAGCTGCGATTGCCACTGTCCTCCATGTCCA
Table 4
SEQ ID No. Amino acids SEQ ID No. Nucléotides
58 CKSCDKTHTCPPCP 73 TGCAAGAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT
59 PGSCDKTHTCPPCP 74 CCCTGCAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCT
60 PKCCDKTHTCPPCP 75 CCCAAGTGCTCiCtjACAAGACCCACACCTGTCCCCCCTGCCCT
61 PK.SCCKTHTCPPCP 76 CCTAAGAGCTGTTGCAAGACCCACACCTGTCCCCCCTGCCCT
62 PKSCDCTHTCPPCP 77 CCCAAGACiCTGCGACTGCACCCACACCTGTCCCCCCTGCCCT
63 PKSCDKCHTCPPCP 78 CCCAAGAGGTGCGACAAGTGCCACACCTGTCCCCCCTGCCCT
64 PKSCDKTHCCPPCP 79 CCCAAGAGCTGCGACAAGACCCACTGCTGTCCCCCCTGCCCT
65 KCDKTHTCPPCP 80 AAGTGCGACAAGACCCACAŒTGTCCCCCCTGCCCT
66 PKSCDCHTCPPCP 81 CCCAAGAGCTGCGACTGCCACACCTGTCCCCCCTGCCCT
67 PKSCDCTHCPPCP 82 CCCAAGAGCTGCGACTGCACCCACTGCCCCCCCTGCCCT
68 PCSCKHTCPPCP 83 CCCTGCAGCTGCAAGCACACCTGTCCCCCCTGCCCT
69 PSCCTHTCPPCP 84 CCTAGCTGCTGCACCCACACCTGTCCCCCCTXiCCCT
70 PSCDKHCCPPCP 85 CCCAGCTGCGACAAGGACTGCTGCCCCCCCTGCCCT
71 PKSCTCPPCP 86 CCCAAGAGCTGCACCTGTCCCCCTTGTCCT
72 PKSCDKCVECPPCP 87 CCCAAGAGCTGCGATAAGTGCGTGGAGTGCCCCCCTTGTCCT
According a first approach, the antîbody will be defined by its heavy chain sequence. More particularly, the antibody of the invention, or one of its functional 10 fragments or dérivatives, is characterized in that it comprises a heavy chain comprising at least one CDR choscn from CDRs comprising the amino acid sequences SEQ 1D Nos. 1 to 3.
The mentioned sequences are the following ones:
SEQ ID No. 1 : GYIFTAYT
SEQ ID No. 2: IKPNNGLA
SEQ ID No. 3: ARSEITTEFDY
According to a preferred aspect, the antibody of the invention, or one of its functional fragments or dérivatives, comprises a heavy chain comprising at least one, preferably two, and most preferably three, CDR(s) chosen from CDR-Hl, CDR-H2 and CDR-H3, wherein:
- CDR-Hl comprises the amino acid sequencc SEQ ID No. I,
- CDR-H2 comprises the amino acid sequence SEQ ID No. 2,
- CDR-H3 comprises the amino acid sequence SEQ ID No. 3.
In a second approach, the antibody will be now defined by its light chain sequence. More particularly, according to a second particular aspect of the invention, the antibody, or one of its functional fragments or dérivatives, is characterized in that it comprises a light chain comprising at least one CDR chosen from CDRs comprising the amino acid sequence SEQ ID Nos. 5 to 7.
The mentioned séquences arc the following ones:
SEQ ID No. 5: ESVDSYANSF
SEQ ID No. 6: RAS
SEQ ID No. 7: QQSKEDPLT
According to another preferred aspect, the antibody of the invention, or one of its functional fragments or dérivatives, comprises a light chain comprising at least one, preferably two, and most preferably three, CDR(s) chosen from CDR-Ll, CDR-L2 and CDR-L3, wherein:
- CDR-Ll comprises the amino acid sequence SEQ ID No. 5,
- CDR-L2 comprises the amino acid sequence SEQ ID No. 6,
- CDR-L3 comprises the amino acid sequence SEQ ID No. 7.
The murine hybridoma capable of secreting monoclonal antibodies according to the présent invention, especially hybridoma of murine origin, was deposited at the CNCM (Institut Pasteur, Paris, France) on 03/14/2007 under the number CNCM 1-3731.
In the présent application, IgGl are preferred to gct effector fonctions, and most preferably ADCC and CDC.
The skilled artisan will recognize that effector fonctions include, for example, Clq bindïng; complément dépendent cytotoxicity (CDC); Fc receptor bînding;
antîbody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down régulation of cell surface reccptors (e.g. B cell receptor; BCR).
The antibodîes according to the présent invention are preferably spécifie monoclonal antibodîes, especially of murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art.
In general, for the préparation of monoclonal antibodîes or their functional fragments or dérivatives, especially of murine origin, it is possible to refer to techniques which are described în particular in the manual “Antibodîes” (Harlow and Lane, Antibodîes: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the technique of préparation from hybridomas described by Kohler and Milsteîn (Nature, 256:495-497, 1975).
The monoclonal antibodîes according to the invention can be obtained, for example, from an animal cell immunized against the c-Met, or one of its fragments contaîning the epitope specifically recognized by said monoclonal antibodîes according to the invention. Said c-Met, or one of its said fragments, can especially be produced according to the usual working methods, by genetic recombination starting with a nuclcic acid sequence containcd in the cDNA sequence coding for the c-Met or by peptide synthesis starting from a sequence of amino acids comprised in the peptide sequence of the c-Met.
The monoclonal antibodîes according to the invention can, for example, be purified on an affinity co lumn on which the c-Met or one of its fragments contaîning the epitope specifically recognized by said monoclonal antibodîes according to the invention has previously been immobilized. More particularly, saîd monoclonal antibodîes can bc purified by chromatography on protein A and/or G, followed or not followed by ion-exchange chromatography aimed at climinatîng the residual protein contaminants as well as the DNA and the LPS, in itself followed or not followed by exclusion chromatography on Sepharose™ gel in order to elimînate the potential aggregates duc to the présence of dîmers or of other multimcrs. In an even more preferred manner, the whole of these techniques can be used simultaneously or successively.
i, G
The antibody of the invention, or a divalent functional fragment or dérivative thereof, consists preferably of a chimeric antibody.
By chimeric antibody, it is intended to îndicate an antibody which contains a natural variable (light chain and heavy chain) région derived from an antibody of a given species in combination with the light chain and heavy chain constant régions of an antibody of a species heterologous to said given species (e.g. mouse, horse, rabbît, dog, cow, chicken, etc.).
The antibodies or their fragments of chimeric type according to the invention can be prepared by using the techniques of genetic recombination. For cxample, the chimeric antibody can be produced by cloning a recombinant DNA containing a promoter and a sequence coding for the variable région of a non-human, especially murine, monoclonal antibody according to the invention and a sequence coding for the constant région of human antibody. A chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-man chimcra, the specificity of this antibody being determined by the variable région derived from the murine DNA and its isotype determined by the constant région derived from the human DNA. For the methods of préparation of chimeric antibodies, it is possible, for example, to refer to the documents Verhoeyn et al. (BioEssays, 8:74, 1988), Morrison et al, (Proc. Natl. Acad. Sci. USA 82:6851-6855, 1984) or US 4,816,567.
More particularly, said antibody, or a functional fragment or dérivative thereof, comprises a chimeric heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 46 or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 46.
SEQ ID No. 46: EVQLQQSGPELVKPGASVKISCKTSGYIFTAYTMHWVRQSLGE SLDWIGGIKPNNGLANYNQKFKGKATLTVDKSSSTAYMDLRSLTSEDSAVYYC ARSE1TTEFDYWGQGTALTVSS
More particularly, said antibody, or a functional fragment or dérivative thereof, comprises a chimeric light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 47 or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 47.
>7 U
SEQ ID No. 47: DIVLTQSPASLAVSLGQRATISCRASESVDSYANSFMHWYQQKP GQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSKEDP LTFGSGTKLEMKR
More particularly, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [IgG2chim], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 22.
In the présent application, the use of square brackets is not necessary and, as en example, the reference [224G11] [IgG2chim] must be considered as identical to 224G1 llgG2chim. In a same way, to indicate that the antibody îs a murine one, the expression murine or the letter m can be added; to indicate that the antibody is a chimeric one, the expression chim or the letter c can be added and ; to indicate that the antibody is a humanized one, the expression hum, hz, Hz or the letter h can be added. As an example, the chimeric antibody 224GlIgG2 can be referred as c224GllIgG2, c224Gl l[IgG2], c[224Gl l]IgG2, c[224Gll][IgG2], 224G11 IgG2chim,
224G1 l[IgG2chim], [224G1 l]IgG2chim or [224G1 l][IgG2chim].
ln another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [TH7chim], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 28.
In the présent application, the reference TH7 must be considered as identical to C7A6-9 or TH7C7A6-9. The symbol Δ means délétion.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224GH] [MHchim], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 23.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224GH] [MUP9Hchim], ί L16258 comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 26.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224GH] [MMCHchim], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 24.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [Cl], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 58.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C2], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 59.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224GU] [C3], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 60.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224GH] [C5], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 61.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C6], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No, 46, a
I light chaîn variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 62,
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C7], comprises a 5 heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 63.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C9], comprises a 10 heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 64.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [Δ1-3], comprises a 15 heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 65.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C7A6], comprises a 20 heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 66.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C6A9], comprises a 25 heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 67, ' In another aspect, a preferred antibody, or a divalent functional fragment or i dérivative thereof, according to the invention and named [224G11] [C2A5-7], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a ; light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a j hinge région comprising the amino acid sequence SEQ ID No. 68.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [€5Δ2-6], comprises a heavy chain variable domain comprising the amino acid sequencc SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 69.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [C9A2-7], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 70.
In another aspect, a preferred antibody, or a divalent functional fragment or dcrivatîve thereof, according to the invention and named [224G11] [Δ5-6-7-8], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequence SEQ ID No. 71.
In another aspect, a preferred antibody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [IgGl/IgG2], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 46, a light chain variable domain comprising the amino acid sequence SEQ ID No. 47, and a hinge région comprising the amino acid sequencc SEQ ID No. 72.
The antibody of the invention, or a divalent functional fragment or dérivative thereof, consiste preferably of a human antibody.
The term “human antibody” includes ail antibodies that hâve one or more variable and constant région derived from human immunoglobulin sequences. In a preferred embodiment, ail of the variable and constant domains (or régions) are derived from human immunoglobulin sequence (fiilly human antibody). In other words, it includes any antibody which hâve variable and constant régions (if présent) derived from human germline immunoglobulin sequences, i.e. which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any techniques for makîng human antibodies known by the man skill in the art.
In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
As example for such transgenic mouse, it can be mentioned the XENOMOUSE™ which is an engineered mouse strain that comprises large fragments of the human îmmunoglobulin locî and is déficient in mouse antibody production (Green at al., 1994, Nature Genetics, 7:13-21). The XENOMOUSE™ produces an adult-like human répertoire of fully human antibodies, and generate antigen-specific human monoclonai antibodies. A second génération XENOMOUSE™ contains approximately 80% of the human antibody répertoire (Green & Jakobovits, 1998, J. Exp. Med., 188:483-495).
Any other technique known by the man skill in the art, such as phage display technique, can also be used for the génération of human antibody according to the invention.
The antibody of the invention, or a divalent functional fragment or dérivative thereof, consists preferably of a humanized antibody.
By the expression “humanized antibody”, it is intended to indicate an antibody which contains CDR régions derived from an antibody of nonhuman origin, the other parts of the antibody molécule being derived from onc (or from several) human antibodies. Moreover, some of the residues of the segments of the skeleton (called FR) can be modified in order to conserve the affinity of the binding (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann ct al., Nature, 332:323-327, 1988).
The humanized antibodies according to the invention or their fragments can be prepared by techniques known to the person skilled in the art (such as, for example, those described in the documents Singer et al., J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bcbbington et al., Bio/Technology, 10:169-175, 1992).
Other humanization method are known by the man skill in the art as, for example, the “CDR Grafting” method described by Protein Design Lab (PDL) in the patent applications EP 0 451261, EP 0 682 040, EP 0 9127, EP 0 566 647 or US ί
J.
Ϊ 24
5,530,101, US 6,180,370, US 5,585,089 and US 5,693,761, The following patent applications can also be mentioned: US 5,639,641; US 6,054,297; US 5,886,152 and US 5,877,293.
More particularly, said antîbody, or a functional fragment or dérivative thereof, comprises a humanized heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 4 or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 4.
SEQ ID No. 4: QVQLVQSGAEVKKPGASVKVSCKASGYIFTAYTMHWVRQAPG QGLEWMGWIKPNNGLANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVY 10 YCARSEITTEFDY WGQGTLVTVSS
More particularly, said antîbody, or a functional fragment or dérivative thereof, comprises a humanized light chain variable domain selected from the group of séquences comprising the amino acid sequence SEQ ID No. 8, 9 or 10 or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 8, 15 9 or 10.
SEQ IDNo. 8: DIVLTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKP GQPPKLLIYRASTRESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSKEDP LTFGGGTKVEÏKR
SEQ IDNo. 9: DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFMHWYQQKP
0 GQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDP
LTFGGGTKVEÏKR
SEQ ID No. 10: DIVMTQSPDSLAVSLGERATINCKSSESVDSYANSFLHWYQQKP GQPPKLLIYRASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEDP LTFGGGTKVEÏKR
More particularly, a preferred antîbody, or a divalent functional fragment or dérivative thereof, according to the invention and named [224G11] [IgG2Hzl], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 8,
0 and a hinge région comprising the amino acid sequence SEQ ID No. 22.
' In another aspect, a preferred antîbody, or a divalent functional fragment or
I dérivative thereof, according to the invention and named [224G11] [IgG2Hz2], $L· comprises a heavy chain variable domain comprising the amino acid sequencc SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 9, and a hinge région comprising the amino acid sequence SEQ ID No. 22.
