WO2010064090A1 - Process for the modulation of the antagonistic activity of a monoclonal antibody - Google Patents

Abstract

The present invention relates to the antibody engineering field and, more particularly, to a process for the screening of antibodies and/or the modulation of the agonistic/antagonistic activity of antibodies. More particularly, the invention concerns a process of improving the antagonistic activity of a monoclonal antibody directed against a specific target molecule, or a divalent functional fragment or derivative thereof, said antibody being capable of inhibiting one or more of the biological activities of said target molecule, wherein said process comprises a stage of reconfiguration of the hinge region consisting of a modification of the amino acid sequence of said hinge region by the deletion, the addition or the substitution of at least one amino acid. The invention also relates to polypeptides useful for such a modulation method and the obtained antibodies.

Description

PROCESS FOR THE MODULATION OF THE ANTAGONISTIC ACTIVITY OFA

MONOCLONAL ANTIBODY

The present invention relates to the antibody engineering field and, more particularly, to a process for the screening of antibodies and/or the modulation of the agonistic/antagonistic activity of antibodies. More particularly, the invention concerns a method for the modulation of the antagonistic activity of a monoclonal antibody, or a divalent functional fragment or derivative thereof by genetic engineering. The invention also relates to polypeptides useful for such a modulation method and the obtained antibodies.

The terms "antibody", "antibodies" or "immunoglobulin" are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies or multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity).

More particularly, such molecule consists in a glycoprotein 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 region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHl, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino -terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immunue system (e.g. effector cells) and the first component (CIq) of the classical complement system. The heavy chains of immunoglobulins can be divided into three functional regions: the Fd region, the hinge region, the Fc region (fragment crystallizable) which are connected by a flexible hinge region. The Fd region 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 effector functions, which include, for example, complement fixation and binding to cognate Fc receptors of effector cells. The hinge region, 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 region. In contrast to the constant regions, the hinge domain is structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.

According to crystallographic studies, the immunoglobulin hinge region can be further subdivided structurally and functionally into three regions: 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 region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges. The lower hinge region joins the amino terminal end of, and includes residues in, the CH2 domain. The core hinge region 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 flexibility. Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may affect the effector functions of the Fc portion of the antibody.

In general, for the preparation of monoclonal antibodies or their functional fragments, of murine origin, it is possible to refer to techniques which are described in particular in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988) or to the technique of preparation from hybridomas described by Kohler and Milstein

(Nature, 256:495-497, 1975). Then, the monoclonal antibodies can, for example, be purified on an affinity column on which the receptor of interest or one of its fragments containing the epitope specifically recognized by said monoclonal antibodies has previously been immobilized. More particularly, said monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion- exchange chromatography aimed at eliminating 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 eliminate the potential aggregates due to the presence of dimers or of other multimers. In an even more preferred manner, the whole of these techniques can be used simultaneously or successively.

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 diabodies, 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 least one of the characteristic CDRs of the original antibody.

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. According to the present 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 reduction. In another manner, the antibody fragments comprised in the present 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 synthesizers such as those supplied by the company Applera, etc.

The term "antagonist" as used herein refers to a molecule which is capable of inhibiting one or more of the biological activities of a target molecule, such as an extracellular or transmembranar receptor. Antagonists may act by interfering with the binding of a receptor to a ligand and vice versa, by decreasing receptor phosphorylation, and/or by incapacitating or killing cells which have been activated by a ligand. The antagonist may completely block receptor- ligand interactions or may substantially reduce such interactions by competition, change of conformation, shedding or downregulation. All such points of intervention by an antagonist shall be considered equivalent for purposes of this invention.

The term "agonist" as used herein refers to any compound, including a protein, a polypeptide, a peptide, an antibody, an antibody fragment, a conjugate, a large molecule, a small molecule, capable of activating one or more of the biological activities of a target molecule.

In the research of therapeutic antibodies, it is often expected to have antibodies as antagonist as possible. Classical examples of antagonist antibodies are Herceptin, Pertuzumab,

Cetuximab, anti- VEGFR or anti-IGF-lR antibodies.