In another aspect, a preferred antibody, or a divalent functional fragment or derivative thereof, according to the invention and named [224GU] [IgG2Hz3], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 10, and a hinge région comprising the amino acid sequence SEQ ID No. 22.
In another aspect, a preferred antibody, or a divalent functional fragment or derivative thereof, according to the invention and named [224GH] [TH7Hzl], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 8, and a hinge région comprising the amino acid sequence SEQ ID No. 28.
In another aspect, a preferred antibody, or a divalent functional fragment or derivative thereof, according to the invention and named [224G11] [TH7z2], comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 9, and a hinge région comprising the amino acid sequence SEQ ID No, 28.
In another aspect, a preferred antibody, or a divalent functional fragment or derivative thereof, according to the invention and named [224G11] [TH7Hz3], comprises a heavy chain variable domain comprising the amino acid sequencc SEQ ID No. 4, a light chain variable domain comprising the amino acid sequence SEQ ID No. 10, and a hinge région comprising the amino acid sequence SEQ ID No. 28.
In another aspect, antibodies of the invention can be described by their total heavy and light chains, respectively.
As example, the antibody [224G11] [lgG2chim] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 50, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 50, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
L·
As another example, the antibody [224GH] [TH7chim] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 51, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 51, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52,
As another example, the antibody [224G11] [Cl] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 88, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 88, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another examplc, the antibody [224G1I] [C2] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 89, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 89, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [C3] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 90, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 90, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [C5] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 91, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 91, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [C6] of the invention comprises a complète heavy chain comprising the amino acid sequence SEQ ID No. 92, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 92, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224GJ l] [C7] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 93, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No, 93, and a complète light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [C9] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 94, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 94, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [Δ1-3] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 95, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 95, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11 ] [C7â6] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 96, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 96, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [C6A9] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ JD No. 97, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 97, and a complète light chain comprising the amino acid sequence SEQ ID No.
AL·
52, or a sequence having at least 80 % identîty after optimum alîgnment with the sequence SEQ ID No. 52.
As another example, the antibody [224GH] [C2A5-7] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 98, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 98, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another cxample, the antibody [224G11] [C5A2-6] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 99, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 99, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224GH] [C9A2-7] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 100, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 100, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another examplc, the antibody [224G11] [Δ5-6-7-8] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. ΙΟΙ, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 101, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80% identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [IgGl/IgG2] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 102, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 102, and a complété light chain comprising the amino acid sequence SEQ ID No. 52, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 52.
As another example, the antibody [224G11] [IgG2Hzl] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 36, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 36 and a complète light chain comprising the amino acid sequence SEQ ID No. 38, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 38.
As another example, the antibody [224G11] [IgG2Hz2] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No, 36, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 36 and a complété light chain comprising the amino acid sequence SEQ ID No. 39, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 39.
As another cxample, the antibody [224G11] [IgG2Hz3] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 36, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 36 and a complété light chain comprising the amino acid sequence SEQ ID No. 40, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 40.
As another example, the antibody [224G11] [TH7Hzl] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No, 37, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 37 and a complété light chain comprising the amino acid sequence SEQ ID No. 38, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 38.
As another example, the antibody [224GH] [TH7Hz2] of the invention comprises a complété heavy chain comprising the amino acid sequence SEQ ID No. 37, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 37 and a complote light chain comprising the amino acid sequence SEQ ID No. 39, or a sequence having at least 80 % identity after optimum alignment with the sequence SEQ ID No. 39.
As another example, the antibody [224GU] [TH7Hz3] of the invention comprises a complète heavy chain comprising the amino acid sequence SEQ JD No. 37, or a sequence having at Ieast 80 % identity after optimum alignment with the sequence SEQ ID No. 37 and a complété light chain comprising the amino acid sequence SEQ ID No. 40, or a sequence having at Ieast 80 % identity after optimum alignment with the sequence SEQ ID No. 40.
Other examples of antibodies, or dérivatives thereof, according to the invention comprises complète heavy chains comprising an amino acid sequence selected in the group consisting of SEQ ID Nos. 88 to 102 (corresponding nucléotide sequences are SEQ ID Nos. 103 to U7).
By “functional fragment” of an antibody according to the invention, it is intended to indicate in particular an antibody fragment, such as Fv, scFv (sc for single chain), Fab, F(ab’)2, Fab’, scFv-Fc fragments or diabodics, or any fragment of which the half-life time would have been increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”) (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)2-PEG or Fab’-PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation in a liposome, said fragments having at Ieast one of the characteristic CDRs of sequence SEQ ID Nos. I to 3 and 5 to 7 according to the invention, and, especially, in that it is capable of exerting in a general manner an even partial activity of the antibody from which it is descended, such as in particular the capacity to recognize and to bind to the c-Met, and, if necessary, to înhîbit the activity of the c-Met.
Preferably, said functional fragments will be constitutcd or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same specificity of bînding as the antibody from which it is descended and a sufficient affinity, preferably at Ieast equal to 1/100, in a more preferred manner to at Ieast 1/10, of that of the antibody from which it is descended, with respect to the c-Met. Such a functional fragment will contain at the minimum 5 amino acids, preferably 6, 7, 8, 9, 10, 12, 15, 25, 50 and 100 consecutive amino acids of the sequence of the antibody from which it is descended.
Preferably, these functional fragments will be fragments of Fv, scFv, Fab, F(ab’)2, F(ab’), scFv-Fc type or diabodies, which generally have the same specificity of binding as the antibody from which they are descended. In a more preferred embodiment of the invention, these fragments arc selected among divalent fragments ? to such as F(ab’)2 fragments. According to the présent invention, antibody fragments of the invention can be obtained starting from antibodies such as described above by methods such as digestion by enzymes, such as pepsin or papain and/or by cleavage of the disulfide bridges by chemical réduction. In another manncr, the antibody fragments comprised in the présent invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesîzers such as those supplied by the company Applied Biosystems, etc.
By “divalent fragment”, it must bc understood any antibody fragments comprising two arms and, more particularly, F(ab’)2 fragments.
By “dérivatives” of an antibody according to the invention, it is meant a binding protein comprising a protein scaffold and at least on of the CDRs selected from the original antibody in order to maintain the binding capacity. Such compounds are well known by the man skilled in the art and will be described in more details in the following spécification.
More particularly, the antibody, or one of its functional fragments or dérivatives, according to the invention is characterized in that said dérivative consiste in a binding protein comprising a scaffold on which at least one CDR has been grafted for the conservation of the original antibody paratopic recognizing properties.
One or several sequences through the 6 CDR sequences described in the invention can be presented on a protein scaffold. In this case, the protein scaffold reproduces the protein backbonc with appropriate folding of the grafted CDR(s), thus allowîng it (or them) to maintain their antigen paratopic recognizing properties.
The man skilled in the art knows how to select the protein scaffold on which at least onc CDR selected from the original antibody could be grafted. More particularly, it is known that, to be selected, such scaffold should display several features as follow (Skerra A., J. Mol. Rccogn., 13, 2000, 167-187):
phylogenetically good conservation, robust architecture with a well known three-dimensional molecular organizatîon (such as, for examplc, crystallography or NMR), small size, no or only low dcgrcc of post-translational modifications,
W t easy to produce, express and purify.
Such protein scaffold can be, but without limitation, structure selected from the group consisting in fibronectin and prcfcrentially the tcnth fibronectin type III domain (FNfhlO), lipocalin, anticalin (Skerra A., J. Biotechnol., 2001, 74(4):257-75), the protein Z dérivâtive from the domain B of staphylococcal protein A, thioredoxin A or any protein with repeated domain such as “ankyrin repeat” (Kohl et al., PNAS, 2003, vol. 100, No.4, 1700-1705), “armadillo repeat”, “leucin-rich repeat” or “tetratricopeptide repeat”.
It could also be mentioned scaffold dérivative from toxins (such as, for example, scorpion, insect, plant or mollusc toxins) or protein inhibitors of neuronal nitric oxyde synthase (PIN),
As non limitative example of such hybrid constructions, it can be mentioned the insertion of the CDR-H1 (heavy chain) of an antî-CD4 antibody, i.e. the 13B8.2 antibody, into one of the exposed loop of the PIN. The binding properties of the obtained binding protein remain similar to the original antibody (Bes et al., BBRC 343, 2006, 334-344). It can also be mentioned the grafting of the CDR-H3 (heavy chain) of an anti-lyzozyme VHH antibody on a loop of ncocarzinostatine (Nicaise et al., 2004).
As above mentioned, such protein scaffold can comprise from 1 to 6 CDR(s) from the original antibody. In a preferred embodiment, but without any limitation, the man skilled in the art would select at least a CDR from the heavy chain, said heavy chain being known to be particularly împlicated in the antibody specificîty. The sélection of the CDR(s) of interest will be évident for the man of the art with known method (BES et al., FEBS letters 508, 2001, 67-74).
As an evidence, these examples are not limitative and any other scaffold known or described must be includcd in the présent spécification.
According to a novel aspect, the présent invention relates to an isolated nucleîc acid, characterized in that it is chosen from the following nucleîc acids:
a) a nucleîc acid, DNA or RNA, coding for an antibody, or one of its functional fragments or dérivatives, according to the invention;
b) a nucleîc acid sequence comprising the sequences SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and the sequences SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17;
VjL
c) a nucieic acid sequence comprising the scquences SEQ ID No. 14 and SEQ IDNo. 18, 19 or 20;
d) the corresponding RNA nucieic acids of the nucieic acids as defined in b) or c);
e) the complementary nucieic acids of the nucieic acids as defined in a), b) and c); and
f) a nucieic acid of at least 18 nucléotides capable of hybridizing under conditions of high stringency with at least one of the CDRs of sequence SEQ ID Nos. 11 to 13 and 15 to 17.
According to still another aspect, the présent invention relates to an isolated nucieic acid, characterizcd in that it is chosen from the following nucieic acids:
- a nucieic acid, DNA or RNA, coding for an antibody, or one of its functional fragments or dérivatives, according to the présent invention and wherein the nucieic sequence coding for the hinge région of said antibody comprises or has a sequence selected from the group consisting of the séquences SEQ ID Nos. 29 to 35 and SEQ ID Nos. 73 to 87.
By nucieic acid, nucieic or nucieic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucléotide sequence, tenus which will be employed indifferently in the présent invention, it is intended to indicate a précisé linkage of nucléotides, which are modified or unmodified, allowing a fragment or a région of a nucieic acid to be defined, containing or not containing unnatural nucléotides, and being able to correspond just as well to a double-stranded DNA, a single-stranded DNA as to the transcription products of said DNAs.
It must also bc understood here that the présent invention does not concern the nucléotide séquences in their natural chromosomal environment, that is to say in the natural state. It conccms sequences which hâve been isolated and/or purified, that is to say that they hâve been selected directly or indirectly, for example by copy, their environment having been at least partially modified. It is thus likewise intended to indicate here the isolated nucieic acids obtained by genetic recombination by means, for example, of host cells or obtained by chemical synthesis.
A hybridization under conditions of high stringency signifies that the température conditions and ionic strength conditions arc choscn in such a way that they allow the maintenance of the hybridization between two fragments of complementary DNA. By way of illustration, conditions of high stringency of the hybridization step for the purposes of defîning the polynucleotidc fragments described above are advantageously the following.
The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (l) prehybridization at 42°C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (l x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% of formamide, 7 % of sodium dodecyl sulfate (SDS), 10 x Denhardt’s, 5 % of dextran sulfate and l % of salmon sperm DNA; (2) actual hybridization for 20 hours at 10 a température dépendent on the size of the probe (i.e.: 42°C, for a probe size > 100 nucléotides) followed by 2 washes of 20 minutes at 20°C in 2 x SSC + 2% of SDS, 1 wash of 20 minutes at 20°C in 0.1 x SSC + 0.1 % of SDS. The last wash is carried out in 0.1 x SSC + 0.1 % of SDS for 30 minutes at 60°C for a probe size > 100 nucléotides. The hybridization conditions of high stringency described above for a polynucleotidc of 15 defined size can be adapted by the person skilled in the art for oligonucleotides of greater or smaller size, according to the teaching of Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).
The invention likewise relates to a vector comprising a nucleic acid according to the présent invention.
The invention aims especially at cloning and/or expression vectors which contain a nucléotide sequence according to the invention.
The vectors according to the invention preferably contain éléments which allow the expression and/or the sécrétion of the translated nucléotide sequences in a determined host cell. The vector must therefore contain a promoter, signais of initiation 25 and termination of translation, as well as appropriate régions of régulation of transcription. It must be able to be maintained in a stable manner in the host cell and can optionally hâve particular signais which specify the sécrétion of the translated protein. These different éléments are chosen and optimized by the person skilled in the art as a function of the host cell used. To this effect, the nucléotide sequences according to the 3 0 invention can be inserted into autonomous réplication vectors in the chosen host, or be intégrative vectors of the chosen host.
L·
Such vectors arc prepared by methods currently used by the person skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, thermal shock, or chemical methods.
The vectors according to the invention arc, for example, vectors of plasmidic or viral origin. They are usefol for transforming host cells in order to clone or to express the nucléotide sequences according to the invention.
The invention lîkewise comprises the host cells transformed by or comprising a vector according to the invention.
The host cell can be chosen from prokaryotic or eukaryotic Systems, for example bacterial cells but lîkewise yeast cells or animal cells, in particular mammalian cells. It is lîkewise possible to use insect cells or plant cells.
The invention likcwisc concems animais, exccpt man, which comprise at least one cell transformed according to the invention.
According to another aspect, a subject of the invention îs a process for production of an antibody, or one of its functional fragments according to the invention, characterized in that it comprises the following stages:
a) culture in a medium and appropriate culture conditions of a host cell according to the invention; and
b) the recovery of said antibodies, or one of their functional fragments, thus produced startîng from the culture medium or said eultured cells.
The cells transformed according to the invention can be used in processes for préparation of recombinant polypeptides according to the invention. The processcs for préparation of a polypeptide according to the invention in recombinant form, characterized in that they cmploy a vcctor and/or a cell transformed by a vector according to the invention, are themselves comprised in the présent invention. Preferably, a cell transformed by a vector according to the invention is eultured under conditions which allow the expression of said polypeptide and said recombinant peptide is recovcred.
As has been said, the host cell can bc chosen from prokaryotic or eukaryotic Systems. In particular, it is possible to identify nucléotide sequences according to the invention, facilitating secrétion in such a prokaryotic or eukaryotic System. A vector according to the invention carrying such a scqucncc can thcrcforc advantagcously bc used for the production of recombinant proteins, intcnded to be secrcted. In effect, the purification of these recombinant proteins of interest will be facilitated by the fact that they are présent in the supematant of the cell culture rather than in the interior of the host cells.
It is likewise possible to préparé the polypeptides according to the invention by chemical synthesis. Such a préparation process is likewise a subject of the invention. The person skilled in the art knows the processes of chemical synthesis, for example the techniques employing solid phases [Steward et al., 1984, Solid phase peptide synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)] or techniques using partial solid phases, by condensation of fragments or by a classical synthesis in solution. The polypeptides obtained by chemical synthesis and being able to contain corresponding unnatural amino acids arc likewise comprised in the invention.
The antibodies, or one of their functional fragments or dérivatives, capable of being obtained by a process according to the invention are likewise comprised in the présent invention.
The invention also conccms the antibody of the invention as a médicament.
The invention likewise concems a pharmaceutical composition comprising by way of active principle a compound consisting of an antibody, or one of its functional fragments according to the invention, preferably mixed with an excipient and/or a pharmaceutically acceptable vehicle.
Another complementary embodiment of the invention consiste in a composition such as described above which comprises, moreover, as a combination product for simultaneous, separate or scquentîal use, an anti-tumoral antibody.
Most preferably, said second anti-tumoral antibody could be chosen through anti-IGF-IR, anti-EGFR, anti-HER2/neu, anti-VEGFR, anti-VEGF, etc., antibodies or any other anti-tumoral antibodies known by the man skilled in the art. It is évident that the use, as second antibody, of functional fragments or dérivatives of above mentioned antibodies is part of the invention.
As a most preferred antibody, anti-EGFR antibodies arc selected such as for example the antibody C225 (Erbitux).
ÿ? L· “Simultaneous use” is understood as meaning the administration of the two compounds of the composition according to the invention in a single and identical pharmaceutical form.
“Separate use” is understood as meaning the administration, at the same time, of the two compounds of the composition according to the invention în distinct pharmaceutical forms.
“Sequcntial use” is understood as meaning the successive administration of the two compounds of the composition according to the invention, each in a distinct pharmaceutical form.
In a general fashion, the composition according to the invention considerably increases the efficacy of the treatment of cancer. In other words, the therapeutic effect of the anti-c-Met antibodîes according to the invention îs potentiated in an unexpected manner by the administration of a cytotoxic agent, Another major subséquent advantage produced by a composition according to the invention conccms the possibility of using lower efficacious doses of active principle, which allows the risks of appearance of secondary effects to be avoided or to be reduced, in particular the effects of the cytotoxic agent.
In addition, this composition according to the invention would allow the expected therapeutic effect to be attaincd more rapidly.
The composition of the invention can also be characterized in that it comprises, moreover, as a combination product for simultaneous, separate or sequential use, a cytotoxîc/cytostatic agent.
By “anti-cancer therapeutic agents” or “cytotoxîc/cytostatic agents”, it is intended a substance which, when administered to a subject, treats or prevents the development of cancer in the subject’s body. As non limitative example of such agents, it can be mentioned alkylating agents, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, chromatin fonction inhibitors, anti-angiogenesis agents, anti-estrogens, antiandrogens or immunomodulators.
Such agents are, for example, cîted in the 2001 édition of VIDAL, on the page devoted to the compounds attached to the cancerology and hematology column “Cytotoxics”, these cytotoxic compounds cited with reference to this document are cited hcrc as preferred cytotoxic agents.
Ά L·16258
More particularly, the following agents are preferred according to the invention.
“Alkylating agent” refers to any substance which can cross-link or alkylate any molécule, preferably nucleic acid (e.g., DNA), within a cell. Examples of alkylating agents include nitrogen mustard such as mcchlorethamine, chlorambucol, melphalen, chlorydrate, pipobromen, prednimustin, disodic-phosphate or estramustine; oxazophorins such as cyclophosphamide, altretamine, trofosfamidc, sulfofosfamide or ifosfamide; aziridines or imine-ethylenes such as thiotepa, triethylenamine or altetramine; nitrosourea such as carmustine, streptozocin, fotemustin or lomustine; alkyle-sulfonates such as busulfan, treosulfan or improsulfan; triazenes such as dacarbazine; or platinum complexes such as cis-platinum, oxaliplatin and carboplatin.
“Anti-metabolites” refer to substances that block cell growth and/or metabolism by intcrfering with certain activities, usually DNA synthesis. Examples of antimetabolites include méthotrexate, 5-fluoruracil, floxuridine, 5-fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosinc arabinoside, 6-mcrcaptopurine (6-MP), 6-thioguanine (6-TG), chlorodcsoxyadenosine, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin and pentostatin.
“Anti-tumor antibiotics” refer to compounds which may prevent or inhibit DNA, RNA and/or protein synthesis. Examples of anti-tumor antibiotics include doxorubicin, daunorubicin, îdarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, and procarbazine.
“Mitotic inhibitors” prevent normal progression of the cell cycle and mitosis. In general, microtubule inhibitors or taxoides such as paclitaxel and docetaxel are capable of inhibiting mitosis. Vinca alkaloid such as vinblastine, vincristine, vindesine and vinorelbine are also capable of inhibiting mitosis.
“Chromatin fonction inhibitors” or “topoisomerase inhibitors” refer to substances which inhibit the normal fonction of chromatin modeling proteins such as topoisomerase I or topoisomerase II. Examples of chromatin fonction inhibitors include, for topoisomerase I, camptothccine and its dérivatives such as topotecan or irinotecan, and, for topoisomerase II, ctoposide, etoposide phosphate and teniposide.
“Anti-angiogenesis agent” refers to any drug, compound, substance or agent which inhîbits growth of blood vessels. Exemplary anti-angiogenesis agents include, but arc by no means limited to, razoxin, marimastat, batimastat, prinomastat, tanomastat, 6L ilomastat, CGS-27023A, halofuginon, COL-3, neovastat, BMS-275291, thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferonalpha,EMD121974, interleuktn-12, IM862, angiostatin and vitaxin.
“Anti-estrogen” or “anti-cstrogenic agent” refer to any substance which rcduccs, antagonîzes or inhibits the action of estrogcn. Examples of anti-estrogen agents are tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole, and exemestane.
“Anti-androgens” or “anti-androgen agents” refer to any substance which reduces, antagonîzes or inhibits the action of an androgen. Examples of anti-androgens are flutamide, nilutamide, bicalutamide, sprironolactone, cyproterone acetate, finasteride and cimitidine.
“Immunomodulators” are substances which stimulate the immune system.
Examples ofimmunomodulators include interferon, interleukin such as aldesleukîne, OCT-43, denileukin diflitox and interleukin-2, tumoral nécrosé fators such as tasonermine or others immunomodulators such as lentinan, sizofiran, roquinimex, pidotimod, pegademase, thymopentine, poly I:C or levamisole in conjunction with 5fluorouracil.
For more detail, the man skill in the art could refer to the manual edited by the “Association Française des Enseignants de Chimie Thérapeutique” and entitled “Traité de chimie thérapeutique”, vol. 6, Médicaments antitumoraux et perspectives dans le traitement des cancers, édition TEC & DOC, 2003.
Can also be mentioned as chemical agents or cytotoxîc agents, all kinase inhibitors such as, for example, gefitinib or crlotinib.
In a particularly preferred embodîment, said composition as a combination product according to the invention is characterized in that said cytotoxîc agent is coupled chemically to said antibody for sîmultaneous use.
In order to facilitate the coupling between said cytotoxîc agent and said antibody according to the invention, it is especially possible to introduce spaccr molécules between the two compounds to be coupled, such as poly(alkylcnc) glycols like polyethylene glycol, or else amino acids, or, in another embodîment, to use active dérivatives of said cytotoxîc agents înto which would hâve been introduced fonctions capable of rcacting with said antibody according to the invention. Thèse coupling techniques are well known to the person skîlled in the art and will not be expanded upon in the présent description.
The invention relates, in another aspect, to a composition characterized in that one, at least, of said antibodies, or one of their functional fragments or dérivatives, is conjugated with a cell toxin and/or a radioélément.
Preferably, said toxin or said radioélément is capable of inhibiting at least one cell activity of cells expressing the c-Met, in a more preferred manner capable of preventing the growth or the prolifération of said cell, especially of totally inactivating said cell.
Preferably also, said toxin is an enterobacterial toxin, especially Pseudomonas exotoxin A.
The radioéléments (or radioisotopes) preferably conjugated to the antibodies employed for the therapy are radioisotopes which émit gamma rays and preferably iodine131, yttrium90, gold199, palladium100, copper67, bismuth217 and antimony211. The radioisotopes which émit beta and alpha rays can likewise be used for the therapy.
By toxin or radioélément conjugated to at least one antibody, or one of its fonctional fragments, according to the invention, it is intended to indîcate any means allowing said toxin or said radioélément to bind to said at least one antibody, especially by covalent coupling between the two compounds, with or without introduction of a linking molécule.
Among the agents allowing binding in a chemical (covalent), electrostatic or noncovalent manner of ail or part of the components of the conjugate, mention may particularly be made of benzoquinone, carbodiimide and more particularly EDC (1ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide hydrochloride), dimaleimide, dithiobisnitrobenzoic acid (DTNB), N-succinimidyl S-acetyl thio-acetate (SATA), the bridging agents having one or more phenylazide groups reacting with the ultraviolets (U.V.) and preferably N-[-4-(azidosalicylamino)butyl]-3’-(2*-pyridyldithio)-propionamide (APDP), N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP), 6-hydrazino-nicotinamide (HYNIC).
Another form of coupling, especially for the radioéléments, can consist in the use of a bifonctional ion chelator.
L·
Among these chelates, it is possible to mention the chelates derived from EDTA (ethylenediaminetetraacetic acid) or from DTPA (diethylcnetriaminepentaacetîc acid) which hâve been developed for binding metals, cspecially radioactive metals, and immunoglobulins. Thus, DTPA and its dérivatives can be substituted by different groups on the carbon chain in order to increase the stability and the rigîdity of the ligand-metal complex (Krejcarek et al. (1977); Brechbiel et al, (1991); Gansow (1991); US patent 4,831,175).
For example diethylenetriaminepentaacetîc acid (DTPA) and its dérivatives, which havc been widely used in medicine and in biology for a long time either in their free form, or in the form of a complex with a metallic ion, havc the remarkable characteristic of forming stable chelates with metallic ions and of being coupled with proteins of therapeutic or diagnostic interest such as antibodies for the development of radioimmunoconjugates in cancer therapy (Meases et al., 1984; Gansow et al., 1990).
Likewise preferably, said at least one antibody forming said conjugate according to the invention is chosen from its functional fragments, especially the fragments amputated of their Fc component such as the scFv fragments.
As already mentioned, in a preferred embodiment of the invention, said cytotoxic/cytostatic agent or said toxîn and/or a radioélément is coupled chemically to at least one of the éléments of said composition for simultaneous use.
The présent invention comprises the described composition as a médicament.
The présent invention moreover comprises the use of the composition according to the invention for the préparation of a médicament.
In another aspect, the invention deals with the use of an antibody, or one of its functional fragments or dérivatives, and/or of a composition as above described for the préparation of a médicament intended to inhibit the growth and/or the prolifération of tumor cells.
Another aspect of the invention consists in the use of an antibody, or one of its functional fragments or dérivatives and/or of a composition, as described above or the use above mentioned, for the préparation of a médicament intended for the prévention or for the treatment of cancer.
Is also comprised in the présent invention a method intended to inhibit the growth and/or the prolifération of tumor cells in a patient comprising the administration bu to a patient in need thereof of an antibody, or one of its functional fragments or dérivatives according to the invention, an antibody produced by an hybridoma according to the invention or a composition according to the invention,
The présent invention further comprises a method for the prévention or the treatment of cancer in a patient in need thereof, comprising the administration to the patient of an antibody, or one of its functional fragments or dérivatives according to the invention, an antibody produced by an hybridoma according to the invention or a composition according to the invention, ln a particular preferred aspect, said cancer is a cancer chosen from prostate cancer, osteosarcomas, lung cancer, breast cancer, endométrial cancer, glioblastoma or colon cancer.
As explained before, an advantage of the invention is to allow the treatment of HGF dépendent and independent Met-activation related cancers.
ln a particular aspect, the invention comprises the use of an antibody, or one of its functional fragments or dérivatives and/or of a composition, as described above or the use above mentioncd, for the préparation of a médicament intended for the prévention or for the treatment of a patient in need thereof having a cancer characterized by overexpression of c-Met (for example, due to a génie amplification of c-Met) resulting in a ligand-independent constitutive activation of said c-Met receptor .
Preferably, said cancer characterized by a génie amplification of c-Met resulting in a ligand-independent constitutive activation of said c-Met receptor is selected from the group consîsting of rénal cell carcinoma and gastric cancer.
The invention, in yet another aspect, encompasses a method of in vitro dîagnosis of Aînesses induced by an overexpression or an underexpression of the c-Met receptor starting from a biological sample in which the abnormal presence of c-Met receptor is suspected, said method being characterized in that it comprises a step wherein said biological sample is contacted with an antibody of the invention, it being possible for said antibody to be, Îf necessary, labeled.
Preferably, said illncsses connected with an abnormal presence of c-Met receptor in said dîagnosis method will be cancers.
Said antibody, or one of its functional fragments, can be présent in the form of an immunoconjugate or of a labelled antibody so as to obtain a détectable and/or quantifiable signal.
The antîbodies labelled according to the invention or their functional fragments include, for example, antîbodies called immunoconjugates which can be conjugated, for example, with enzymes such as peroxidase, alkaline phosphatase, beta-D-galactosidase, glucose oxydase, glucose amylase, carbonic anhydrase, acetylcholinestérase, lysozyme, malate dehydrogenase or glucose 6-phosphate dehydrogenase or by a molécule such as biotin, digoxygenin or 5-bromodcoxyuridinc. Fluorescent labels can be likewise conjugated to the antîbodies or to their functional fragments according to the invention and especially include fluoresceîn and its dérivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for “Green Fluorescent Protein”), dansyl, umbelliferone etc. In such conjugates, the antîbodies of the invention or their functional fragments can be prepared by methods known to the person skilled in the art. They can be coupled to the enzymes or to the fluorescent labels directly or by the intermediary of a spacer group or of a linking group such as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or în the presence of coupling agents such as those mentioned above for the therapeutic conjugates. The conjugates containing labels of fluoresceîn type can be prepared by réaction with an isothiocyanate.
Other conjugates can likewise include chemolumincscent labels such as luminol and the dîoxetanes, bio-luminescent labels such as luciferase and lucîferin, or else radioactive labels such as iodinc123, iodinc125, iodinc126, iodine133, bromine77, technetium99 1, indium111, indium113 1, gallium67, gallium68, ruthénium95, ruthénium97, ruthénium103, ruthénium105, mercury107, mercury203, rhénium99 1, rhénium101, rhénium105, scandium47, tellurium1211, tellurium122m, tcllurium125 1, thulium165, thulium167, thulium168, fluorine18, yttrium199, iodine131. The methods known to the person skilled in the art existîng for coupling the therapeutic radioisotopes to the antîbodies either directly or via a chelating agent such as EDTA, DTPA mentioned above can be used for the radioéléments which can be used in diagnosis. It is likewise possible to mention labclling with Na[I125] by the chloramine T method [Hunter W.M. and Greenwood F.C. (1962) Nature 194:495] or else with technetium99 1 by the technique of Crockford et al.
(US patent 4,424,200) or attachcd via DTPA as described by Hnatowich (US patent 4,479,930).
Thus, the antibody, or a functional fragment or dérivative thereof, according to the invention can be employed in a process for the détection and/or the quantification of an overexpression or of an underexpression, preferably an overexpression, of the c-Met receptor in a biological sample, characterized in that it comprises the following steps:
a) the contacting of the biological sample with an antibody, or a functional fragment or dérivative thereof, according to the invention; and
b) the démonstration of the c-Met/antibody complex possibly formed.
In a particular embodiment, the antibody, or a functional fragment or dérivative thereof, according to the invention, can be employed in a process for the détection and/or the quantification of the c-Met receptor in a biological sample, for the monitoring of the efficacy of a prophylactic and/or therapeutic treatment of c-Met-dependent cancer,
More generally, the antibody or a functional fragment or dérivative thereof, according to the invention can be advantageously employed in any situation where the expression of the c-Met- receptor must be observed in a qualitative and/or quantitative manner.
Preferably, the biological sample is formed by a biological fluid, such as sérum, whole blood, cells, a tissue sample or biopsies of human origin.
Any procedure or conventional test can be employed in order to carry out such a détection and/or dosage. Said test can be a compétition or sandwich test, or any test known to the person skilled in the art dépendent on the formation of an immune complex of antibody-antigen type. Following the applications according to the invention, the antibody or a fonctîonal fragment or dérivative thereof can be immobilized or labelled. This îmmobilization can be carried out on numerous supports known to the person skilled in the art. These supports can especially include glass, polystyrène, poly-propylene, polyethylene, dextran, nylon, or natural or modified cells. These supports can be either soluble or insoluble.
By way of example, a preferred method brings into play immunoenzymatic processes according to the EL1SA technique, by immunofluorescence, or radioimmunoassay (R IA) technique or équivalent.
« L·16258
Thus, the présent invention likewise comprises the kits or sets necessary for carrying out a method of diagnosis of illnesses induced by an overexpression or an underexpression of the c-Met receptor or for carrying out a process for the détection and/or the quantification of an overexpression or of an underexpression of the c-Met receptor in a biological sample, preferably an overexpression of said receptor, characterizcd in that said kit or set comprises the following éléments:
a) an antibody, or a functional fragment or dérivative thereof, according to the invention;
b) optionally, the reagents for the formation of the medium favorable to the immunological reaction;
c) optionally, the reagents allowing the démonstration of c-Met/antibody complexes produced by the immunological reaction.
A subject of the invention is likewise the use of an antibody or a composition according to the invention for the préparation of a médicament intended for the spécifie targeting of a biologically active compound to cells expressing or overex pressing the cMet receptor.
It is intended here by biologically active compound to indicate any compound capable of modulating, especially of inhibiting, cell activity, in particular their growth, their prolifération, transcription or gene translation.
A subject of the invention is also an in vivo diagnostic reagent comprising an antibody according to the invention, or a functional fragment or dérivative thereof, preferably labellcd, especially radio labellcd, and its use in medical imaging, in particular for the détection of cancer connected with the expression or the overexpression by a cell of the c-Met receptor.
The invention likewise relates to a composition as a combination product or to an anti-c-Met/toxin conjugate or radioélément, according to the invention, as a médicament.
Preferably, said composition as a combination product or said conjugate according to the invention will be mixed with an excipient and/or a pharmaceutically acceptable vehicle.
In the présent description, pharmaceutically acceptable vehicle is intended to indicate a compound or a combination of compounds entering into a pharmaceutical
VL· il composition not provoking secondary reactions and which allows, for examplc, facilitation of the administration of the active compound(s), an increase in its lifespan and/or in its efïicacy in the body, an increase in its solubility in solution or else an improvement in its conservation. These pharmaceutically acceptable vehicles arc well known and will be adapted by the person skilled in the art as a fonction of the nature and of the mode of administration of the active compound(s) chosen.
Preferably, these compounds will be administered by the systemîc route, in particular by the intravenous route, by the intramuscular, intradermal, intraperitoneal or subeutaneous route, or by the oral route. In a more preferred manner, the composition comprising the antibodies according to the invention will be administered several times, in a sequential manner.
Their modes of administration, dosages and optimum pharmaceutical forms can bc determined according to the criteria generally taken into account in the establishment of a treatment adapted to a patient such as, for cxample, the âge or the body weight of the patient, the scriousness of his/her general condition, the tolérance to the treatment and the secondary effccts noted.
Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures wherein:
Figure l : Effect of irrelevant IgGl Mabs from mouse and human origin and PBS on c-Met receptor phosphorylation on A549 cells.
Figures 2A and 2B: Effect of murine and humanized 224G11 Mabs produced as a human IgGl/kappa isotype on c-Met receptor phosphorylation on A549 cells. Figure 2A: agonist effect calculated as percentagc versus maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure 2B: antagonist effect calculated as percentage of inhibition of the maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 3A and 3B: Comparison between murine 224G11 Mab and chimeric 224G11 Mabs containing various engîneered hinge régions, on c-Met receptor phosphorylation on A549 cells.
Figure 3A: agonist effect calculated as percentagc versus maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure 3B: antagonist effect calculated as percentagc of inhibition of the maximal
PL· stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 4 A and 4B: Comparison between murine 224G11 Mab and chimeric and humanized 224G11 Mabs produced as a human TgG2/kappa isotype, on c-Met receptor phosphorylation on A549 cells.
Figure 4A: agonist effect calculated as percentage versus maximal stimulation of c-Met phosphorylation by HGF [ 100 ng/ml].
Figure 4B; antagonist effect calculated as percentage of inhibition of the maximal stimulation of c-Met phosphorylation by HGF [ 100 ng/ml].
Figures 5A and 5B: Comparison between murine 224G11 Mab and chimeric and humanized 224G11 Mabs produced as an engineered hinge mutant TH7IgGl /kappa, on c-Met receptor phosphorylation on A549 cells.
Figure 5A: agonist effect calculated as percentage versus maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure 5B: antagonist effect calculated as percentage of inhibition of the maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figures 6A and 6B, Figures 7A and 7B, Figures 8A and 8B, Figures 9A and 9B, Figures 10A and 10B: BRET models with Figures A: c-Met dimerization model; and Figures B: c-Met activation model.
Figure 11 : c-Met récognition by chimeric and humanized 224G11 forms.
Figure 12: Effect of murine and chimeric antibodies on HGF-induced prolifération of NCI-H441 cells in vitro. NCI-H441 cells were plated in serum-frec medium. 24 hours after plating m224Gll and [224Gll]chim were added either in absence or in presence of HGF. Black arrows indicatc the wells plated with cells alone either in absence or in presence il of HGF. A murine IgG l (mlgGl) was introduced as an isotype control.
Figure 13: In vivo comparison of murine and IgGl chimeric 224GU Mabs on the NCI-H441 xenograft model.
Figures 14A and 14B: Effect of the murine 224GU Mab and of various chimeric and humanized versions of this antibody on HGF-induced prolifération of NCI-H441 cells in vitro. NCI-H441 cells were plated in serum-free medium. Twenty four hours after plating antibody to be tested were added either in absence or in présence of HGF. In panel (Figure 14A), the murine m224Gll, chimeric IgGl [224Gll]chim, humanized IgGI [224G11] [Hzl], [224G11] [Hz2], [224G11] [Hz3] versions were shown. In panel (Figure 14B), the murine m224Gl 1 and various chimeric IgGI forms ([224G11] chim, [224GU] [MH chim], [224G11] [MUP9H chim], [224G11] [MMCH chim], [224G11] [TH7 chim]) were prcscnted. Black arrows indicate the wells plated with cells alone either in absence ü or in presence ii of HGF. A murine IgGI was introduced as a négative control for agonist activity. The m5D5 was used as a dose-dépendent full agonist control.
Figure 15; Effect of the murine 224G11 Mab and of various chimeric and humanized versions of this antibody on HGF-induced prolifération of NCI-H441 cells in vitro. NCI-H441 cells were plated in sérum-free medium. Twenty four hours after plating antibody to be tested were added either in absence or in presence of HGF. The murine m224Gll, [224G11] chim, [224G11] [TH7 chim]) IgGI chimeric forms and [224G11] [TH7 Hzl], [224G11] [TH7 Hz3],) were presented. Black arrows indicate the wells plated with cells alone either in absence Ιέ or in presence ùk of HGF. A murine IgGI was introduced as a négative control for agonist activity. The m5D5 was used as a dose-dependent full agonist control.
Figures 16A-16C: In vivo comparison of murine, chimeric and humanized 224G11 Mabs on the NCI-H441 xenograft model.
Figure 17A: agonist effect calculated as percentage versus maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure I7B; antagonist effect calculated as percentage of inhibition of the maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml].
Figure 18: BRET models with c-Met activation model.
Figures 19A-19B: Effect of m224Gll and h224Gll on c-Met dégradation on A549 cells. A) Mean of 4 independent experiments +/- s.e.m. B) Western blot image représentative of the 4 independent experiments performed.
Figures 20A-20B: Effect of m224Gl 1 and h224Gl 1 on c-Met dégradation on NCI-H441 cells. A) Mean of 4 independent experiments +/- s.e.m. B) Western blot image représentative of the 4 independent experiments performed.
Figure 21 : Set up of an ELISA to evaluate c-Met shedding.
Figure 22: In vitro évaluation of c-Met shedding on NCI-H441 cells treated for 5 days with m224G 11. mlgG 1 is an îrrclcvant antibody used as an isotype control.
Figure 23: In vitro évaluation of c-Mct sheddîng on amplified Hs746T, MKN45 and EBC-l cell lines treated for 5 days with m224Gl l. mlgGl is an irrelevant antibody used as an isotype control. PMA is a sheddîng inducer used as a positive control.
Figure 24: In vitro évaluation of c-Met sheddîng on NCI-H441 and amplified Hs746T, MK.N45 and EBC-l cell lines treated for 5 days with m224Gl l. mlgGl is an irrclevant antibody used as an isotype control. PMA is a sheddîng inducer used as a positive control.
Figure 25: Study of intrinsic phosphorylation of h224Gl l on Hs746T cell line.
Figures 26A-26B: Study of intrinsic phosphorylation of h224Gl l on NCI-H441 cell line. A) phospho-ELISA and B) Western analysis.
Figures 27A-27B: Study of intrinsic phosphorylation of h224GU on Hs578T cell line. A) phospho-ELISA and B) Western analysis.
Figures 28A-28B: Study of intrinsic phosphorylation of h224Gl l on NCI-H125 cell line. A) phospho-ELISA and B) Western analysis.
Figures 29A-29B: Study of intrinsic phosphorylation of h224Gl l on T98G cell line. A) phospho-ELISA and B) Western analysis.
Figures 30A-30B: Study of intrinsic phosphorylation of h224Gl l on MDA-MB231 cell line. A) phospho-ELISA and B) Western analysis.
Figures 31A-31B: Study of intrinsic phosphorylation of h224Gll on PC3 cell line. A) phospho-ELISA and B) Western analysis.
Figure 32: Study of intrinsic phosphorylation of h224Gl l on HUVEC cells.
Figure 33: In vivo comparison of the wild type murine 224G11 antibody with a chimeric hinge-engineered 224G1 l[C2D5-7] Mabs on the NCI-H441 xenograft model.
Figures 34A-34B: ADCC induction by h224Gll on both Hs746T and NCIH44l cells. ;iCr-labelcd Hs746T (A) or NCI-H441 (B) cells loaded (bold squares) or not (empty squares) with h224Gl l were mixed with different ratio of human NK cells and incubated for 4 hr. Cells were harvested and cpm of 51Cr reieased by lysis was counted. The results are plotted as percentage of lysis against the effector/target cell ratio. NL for non loaded cells.
Figures 35A-35C: h224Gll staining in tumor xenograft which expressed various ievel of c-Met (A: Hs746T amplified cell line for c-Met, B: NCI-H441 high ievel of c-Mct expression and C: MCF-7 low level of c-Mct).
vvL
Figures 36A-36D: In vivo activity of h224Gl l on the Hs746T xenograft model. A) FACS analysis showing c-Met expression on Hs746T cells compared to other cell Unes expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 37A-37D: In vivo activity of h224G 11 on the MK.N-45 xenograft model. A) FACS analysis showing c-Met expression on MK.N45 cells compared to other cell fines expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 38A-38D: In vivo activity of h224Gl l on the EBC-l xenograft model. A) FACS analysis showing c-Met expression on EBC-l cells compared to other cell fines expressing c-Met. B) QRTPCR showing c-Met amplification. C) c-Met phosphorylation in absence or in presence of HGF. D) In vivo model.
Figures 39A-39H: IHC examination of Hs746T tumors in mi ce treated with the h224G 11 Mab compared to control-treated tumors. (A) and (B) panels correspond to isotype control antibodies. (C) and (D) show the expression of c-Met on tumor sections from mice treated with PBS or h224Gll respectively demonstrating that a chronic treatment with h224G 11 induced a dramatic down régulation of c-Met. Ki-67 staining is significantly decreased on treated tumors (F) compared to PBS control samples (E). A significant apoptosis was observed in tumor treated with h224Gll (H) while no apoptosis was noticed in control tumors (G).
Example 1: Génération of antibodies against c-Met
To generate antî-c-Met antibodies 8 weeks oid BALB/c mice were immunized eîther 3 to 5 fîmes subcutaneously with a CHO transfected cell line that express c-Met on its plasma membrane (20x106 cells/dose/mouse) or 2 to 3 fîmes with a c-Met extracellular domain fusion protein (10-15 μg/dose/mouse) (R&D Systems, Catalog # 358MT) or fragments of this recombinant protein mixed with complété Frcund adjuvant for the first immunization and incomplète Freund adjuvant for the following ones. Mixed protocols in which mice reccived both CHO-cMet cells and recombinant proteins were also performed. Three days before cell fusion, mice were boosted î.p. or i.v. with the recombinant protein or fragments. Then spleens of mice were collected and fused to SP2/0-Agl4 myeloma cells (ATCC) and subjected to HAT sélection. Four fusions were performed. In general, for the préparation of monoclonal antibodies or their functional fragments, especially of murine origîn, it is possible to refer to techniques which are described in particular in the manual “Antibodies” (Harlow and Lanc, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the technique of préparation of hybridomas described by Kohler and Milstein (Nature, 256:495-497, 1975).
Obtained hybridomas were initial ly screened by ELIS A on the c-Met recombinant protein and then by FACS analysis on A549 NSCLC, BxPC3 pancreatic, and U87-MG glioblastoma cell lines to be sure that the produced antibodies will be able to also recognize the native receptor on tumor cells. Positive reactors on these 2 tests were ampli fied, cloned and a set of hybridomas was recovered, purified and screened for its ability to inhibit in vitro cell prolifération in the BxPC3 model.
For that purpose 50 000 BxPC3 cells were plated in 96 well plates in RPM1 medium, 2 mM L. Glutamine, without SVF. 24 hours after plating, antibodies to bc tested were added at a final concentration ranging from 0,0097 to 40 pg/ml 60 min before addition of 100 ng/ml of hHGF. After 3 days, cells were pulsed with 0.5 pCi of [^HJthymidinc for 16 hours. The magnitude of [3H]thymidine incorporated into trichloroacetic acid-insoluble DNA was quantified by liquid scintillation counting. Results were expressed as raw data to really evaluate the intrinsic agonistîc effect of each Mab.
Then antibodies inhibiting at least 50% cell prolifération were evaluated for their activity on c-Met dimerization and activation BRET analysis on transfectcd cells, c-Met receptor activity was quantified by measuring the Gabl signalling molécule recruitment on activated c-Met. For that purpose, CHO stable cell lines expressing C-Met-Rluc or C-Met-Rluc and C-Met-Kl 100A-YFP for c-Met dimerization or C-Met-Rluc and a mutated form of Gabl [Maroun et al., Mol. Cell. Biol., 1999, 19:1784-1799] fused to YFP for c-Met activation were generated. Cells were distributed in white 96 well microplates in DMEM-F12/FBS 5 % culture medium one or two days before BRET experiments. Cells were first eultured at 37°C with CO2 5 % in order to allow cell attachment to the plate. Cells were then starved with 200 μΐ DMEM/well ovemight. Immediately prior to the experiment, DMEM was removed and cells quickly washed with PBS. Cells were incubated in PBS in the presence or absence of antibodies to be tested or reference compounds, 10 min at 37°C prior to the addition of coelentcrazine 52 with or without HGF in a final volume of 50 μΐ. After incubation for further 10 minutes at 37°C, light-cmîssion acquisition at 485 nm and 530 nm was initiated using the
Mithras luminometer (Berthold) (ls/wave length/well repeated 15 times).
BRET ratio has been defined previously [Angers et al., Proc. Natl. Acad. Sci. 5 USA, 2000, 97:3684-3689] as: [(émission at 530 nm) - (émission at 485 nm) X Cf] / (émission at 485 nm), where Cf corresponds to (émission at 530 nm) / (émission at 485 nm) for cells expressing Rluc fusion protein alone in the same experimental
I conditions. Simplîfyîng this équation shows that BRET ratio corresponds to the ratio 530/485 nm obtained when the two partners were présent, corrected by the ratio 10 530/485 nm obtained under the same experimental conditions, when only the partner fused to R. reniformis lucîferase was présent in the assay. For the sake of readabîlity, results are expressed in mîlliBRET units (mBU); mBU corresponds to the BRET ratio multiplied by 1000.
After this second in vitro test, the antîbody 224G11 i) without intrinsic activity 15 as a whole molécule in the functional test of prolifération, ii) inhibiting significantly BxPC3 prolifération and iii) inhibiting c-Met dimerization was selected. In the experiments, the 5D5 Mab, generated by Genentech, and avaîlable at the ATCC, was added as a control for the intrinsic agonistic activity.
Example 2: Humanization process of mouse 224G11 Mab by CDR-grafting
Humanizatîon of the light chain variable domain (VL)
As a preliminary step, the nucléotide sequence of 224G11 VL was compared to the murine germlîne gene sequences încluded in the IMGT database (http://imgt.cines.fi·). Murine IGKV3-5*O1 and IGKJ4*01 germlîne genes showing a 25 sequence identity of 99.31 % for the V région and 94.28 % for the J région, respectîvely, hâve been identified. Regarding these high homologies, the 224G11VL nucléotide sequence has been used directly to search for human homologies, instead of corresponding mouse germlines.
In a second step, the human germline gene displaying the best identity with the 30 224G11VL has been scarched to identîfy the best human candidate for the CDR grafting. For optimization of the sélection, alignments between the amino acid séquences have been performed. The human IGKV4-l*01 germline gene yicldcd a sequence identity of 67.30 %, but showed a different iength for CDRl (10 amino acids in 224G11 VL and 12 amino acids in IGKV4-l*0l). For the J région, the human !GKJ4*02 germline genc (sequence identity of 77.14 %) was selected.
ln a ncxt step, mouse 224G11 VL CDR régions were engrafted into the above selected human framework sequences. Each amino acid position was analyzed for several criteria such as participation in VH/VL interface, in antigcn binding or in CDR structure, localization of the residue in the 3D structure of the variable domain, CDR anchors, residues belonging to the Vernier zone. Three humanized versions, corresponding to SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10 were constructed, and containing respectively four (4, 39, 40, 84), two (39, 40) or one (40) murine residues in their FR régions and the CDRs corresponding to mouse 224G11 VL.
2°) Humanization of the heavy chain variable domain (VH)
As a prelîminary step, the nucleotidic sequence of the 224GH VH was compared to the murine germline genes sequences included in the IMGT database (http.7/imgt,cines.fr).
Murine IGHVi-l8*0l, lGHD2-4*0l and IGHJ2*0l germline genes with a sequence identity of 92.70 % for the V région, 75.00 % for the D région and 89.36 % for the J région, respectively, hâve been identified. Regarding these high homologies, it has been decided to use directly the 224GHVH nucléotide sequences to search for human homologies, instead of corresponding mouse germlines.
ln a second step, the human germline gene displaying the best identity with the 224GH VH has been scarched to identify the best human candidate for the CDR graftîng. To this end, the nucleotidic sequence of 224G11 VH has been aligned with the human germline genes sequences belonging to the IMGT database. The human IGHVl2*02 V sequence exhibited a sequence identity of 75.00 % at the nucléotide level and 64.30 % at the amino acid level. Looking for homologies for the J région led to the identification of the human IGHJ4*04 germline gene with a sequence identity of 78.72 %.
In a next step, mouse 224G11 VH CDR régions were engrafted into the above selected human framework sequences. Each amino acid position was analyzed for several criteria such as participation in VH/VL interface, in antigcn binding or in CDR structure, localization of the residue in the 3D structure of the variable domain, CDR anchors, residues belonging to the Vernier zone. One fully humanized form, corresponding to SEQ ID 4 was constructed; it contains exclusively human residues in its FR régions and the CDRs corresponding to mouse 224G11 VH.
Example 3: Engineering of improved hinge mutants
It is well known by the skilled artisan that the hinge région strongly participâtes in the flexibility of the variable domain of immunoglobulîns (see Brekke et al., 1995; Roux et al., 1997). During the chimerization process of 224G11 Mab, the mouse constant domain IGHGl was replaced by the équivalent IGHGl portion of human 10 origin. Since the amino acid sequence of the hinge région were highly divergent, “murinization” of the hinge région was performed in order to keep its length and rigidîty. Since the human 1GHG2 hinge région corresponds to the closest homologue of the mouse IGHGl hinge, this sequence was as well considered. A sériés of 7 different hinge sequences were constructed (SEQ ID Nos. 22 to 28) by incorporating portions of 15 the mouse IGHGl and the human IGHG2 hînges into the human IGHGl hinge portion.
Another séries of hinge mutants was designed and constructed (SEQ ID Nos. 58 to 72) to evaluate the influence of either an additionaî cysteine and its position along the hinge domain, délétion of t, 2, 3 or 4 amino acids along the hinge domain and a combination of these two parameters (cysteine addition and amino acid délétion).
Example 4: Production of humanized 224G11 Mab and engineered hinge Mab formats
Ail above described Mab forms contaîning either chimeric, humanized and/or engineered hinge régions were produced upon transient transfection and by using the 2 5 HEK293/EBNA System with a pCEP4 expression vcctor (InVitrogen, US).
The entire nucléotide sequences corresponding to the humanized versions ofthe variable domain of 224G11 Mab light (SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20) and heavy (SEQ ID No. 14) chains were synthesized by global gene synthesis (Genecust, Luxembourg). They were subcloned into a pCEP4 vector (InVitrogen, US) 30 carrying the entire coding sequence of the constant domain [CH1 -Hinge-CH2-CH3] of a human IgGl or IgG2 immunoglobulîn. Modification of the hinge région was performed by cxchanging a {Nhell-Bcll} restriction fragment by the équivalent portion carrying the desired modifications, each respective {Nhel-Bcll} fragment being synthesized by global gene synthesis (Genecust, LU). Ail cloning steps were performed according to conventional molecular biology techniques as described in the Laboratory manual (Sambrook and Russcl, 2001) or according to the supplier’s instructions. Each genetic construct was fully validated by nucléotide sequencing using Big Dyc terminator cycle sequencing kit (Applied Biosystems, US) and analyzed using a 3100 Genetic Analyzer (Applied Biosystems, US).
Suspension-adapted HEK293 EBNA cells (InVitrogen, US) were routinely grown in 250 ml flasks in 50 ml of serum-frec medium Excell 293 (SAFC Biosciences) supplemented with 6 mM glutamine on an orbital shaker (110 rpm rotation speed). Transient transfection was performed with 2.106 cells/ml using linear 25 kDa polyethyleneimine (PEI) (Polysciences) prepared in water at a final concentration of 1 mg/ml mixed and plasmid DNA (final concentration of 1.25 gg/ml for heavy to light chain plasmid ratio of 1:1). At 4 hours post-transfection, the culture was diluted with one volume of fresh culture medium to achieve a final cell density of 106 cells/ml. Cultivation process was monitored on the basis of cell viability and Mab production. Typically, cultures were maintained for 4 to 5 days. Mabs were purified using a conventional chromatography approach on a Protein A resin (GE Healthcare, US).
Ail different forms of Mabs were produced at levels suitable with functional évaluations. Productivity levels are typically ranging between 15 and 30 mg/1 of purified Mabs.
Example 5: Evaluation of c-Met phospshorylatîon status by a Phospho-cMet-specific ELISA assay
This functional assay allows to monitor modulation c-Met phosphorylation status either by Mabs alone or in the co-prescnce of HGF.
A549 cells were seeded in a 12MW plate in complété growth medium [F12K + 10 % FCS]. Cells were starved for 16 hours before stimulation with HGF [100 ng/ml], and each Mab to be tested was added at its final concentration of 30 .ug/ml 15 minutes prior to ligand stimulation. Ice-cold lysis buffer was added 15 minutes after the addition of HGF to stop the phosphorylation réaction. Cells were scaped mechanically and cell lysâtes were collected by centrifugation at 13000 rpm for 10 min. at 4°C and correspond to the supematant phase, Protein content was quantified using a BCA kit (Pierce) and stored at -20°C until use. The phosphorylation status of c-Met was quantified by ELISA. A goat anti-c-Mct Mab (R&D, ref AF276) was used as a capture antibody (ovemight coating at 4°C) and after a saturation step with a TBS-BSA 5% buffer (l hour at room température (RT)), 25 μ g of protein lysâtes were added to each well of the coated 96MW plate. After a 90 minutes incubation at RT, plates were washed four time and the détection antibody was added (anti-phospho-c-Met Mab, directed against the phopshorylated Tyr residues at position 1230, 1234 and 1235). After an additional 1 hour incubation and 4 washes, an anti-rabbit antibody coupled to HRP (Biosource) was added for 1 hour at RT, and the luminescence détection was performed by adding Luminol. Luminescence rcadings were on a Mithras LB920 multimodc plate reader (Bcrthold).
Both basal and HGF [100 ng/ml]-induced c-Met receptor phosphorylation level were unaffected neither by PBS treatment, nor by the addition of mouse or human Mabs which do not target human c-Met receptor (Figure 1 ). On the other hand, mouse (m) 224G11 Mab strongly inhibited HGF [100 ng/ml]-induced c-Met phosphorylation (Figure 2B) without altering by îtself receptor phosphorylation (Figure 2A). Surprisingly, the chimeric form of 224G11 Mab (224G11 chim/IgG 1 ), meaning variable domain (VH+VL) fforn m224Gl 1 combined with human constant domain IgGl/kappa yielded strong (17 % of maximal HGF effect, Figure 2A) agonist activity associated with a reduced antagonist efficacy (54 % inhibition of HGF maximal effect compared to the m224Gl 1 that yields 75% inhibition of HGF maximum effect, Figure 2B). Three humanized forms of 224G11 Mab, [224Gll]Hzl/IgGl, [224G1 l]Hz2/IgGl and [224G1 l]Hz3/IgGI, also constructed on a human IgGl/kappa backbone, yielded also decreased antagonist efficacy and significant agonist activity (11 to 24 % of maximal HGF level) as compared to mouse 224G11 (Figures 2A and 2B), A sériés of engineered versions of the heavy chain hinge domain were constructed and assayed in the c-Met receptor phosphorylation assay. As shown in Figure 3A, an important réduction of the agonist effect associated with the hlgGl/kappa isotype was observed for both the IgG2based construct and for engineered IgGl/kappa constructs [MH, MUP9H and TH7]. A concomitant increase in antagonist efficacy was as well obtained. The hlgGI/kappabased TH7 hinge mutant, with the most human scqucncc, was selected to complète the humanization process. In a next step, three humanized versions of 224GJ l Mab variable domain were generated by combination to either a human IgG2/kappa or an IgGl/kappa-based TH7 engineered hinge constant domain. For the hlgG2/kappa humanized constructs, the humanized version Hz3 yielded strong agonism (Figure 4A), and for ail three humanized versions, the antagonist efficacy was below that observed with murine 224G11 Mab and comparable to the chimeric hlgGl-based Mab (56-57 % inhibition of HGF effect, Figure 4B). On the other hand, combination of the three humanized versions Hzl, Hz2 or Hz3 to the engineered IgGl/TH7 mutant almost fully restored the properties of mouse 224G11 Mab in terms of weak agonîst activity (5-6 % of HGF effect) and strong antagonist efficacy (68 to 72 % inhibition of HGF effect) of c-Met receptor phosphorylation (Figures 5A and 5B). These variants were highly improved as compared to chimeric IgG 1-based 224G11 Mab but also to lgG2-based humanized forms.
A second sériés of engineered versions of the heavy chain hinge domain was constructed and assayed in the c-Met receptor phosphorylation assay. As shown in figure 17A, ail those new versions (c224Gl 1[C2], c224Gll[C3], c224Gll[C5], c224Gll[C6], C224G11[C7], c224Gl 1[Δ1-3], c224Gll[C7A6], c224Gll[C6A9], c224Gll[C2A5-7], c224Gl l[C5A2-6], c224G 11 [C9A2-7] and c224GH[A5-6-7-8J) exhibited wcaker agonist effect than c224Gll since their agonism activities are compriscd between 6 and 14% of the HGF effect compared to 23% for c224Gl 1. As c224Gl 1[TH7], ail those new versions exhibited a concomitant increase in antagonist efficacy [figure I7B], Those results showed that engineering of the heavy chain domain by point mutation and/or délétion could modify agonistic/antagonîstîc properties of an antibody.
Example 6: BRET analysis
In a first set of experiments, it had been control that irrelevant mouse IgGl, human IgGl and human IgG2 had no effect of HGF induced BRET signal in both BRET models (représentative experiment out of 12 independent experiments; Figure 6). These Mabs are forthwith cited as controls.
The effect of a IgGl chimeric form of mouse 224G11 Mab ([224Gll]chim) on both c-Met dimerization and c-met activation BRET model was evaluated. Whîle mouse
Us
224G11 Mab inhibited 59.4% of the HGF induced BRET signal on c-Met dimerization mode!, [224G1 ljchim Mab inhibited only 28.9% (Figure 7 A). [224Gll]chim antibody was also less effective in inhibiting HGF induced c-Met activation since [224G1 ljchim and m224Gll antibodies inhibited respectively 34.5% and 56.4% of HGF induced BRET signal (Figure 7B). Moreover, m224Gl l alone had no effect on c-Met activation while [224G1 ljchim had a partial agonist effect on c-Met activation corresponding to 32.9% of the HGF induced signal. This partial agonist effect of the [224G1 ljchim was also seen on c-Met dimerization BRET model since [224G1 ljchim alone induced a BRET increase corresponding to 46.6% of HGF-induccd signal versus 21.3% for m224Gll (Figure 7A).
In Figures 8A and 8B, hinge mutated chimeric forms of 224G11 antibody showed a greater inhibitory effect on HGF induced BRET signal than [224G1 ljchim since they showed a 59.7%, 64.4%, 53.2% and 73.8% inhibition of the HGF induced activation BRET signal (Figure 8B) and 61.8%, 64.4% 52.5% and 64.4% inhibition of the HGF induced c-Met dimerization BRET signal (Figure 8A) for [224G11][MH chim], [224G11][MUP9H chim], [224G11][MMCH chim] and [224G11][TH7 chim] respectively. Contrary to [224G1 ljchim, which had a partial agonist effect on c-Met activation, hinge mutated chimerical forms of 224G11 antibody showed no significant effect on c-Met activation alone (5.1%, 7.6%, -2.0% and -6.9% respectively) as observed for m224Gll.
In Figure 9B, like the [224G11] [TH7 chim], the 3 humanized versions of 224G11 IgGl antibody with the TH7 hinge induced no significant increased of BRET signai in activation model when tested alone and showed a strong inhibition of HGF induced BRET signal: 59.9%, 41.8% and 57.9% for the Hzl, Hz2 and Hz3 forms respectively. Moreover, [224G11] [TH7 Hzl], [224G11] [TH7 Hz2] and [224G11] [TH7 Hz3] inhibited HGF induced BRET signal on dimerization model of 52.2%, 35.8% and 49.4% respectively (figure 9A).
Contrary to [224G1 ljchim, the chimeric form of 224G11 IgG2 antibody ([224G11] [IgG2 chim]) showed no partial agonist effect alone and inhibited 66.3% of the HGF effect on c-Met activation model (Figure 10B). On c-Met dimerization model, [224G11] [IgG2 chim] inhibited 62.4% of the HGF induced BRET signal (Figure 10A).
The agonist cfficacy of the second séries of cngincercd versions of the heavy
V chain hinge domain was evaluated in c-Met activation BRET model (Figure 18). In contrast to c224Gll, which had a partial agonist effect on c-Met activation, c224Gll[C2], c224Gll[C3], c224Gll[C5], c224Gll[C6], c224Gll[C7], c224Gll[Al-3], c224Gl I[C7A6], c224Gll[C6A9], c224Gll[C2A5-7], c224Gll[C5A2-6], c224Gl l[C9A2-7] and c224Gl 1[Δ5-6-7-8] hinge mutated chimeric forms of 224G11 antibody showed no significant effect on c-Met activation alone.
Example 7: c-Met récognition by chimeric and humanized 224G11 forms
A direct EU SA has been set up to détermine the binding ability of the various chimeric and humanized forms on the recombinant c-Met. Briefly recombinant dimeric c-Met from R&D Systems was coated at 1.25 pg/ml on 96-well Immunlon II plates. After an overnight incubation at 4°C, wells were saturated with a 0.5% gelatinc/PBS solution. Plates were then incubated for 1 hour at 37°C before addition of 2 fold dilutions of antibodies to be tested. Plates were incubated an additional hour before addition of a goat antî-mousc IgG HRP for dctccting the murine antibody and a goat anti-human Kappa light chain HRP for chimeric and humanized antibody récognition. Plates were incubated for one hour and the peroxydase substrate TMB Uptima was added for 5 mn before neutralization with H^SOj IM, Results presented in Figure 11 showed that all tested forms were comparable for c-Met récognition.
Example 8: Effect of murine and chimeric 224G11 on HGF-induced prolifération of NCI-H441 cells in vitro
NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% LGlutaminc (Invitrogen corporation). For prolifération assays, cells were split 3 days before use so that they were in the confluent phase of growth before plating, NCI-H441 cells were plated in 96-well tissue culture plates at a density of 3,75xl04 cells/well in 200 μΐ of sérum free medium (RPMI 1640 medium plus 1% L-Glutamine). Twenty four hours after plating, antibodies to be tested were added to NCI-H441 and incubated at 37°C for thirty minutes before adding HGF at a final concentration of 400 ng/ml (5 nM) for 142 additional hours, The dose range tested for each antibody is from 10 to 0.0097 Lig/ml (final concentration in each well). In this experiment, a murine IgGl Mab
AL· *
was added as a murine isotype control and the tested antibodies were the following one: m224Gl l and its human IgGl chimeric form îdentified as [224G1 ljchim. Wells plated with cells alone -/+ HGF were also included. Then cells were pulsed with 0,25 pCi of ['HjThymidinc (Amersham Biosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of [3H]Thymidine incorporated in trichloroacctic acid-insoluble DNA was quantified by liquid scintillation counting. Results are expressed as non transformed cpm data to better evaluate the potential intrinsic agonist actîvity that could occur with anti-c-Met Mabs when added alone to tumour cell.
Results described in Figure 12 demonstrated that, as expected, the murine antibody m224Gll displayed no agonist effect when added alone to cancer cells whatever the tested dose. No signîficant inhibition of the HGF-induced prolifération was observed with the isotype control regarding to the cpm variations observed for this compound in this experiment. When added alone, the m224Gl 1 antibody did not show any agonist effect compared to the mlgGl îsotype control Mab or cells alone. A dose dépendent antî-proliférative activitics rcaching 78% was observed for m224Gll (% inhibition calculation: 100-[(cpm cells+Mab to be tested-mean cpm background mlgGl) x 100 / (mean cpm cells + HGF- mean cpm cells alone)]). Surprisingly, the chimeric form of the 224G11 Mabs induced a signîficant, dose dépendent agonist effect when added alone. This agonist effect had an impact on the in vitro inhibition of HGFinduced prolifération that shifted from 78% for the murine 224G11 to 50% for its chimeric form. To détermine whether such “lower” in vitro intrinsic agonist actîvity was compatible with an unchanged in vivo effect, both m224Gl 1 and [224G1 ljchim were produced for in vivo testing. As, in previous studies, the 30 pg/mice dose had demonstrated a signîficant in vivo actîvity, that dose was selected for in vivo évaluation.
Example 9: In vivo comparison of murine and chimeric 224G11 Mabs on the NCI-H441 xenograft model
NCI-H441 is derived from papillary lung adenocarcinoma, expresses high levels of c-Met, and demonstrates constitutive phosphorylation of c-Met RTK
To evaluate the in vivo effect of antibodies on the NCI-H441 xenograft model, six to eight weeks old athymie mîce were housed in sterilized filter-topped cages, maintained in stérile conditions and manipulatcd according to Frcnch and Europcan
H guidelines. Mice were înjected subcutaneously with 9x106 cells. Then, six days after cell implantation, tumors were measurable (approximately 100 mm3), animais were divided into groups of 6 mice with comparable tumor size and treated first with a loading dose of 60 pg of antibody/mice and then twice a week with 30 pg/dose of each antibody to be tested. The mice were followed for the observation of xenograft growth rate. Tumor volume was calculated by the formula: π (Pi)/6 X length X width X height. Results described in Figure 13 demonstrate that the murine Mab devoîded of agonist activity in vivo behave, as cxpcctcd, as potent antagonist even at the low tested dose. In contrast to what observed with the murine Mab, the chimeric one displayed a very transient in vivo activity and tumor completely escaped to the treatment at D20 post cell injection. This experiment demonstrates clearly that the increase of in vitro agonist effect that resulted in a decrease of antagonist activity was also responsible for a significant in vivo loss of antagonist activity,
Example 10: Effect of the murine 224G11 Mab and of various chimeric and humanized versions of this antibody on HGF-induced prolifération of NCI-H441 cells in vitro
NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium (Jnvitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% LGlutaminc (Invitrogen Corporation). For prolifération assays, cells were split 3 days before use so that they were in the confluent phase of growth before plating. NCI-H441 cells were plated in 96-well tissue culture plates at a density of 3.75x104 cclls/wcll in 200 μΙ of sérum free medium (RPMI 1640 medium plus 1% L-Glutamine). Twenty four hours after plating, antibodies to be tested were added to NCI-H441 and incubated at 37°C for thirty minutes before adding HGF at a final concentration of 400 ng/ml (5 nM) for 142 additionai hours. The dose range tested for each antibody is from 10 to 0.0097 pg/ml (final concentration in each well). In this experiment, murine IgGl Mab was added as a murine isotype control and as an agonist négative control. The tested antibodies were the following one: î) m224Gll, ii) its human IgGl chimeric forms respectively identifîed as [224G11] chim, [224G11] [MH chim], [224G11] [MUP9H chim], [224GI1] [MMCH chim], [224G11] [TH7 chim] iii) its humanized IgGl forms respectively described as [224G11] [Hzl], [224G11] [Hz2], [224G11] [Hz3], Wells plated with cells alone -/+ HGF were also included. The 5D5 whole antibody from Genentech commercially available at the ATCC as an hybridoma cell line was introduced as a fiill agonist positive control and thereafter called m5D5. Then cells were pulscd with 0.25 pCi of [ 'HJThyrnidinc (Amersham Biosciences AB, Uppsala, Swcden) for 7 hours and 30 minutes. The magnitude of ['HjThymidinc incorporated in trichloroacetic acid-insoluble DNA was quantified by liquid scintillation counting. Results are expressed as non transformed cpm data to better evaluate the potential intrinsic agonist activity that could occur with anti-c-Met Mabs when added alone to tumour cell.
Results described in Figure 14A demonstrated that as expected neither the isotype control nor the m224Gll displayed any agonist activity on NCI-H44I prolifération. The isotype control was without effect on HGF-induced cell prolifération whereas m224Gll showed a 66% inhibition when added at the final concentration of l0pg/ml. The m5D5 used as an agonist control showed, as expected, a full dose dépendent agonist effect when added alone to the cells. As already observed, the [224G11] chim Mab displayed a significant dose- dépendent agonist effect and, a decreased inhibitory activity of this chimeric form was observed: 19% instead of 66% for the murine form. When added alone, the 3 IgGl humanized Mabs demonstrated dose dépendent agonist effects compared to the m224Gll form. [224G11] [Hzl], [224G11] [Hz2] and [224G11] [Hz3] had comparable antagonist activities about 46, 30 and 35%. These activities are significantly lower than the one observed for m224Gl 1. In Figure 14B, various IgGl chimeric forms were tested. Compared to [224G11] chim form which displayed a dose-dependent agonist effect when added alone to NCI-H441 cells, the [224G11] [MH chim], [224G11] [MUP9H chim], [224G11] [MMCH chim], [224G11] [TH7 chim] forms were without significant intrinsic agonist effect. Their antagonist activity was higher than the one observed for the m224G 11 Mab (57%) with inhibitions reaching 79, 78, 84 and 93% respectively for [224G11] [MH chim], [224G11] [MUP9H chim], [224G11] [MMCH chim] and [224G1I] [TH7 chim].
Example 11: In vitro effect of various IgGl humanized form of the 224G11 Mab
NC1-H441 cells from ATCC were routîncly eultured in RPMI 1640 medium (Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogcn Corporation), l% L! Glutamine (Invitrogen Corporation). For prolifération assays, cells were split 3 days i
i before use so that they were in the confluent phase of growth before plating. NCI-H441 , cells were plated in 96-well tissue culture plates at a density of 3.75χ104 cells/well in j 5 200 μΐ of sérum free medium (RPMI 1640 medium plus l% L-Glutamine). Twenty four ΐ
; hours after plating, antibodies to be tested were added to NCI-H441 and incubated at j 37°C for thirty minutes before adding HGF at a final concentration of 400 ng/ml (5 nM) ! for 142 additional hours. The dose range tested for each antibody is from 10 to
I 0.0097 pg/ml (final concentration in each well). In this experiment, murine IgGl Mab was added as a background négative control for agonist activity and the tested antibodies were the following one: i) m224Gll, îi) its human IgGl chimeric forms rcspcctively identified as [224G11] chim, [224G11] [TH7 chim] iiî) îts humanized IgGl i
j forms respectively described as [224G11] [TH7 Hzl], [224G11] [TH7 Hz3]. Wells [ plated with cells alone -/+ HGF were also included. The 5D5 whole antibody from
15 Genentech commercially availablc at the ATCC as an hybridoma cell line was introduced as a full agonist positive control and thereafier called m5D5. Then cells were pulsed with 0.25 pCi of pHJThymidine (Amersham Biosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of [;'H]Thymidinc incorporatcd in trichloroacetic acid-insoluble DNA was quant ified by liquid scintillation counting.
Results are expressed as non transformed cpm data to better evaluate the potentîal intrinsic agonist activity that could occur with anti-c-Met Mabs when added alone to tumour cell.
Figure 15 showed that the m224GU Mab displayed the usual inhibitory effect (74% inhibition). The chimeric IgGl form [224G11] chim had as expected a dose 2 5 dépendent intrinsic agonist effect and a lower antagonist effect compared to the murine form: 33% versus 74% inhibition, The [224G11] [TH7 chim] had a very weak agonist activity in this experiment. However it displayed a high inhibitory effect (81 %) close to the one noticed for the murine Mab. The 2 humanized forms had no intrinsic agonist effect and had an antagonist activity close to the ones observed for the murine Mab or 30 the [224G11] [TH7 chim] with respectively 67 and 76% inhibition for [224G11] [TH7 Hzl] and [224G11] [TH7 Hz3].
Example 12: ln vivo comparison of murine, chimeric and humanized
224G11 Mabs bearing either the wild type or the TH7- engineered hinge (NCIH441 xenograft model).
NCI-H441 is derived from papillary lung adenocarcinoma, expresses high levels of c-Met, and demonstrates constitutive phosphorylation of c-Met RTK.
To evaluate the necessity of hinge engineering to save in vivo activity of the 224G11 murine antibody, six to eight weeks old athymie mîce were housed in sterilized filtcr-topped cages, maintaîned in stérile conditions and manipulated according to French and Europcan guidelînes. Mice were injected subcutaneously with 9xl06 NCi10 H441 cells. Then, six days after cell implantation, tumors were measurable (approximately 100 mm’), animais were divided into groups of 6 mice with comparable tumor size and treated first with a loading dose of 2 mg of antibody/micc and then twice a week with a 1 mg/dose of each antibody to be tested. Ten antibodies were evaluated in this experiment including the m224Gll, the chimeric form displaying the wild type 15 hinge (c224Gll), the TH7-engineered chimeric form (224G11[TH7 chim]), three humanized form bearing the wild type hinge (224Gll[IgGl Hzl], 224GU[IgGI Hz2] and 224Gll[IgGl Hz3]) and the three corresponding TH7-engineered forms (224G11[TH7 Hzl], 224G11[TH7 Hz2] and 224G11[TH7 Hz3]). Mice were followed for the observation of xenograft growth rate.
Tumor volume was calculatcd by the formula: π (Pi)/6 X lcngth X width X height.
Results described in figure 16 demonstrate that the murine Mab devoid of any agonist activity in vitro behave, as expected, as potent in vivo antagonist. In contrast to what observed with the murine Mab, both chimeric and humanized forms bearing the 25 wild type hinge displayed only a very transient in vivo activity. In any cases the substitution of the wild type hinge by the TH7-engineered one resulted in a complété restoration of the in vivo activity observed with murine antibodies. This experiment demonstrates clearly that the increase of in vitro agonist effect that resulted in a decrease of antagonist activity was also rcsponsiblc of a signîficant in vivo loss of 30 antagonist activity. It also demonstrates that the use of a TH7-engineered région instead of the wild type one is needed for keeping the in vivo properties of the murine Mab.
VvL
Example 13: Effect of m224Gll and its humanized form 11224G11 on c-Met down régulation in vitro in the following examples, for the avoidance of doubt, the expression h224Gl I refers to the humanized form 224G11 [TH7 Hz3] of the antibody of the invention.
Two cell lines hâve been selected to address the activity of antî-c-Met antibodies on c-Met receptor dégradation. A549 (#HTB-174) and NCI-H441 (#CCL-185) are two NSCLC cell lines from the ATCC collection. NCI-H441 cells were seedcd in RPMI 1640 + 1% L-glutamine + 10% heat-inactivated FBS, at 3x10* cells/cm2 in six-well plates for 24 h at 37°C in a 5% CO? atmosphère. A549 cells were seeded in F12K + 10% heat-inactivated FBS, at 2x104 cells/cm2 in six-well plates for 24 h at 37°C in a 5% CO2 atmosphère.
Then, cells were washed twice with phosphate buffer saline (PBS) before being serum-starved for 24 additional hours. Anti-c-Met antibodies (10 pg/ml), irrelcvant mïgGl(10 pg/ml), or HGF (400 ng/mL) were added in sérum-free DMEM medium at 37°C. After eîthcr 4 hours or 24 hours of incubation, the medium was gently removed and cells washed twice with cold PBS. Cells were lyscd with 500 pL of ice-cold lysis buffer [50 mM Tris-HCl (pH 7.5); 150 mM NaCl; 1% Nonidet P40; 0.5% deoxycholate; and 1 complété protease inhibitor cocktail tablet plus 1% antiphosphatases]. Cell lysâtes were shaken for 90 min at 4°C and cleared at 15 000 rpm for 10 minutes. At this stage, cell lysâtes could be stored at -20°C until nceded for western blot analysis. Protein concentration was quantified using BCA. Whole cell lysâtes (5pg in 20 pl) were separated by SDS-PAGE and transferred to nitroceliulose membrane. Menbranes were saturated for 1 h at RT with TBS-Tween 20 0.1%(TBST); 5% non-fat dry milk and probed with anti-c-Met antibody (dilution 1/1000) ovemight at 4°C in TBST-5% nonfat dry milk. Antibodies were diluted in tris-buffered saline-0.1% tween 20 (v/v) (TBST) with I % non-fat dry milk. Then, membranes were washed with TBST and incubatcd with peroxydase-conjugated secondary antibody (dilution 1:1000) for 1 h at RT. Immunoreactive proteins were visualized with ECL (Pierce # 32209). After c-Met visualization, membranes were washed once again with TBST and incubated for 1 h at RT with mouse anti-GAPDH antibody (dilution 1/200 000) in TBST-5% non-fat dry milk. Then, membranes were washed in TBST and incubated with peroxydaseconjugated secondary antibodies, for 1 h at RT. Membranes were washed and GAPDH was revealed using ECL. Band intensif y was quanti fîed by densitometry.
Results presented in figures 19 A and 20A demonstrated that both m224G 11 and h224Gl 1 arc able to significantly downregulate c-Met, in a dose-dependant way, in both A549 and NCI-H441 cell fines. The downregulation is already signifïcant after a 4 hour incubation time and still increased at 24 hour. Histograms presented in figures 19A and 20A corresponds to mean values or respectively 4 and 3 independcnt experiments. Western blot images corresponding to one signifïcant expcriment were included in figures 19B and 20B.
Example 14: Effect of m224Gll and its humanized form h224Gll on c-Met shedding in vitro
Soluble shedded forms of the c-Met receptor occur naturally in the sérum of mice xenografted with human tumor or in sérum of human patient carrying tumors expressing c-Met. Moreover, antibodies directed against c-Met such as the DN30 Mab, are described as shedding inducers of c-Met in in vitro experiments. To déterminé whether the m224Gl 1 as such a property, cells were seeded in six-well plates in 10% FCS medium. When they reached approximately 80% confluence, medium was removed and fresh complété culture medium +/- compounds to be tested was added. Cells were incubated 72 additional hours with either m224Gll, an isotype control mlgGl or PBS. PMA (phorbol meristate acetate) was introduced as a shedding inducer. HGF was also tested on cells to détermine the impact of c-Met ligand on naturel occurring shedding. Then supematants were collected and filtered on 0.2 gm before use in an ELISA test which soluble forms of c-Met were captured with an anti-c-Met antibody that does not rccognize the same epitope as either m224Gl 1 or the cl 1E1 (Figure 21). Moreover, cells from each well were washed once with PBS and lysed to déterminé protein concentration. For the ELISA, 224D10 was used as a capture antibody and after plate saturation, filtered supematants from six well plates were added in the ELISA test. A monomeric c-Met form was used as a positive control. After supematant incubation, plates were washed to remove the unbound c-Met and cil El was used to detect c-Met captured by the 224G11 Mab. The révélation of the test was finally performed by addition of an HRP-conjugated anti-hFc polyclonal antibody.
Results shown in figure 22 indicatc that a naturel shedding of c-Met occurrcd
ÎvL when cells were cultured for 72 hours in vitro. No effect of the mlgG l was observed.
However, the addition of m224G 11 seemed to inhibit c-Met shedding. These results were confirmed for 3 other cells fines (Hs746T, EBC1 and MKN45) in figure 23. In that second experiment, the PMA, added as a positive shedding inducer, increased significantly, as expected, c-Met shedding at least in 2 cell lines (Hs746T and MKN45).
Finally, in a third experiment (Figure 24), HGF was introduccd as a control. No additional shedding was induccd by HGF compared as cells alone or cells + mlgGl. Once again, a significant inhibition of c-Met shedding was observed with m224Gl 1.
Example 15: Intrinsic effect of h224Gll Ab on various cell lines
In previous experiments described in this patent, it has been demonstrated that in contrast to what was observed with other antibodies such as 5D5, the m224Gl 1 and its humanized form h224Gl 1 do not display significant intrinsic activity tumor cell lines. To extend this property to other cell lines, western blot and phospho-ELISA 15 experiments were performed with the antibody alone, added for various times, on a set of cancer cell lines, with variable levels of c-Met expression, including Hs746T, NCIH441, Hs578T, NCI-H125, T98G, MDA-MB-231, PC3. The same test was also performed in a normal cell: HUVEC.
Method for the phospho cMet ELISA assay was already described in example 5 20 of the présent patent application. For the western analysis, protein lysâtes were made from pelletcd cells by incubation in lysis buffer with proteases and phosphatase inhibitors [lOnM Tris (pH 7.4), 150 mM NaCI, ImM EDTA, 1 mM EGTA, 0.5% Nonidet P40, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 2 mM PMSF, 10 mg/ml leupeptin, 10 mg/ml aprotinin] at 4°C. Protein 25 lysâtes were cleared of cellular débris by centrifugation, resolved by electrophoresis on 8% SDS-PAGE gels, and electrotransferred to a nitrocellulose membrane. For c-Met experiments, lysâtes were immunoprecipitated for spécifie protein of interest before electrophoresis and transfer.
Results presented in figures 25 to 32 demonstrate once again that no intrinsic 3 0 activity of the h224Gl 1 antibody was observed in the tested cells.
Example 16: In vivo comparison of the murine wild type 224G11 with a chimeric hinge-engineered 224G11 form described as 224GU|C2D5-7] (NCI-H441 xenograft model)
NC1-H441 is dcrivcd from papillary lung adenocarcinoma, expresses high levels of c-Met, and demonstrates constitutive phosphorylation of c-Met RTK.
To evaluate the necessity of hinge engineering to save in vivo activity of the 224G11 murine antibody, six to eight weeks old athymie mice were houscd in sterilized filter-topped cages, maintained in stérile conditions and manîpulated according to French and European guîdelines. Mice were injectcd subcutaneously with 9x106 NCIH441 cells. Then, six days after cell implantation, tumors were measurable (approximately 100 mm3), animais were dîvîded into groups of 6 mice with comparable tumor size and treated first with a loading dose of 2 mg of antibody/mice and then twice a week with a 1 mg/dose of each antibody to be tested. Mice were followed for the observation of xenograft growth rate. Tumor volume was calculated by the formula: π (Pi)/6 X length X width X height. Results described in figure 33 demonstrate that the murine Mab devoid of any agonist activity în vitro behave, as expected, as a potent in vivo antagonist. As suggested by the results obtained in vitro, in phosphorylation assays, the c224Gl l[C2D5-7] hinge-engineered antibody, that did not display a significant agonist effect, demonstrate a strong in vivo activity, comparable to the one of the m224Gl 1 on the NCI-H441 xenograft model.
Example 17: Evaluation of h224Gll in an ADCC test
As h224Gll is of IgGI isotype, ADCC could be part of its in vivo efficacy in human. An in vitro [5lCr] release cytotoxicity assay was performed using either Hs746T or NCI-H441 cells as target cells and NK cells purified from human peripheral blood mononuclear lymphocytes.
Briefly, one million Hs746T or NCI-H441 target cells were incubated with or without 20 pg of h224Gll Ab in présence of 100 pCi of 51Chromium (Pcrkin Elmcr) for 1 hr. Then, 4 X 103 cells were plated with an încreasing number of human natural kîller (NK) cells isolated from peripheral blood mononuclear cells (PBMC) using a négative sélection (Stemcell Technologies). Cells were incubated together for 4
I
additionaî hours at 37°C, Percent of cell lysis was calculated following the formula:
[(experimental 51 Cr release - spontancous 51 Cr relcase)/(full SICr release - spontaneous 51Cr release)] X 100. Spontaneous release représente the counts obtained when the target cells were cultured in absence of natural killcr cells. Full release represents the counts obtained when the target cells were lysed with 1% Triton X-100. h224Gll significantly enhanced lysis of both Hs746T (Figure 34A) and NCI-H441 (Figure 34B) cells by 62.9% and 63.2%, respectively, at a ratio NK/Target cells of 100.
Example 18: Immunohistochemical Studies (IHC)
Procedures of Paraffïn Embedded Tumors IHC Staining: 8 to 12 μΜ sections of frozen tumor were and immcdîately fixed in pre cooled acetone -20°C for 3 minutes.
Slides were then cooled at room température for 30 minutes to 1 hour. After 2 washes in PBS the Endogcnous peroxidase activîty was blocked using Peroxidase Blocking
Reagent (Dako K.4007) for five minutes. Sections were washed with PBS and incubated in avidin/biotin blocking reagent (Dako X0590) just before saturation of the non spécifie sites in PBS-BSA 4% for 30 minutes at room température. Then, slides were incubated with the biotinylated h224Gl 1 (50 to 10 gg/ml) or human biotinylated IgG 1/kappa (50 to 10 pg/ml, the Binding Site) as négative control 2 hours at room température.
Sections were washed with PBS and incubated with Strcptavidin-peroxydase complex universal (Dako K0679) for 30 to 45 minutes. 3-Amino-9-Ethylcarbazole was used for development of a red reaction product (Sigma). The slides were immersed în hematoxylin for 4 minutes to counterstain (Dako S3309).
Results are represented in Figures 35A-35C.
h224Gl 1 different ially stains the cell membrane of various tumor types. In this immunohistochemistry procedure, the red réaction product correlates to positive staining of the cell membrane and lack of red réaction product correlates to négative staining and no visualization of the cell membrane. The IgG control, human IgG 1/kappa is an isotype matched control.
Example 19: In vivo activîty of anti-c-Met Mabs on three cell lines amplificd for c-Met and xenografted in mice.
Hs746T (ATCC) and MKN45 (JCRJ3), two stomach carcinoma cells, and, EBCl (JCRB), a lung squamous cell carcinoma, arc 3 cell lines that over-express c-Met as shown by FACS analysis in Figures 36A, 37A and 38A. These cell lines are also described in the literature as amplified cell lines for c-Met. To confirm this latter point, c-Met genc amplification was determined by quantitative rcal-time PCR using AACt method. Fifty ng of genomic DNA from each indicated cell line, extracted using the QIAamp DNA midi kit (Qiagen, Les Ulis, FR) was used. The reference gene was DNA topoisomerase III alpha located on chromosome 17. Primers for c-Met gene were located on exon 14. The corresponding primers for both genes are those described by Smolen et al. (PNAS, 2006, 103:2316-2321) and yielded similar PCR efficiencîes. The QPCR protocol consists in a 10 min cycle at 95°C, followed by 40 successive cycles of 15 sec at 95°C/45 sec at 95°C. Data were first normalized to the reference gene, and then each cell line was normalized to MCF-7 cells containing a single copy of c-Met gene per haploid genome. The results presented in figures 36B, 37B and 38B demonstrate that the 3 cell lines display a significant amplification of c-Met ranking from 5 to 10-fold gcne amplification.
As it has been described that such cells are constitutively activated for c-Met, a Western blot assay was performed to détermine the level of c-Met phosphorylation of each cell line either in absence or in presence of HGF (50 ng/ml). For that purpose cells were eultured ovemight in sérum-free medium and then treated or not with 50 ng/ml of HGF for 2 min. Cells were lyscd with lysis buffer supplemented with protease inhibitors. Cell lysâtes were incubatcd at 4°C for 90 min, then centrifuged and supematants were collected. Protein concentrations were determined using the BCA protein assay (Pierce) and samples were immunoblotted. Antibodies used include the anti-c-Met antibody, clone 25H2 from Cell Signaling and the anti-P-c-Met Y1230, 1234, 1235 from Biosource. Immunoblottîng with the phospho-specific c-Met antibody against Y1230/1234/1235 showed strong baseline phosphorylation of the receptor in each amplified cell line (Figures 36C, 37C and 38C). No additional phosphorylation is observed when HGF was added to these cells. This last observation confions that c-Met was activated in an HGF-independent way in Hs746T, MKN45 and EBC-1 cell lines.
In order to test our anti-c-Met antibodies in vivo on ligand-independent cell lines, Hs746T, MKN45 and EBC-1 cells wcrc routincly eultured in complète DMEM, ViL .
I i
RPMI 1640 or EMEM medium respectîvely, Cells were split two days before engraftment so that they were in cxponential phase of growth, Seven million cells were engrafted to either SCID (for Hs746T and EBC-l) or CDl Swiss nude mice (for i
MKN45). Five to seven days after implantation, tumors were measurable and animais were divided into groups of 6 mice with comparable tumor size. Mice were treated ΐ.ρ.| with a loading dose of 2 mg of h224Gl l Mab /mouse and then twice a week with l mg of antîbody/mouse. Tumor volume was measured twice a week and calculated by the
I formula: π/6 X length X width X height. Statistical analysis was performed at each:
measured time using a Mann-Whitncy test,
This experiment demonstrates that h224Gl 1 reduced signifîcantly tumor growth of Hs746T, MKN45 and EBC1 (Figures 36D, 37D and 38D), indicating that the:
humanized antîbody is able to inhibit tumor growth of gastric and lung ligand-I independent cell lin es, Before these results were obtained, it would not hâve been apparent how the presence of the modified hinge in this humanized antîbody would affect its ligand-independent inactivation of c-Met, because the altered hinge also affccted its agonist/antagonîst properties.
Example 20: Ex vivo analysis of b224Gll activity on the Hs746T xenograft mode! in SCID mice.
In order to détermine more precisely mcchanisms of action that resulted in
Hs746T tumor xenograft inhibition, the effect of 3 i, p. injections of h224Gll was i evaluated on tumor mitotic index (Ki67), apoptosis (activated caspase 3) and c-Met expression using immunohistochemistry (1HC) methods. Three mice were analyzed for each condition, A significant down régulation of c-Met was observed on treated tumors
I 25 compared to control one (Fig,39C and D respectîvely). Panel 39A and B corresponds to : isotypes contrôle. In addition a significant decrcase in Ki67 levels (Figures 39E and F) and an increase in activated caspase 3 staining (Figures 39G and H) was noticed on ail ; treated tumors indicating that h224G 11 was able to inhibit signifîcantly cell prolifération and to inducc cell apoptosis within the tumor, i
i

Claims (21)

1. A monocional antibody, or a divalent functional fragment or dérivative thereof, capable to inhibit the c-Met dimerization, said antibody comprising a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3 with respectively the amino acid sequences SEQ ID Nos. 1, 2 and 3; and a Üght chain comprising CDR-L1, CDR-L2 and CDR-L3 with respectively the amino acid sequences SEQ ID Nos. 5, 6 and 7, said antibody further comprising a hinge région comprising the amino acid sequence SEQ ID No. 56;
for use for the prévention or the treatment of a patient in need thereof having a cancer characterized by ligand-independent activation of c-Met.
2. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, characterized in that said hinge région comprises the amino acid sequence SEQ ID No. 57.
3. The antibody of claim 1, or a divalent fijnctional fragment or dérivative thereof, characterized in that said hinge région comprises the amino acid sequence SEQ ID No. 21.
4. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, characterized in that said hinge région comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 22 to 28 and SEQ ID Nos. 58 to 72.
5. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, for use for the prévention or the treatment of a patient in need thereof having a cancer characterized by overexpression of c-Mct.
6. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, for use for the prévention or the treatment of a patient in need thereof having a cancer characterized by overexpression of c-Mct resulting from génie amplification of c-Met.
7. The antibody of claim 6, or a divalent functional fragment or dérivative thereof, for use for the prévention or the treatment of a patient in need thereof having a cancer characterized by overexpression of c-Met resulting from génie amplification of c-Met and resulting in ligand-independent activation of c-Met.
8. The antibody of claim 6, or a divalent functional fragment or dérivative thereof, for use for the prévention or the treatment of a patient in need thereof having a cancer characterized by overexpression of c-Met resulting from génie amplification of c-Met, wherein said cancer is selected from the group consisting of rénal cell carcinoma
10 and gastric cancer,
9. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, characterized in that it consîsts of a chimeric antibody.
10. The antibody of claim 1, or a divalent functional fragment or dérivative
15 thereof, characterized in that it consîsts of a human antibody.
11. The antibody of claim 1, or a divalent functional fragment or dérivative thereof, characterized in that it consiste of a humanized antibody,
20
12. The antibody of claim 11, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence comprising the amino acid sequence SEQ JD No, 4; and a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No, 8, 9 or 10,
25
13. The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 8; and a hinge région comprising the amino acid sequence SEQ ID No. 22.
14, The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence ^1/ comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 9; and a hinge région comprising the amino acid sequence SEQ ID No. 22.
5 15. The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 10; and a hinge région comprising the amino acid sequence SEQ ID No. 22.
16. The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 8; and a hinge région
15 comprising the amino acid sequence SEQ ID No. 28.
17. The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of
20 sequence comprising the amino acid sequence SEQ ID No. 9; and a hinge région comprising the amino acid scqucncc SEQ ID No. 28.
18. The antibody of claim 12, or a divalent functional fragment or dérivative thereof, characterized in that it comprises a heavy chain variable domain of sequence
25 comprising the amino acid sequence SEQ ID No. 4; a light chain variable domain of sequence comprising the amino acid sequence SEQ ID No. 10; and a hinge région comprising the amino acid sequence SEQ ID No. 28.
19. The antibody of claims 1 to 18 as a médicament.
20. A composition for the prévention or the treatment of a patient in need thereof having a cancer characterized by ligand-independent activation of c-Met, said composition comprising by way of active principle a compound consisting of an antibody, or a divalent functional fragment or dérivative thereof, as claîmed in one of
5 claims 1 to 19.
21. The composition of claim 20 as a médicament.
22. The use of an antibody, or a divalent functional fragment or dérivative
10 thereof, of claims 1 to 19 or of a composition of claims 20 or 21 for the préparation of a médicament for the prévention or the treatment of a patient in need thereof having a cancer characterized by ligand-independent activation of c-Met,
76 pages «L
OA1201200490 2010-06-01 2011-06-01 Novel anti-cMet Antibody. OA16258A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US12/791681 2010-06-01

Publications (1)

Publication Number Publication Date
OA16258A true OA16258A (en) 2015-04-10

Family

ID=

Similar Documents

Publication Publication Date Title
AU2017203929B2 (en) ANTI-cMET ANTIBODY
AU2011260286B2 (en) Novel anti-cMet antibody
US20230406940A1 (en) ANTI-cMET ANTIBODY
US20130216527A1 (en) Novel anti-cmet antibody
OA16258A (en) Novel anti-cMet Antibody.