As a particular example, it can be mentioned the anti-c-Met 5D5 antibody generated by Genentech [WO 96/38557] which behaves as a potent agonist when added alone in various models. In order to solve this technical problem, this antibody had to be engineered as a Fab fragment or as a monovalent antibody (one-armed 5D5) to have an antagonistic activity. As a consequence, such antibody can not be considered as an antibody, but a fragment, and does not present all advantages due to the "full antibody" format (no effector functions, reduced clearance and half-life [2 times faster than traditional bivalent antibodies as described in Poster 411 at the 20^th EORTC-NCI- AACR symposium, Geneva, October 21 -24, 2008]).

The skilled artisan will recognize that effector functions include, for example, CIq binding; complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors (e.g. B cell receptor; BCR) and prolonging half-life through incorporation of the salvage receptor binding ligand (FcRn) as described in, for example, U.S. Patent No. 5,739,277 issued Apr. 14, 1998.

One of the inventive aspects of the present invention is to solve such technical problems, i.e. improving the antagonistic activity of an antibody while preserving a "full divalent" format. It must be mentioned herein that the invention can be applied to modulate the agonistic/antagonistic activity of human antibodies obtained by immunization of "human mice" (genetically modified mice that produce human immunoglobulins) or using phage display techniques to build whole antibodies from selected scFv, Fab or any other equivalent fragments.

Another classical technical problem can be met in the course of chimerization and/or humanization of a murine antibody. It is well known by the man skilled in the art that, if the chimerization and/or humanization process of a murine antibody, is quite easy in the theory, it is not so easy to manage the chimerization and/or humanization of such a murine antibody without losing all or part of initial properties. Chimeric or humanized antibody may lose part of its ADCC, CDC, antagonistic/agonistic, binding, (TBC) ... activities. The present invention concerns, more particularly, the modification of agonistic/antagonistic activity of a murine antibody after a chimerization and/or humanization process.

As a particular example, a set of anti-cMet antibodies, thereafter described as 224Gl 1, 2274H1 and 1 IEl, that behave as potent antagonist murine antibodies became partial agonists when chimerized on a human IgGl format. This shift from potent antagonists to partial agonists resulted in a complete loss of in vivo activity in xenograft models.

The present invention intends to solve these problems and relates more particularly to a process of improving the antagonistic activity of a monoclonal antibody directed against a specific target molecule, or a divalent functional fragment or derivative thereof, said antibody being capable of inhibiting one or more of the biological activities of said target molecule, wherein said process comprises a stage of reconfiguration of the hinge region consisting of a modification of the amino acid sequence of said hinge region by the deletion, the addition or the substitution of at least one amino acid. It is clear that the expression "improving the antagonistic activity" must be interpreted in its broadest sense, i.e. as the wanted result. Mechanistically, such result can be obtained by an improvement of the intrinsic antagonistic activity and/or a decrease of the intrinsic agonistic activity of an antibody.

More particularly, basic definitions of terms in quantitative pharmacology are based on the updated recommendations given by the International Union of

Pharmacology (IUPHAR) Committee on receptor Nomenclature (see Neubig et al., 2003). The term 'agonist' stands for a ligand (any type of molecule) that binds to a receptor and alters the receptor state resulting in a stimulatory or increased biological response. Agonists can act as full agonists or partial agonists:

- Full agonist: when the receptor stimulus induced by an agonist reaches the maximal response capability of the system, then it will produce the system maximal response and be a full agonist in that system. Several agonists may elicit the same maximal response, they are all full agonists in that experimental system.

- Partial agonist: a molecule that in a given tissue, under specified conditions, cannot elicit as large an effect (even when applied at high concentration, so that all the receptors should be occupied) as can a full agonist acting through the same receptors in the same system. Partial agonists are generally also partial antagonists since in the co- presence of a full agonist, they reduce the maximal response of the said full agonist to their own maximal response. This designation of full vs. partial agonist is system- dependent, and a full agonist for one system or measurement may be a partial agonist in another.

The term 'antagonist' stands for a molecule that reduces the action of another drug, generally an agonist. Many antagonists act at the same receptor macro molecule as the agonist.

- The efficacy of antagonism can be full antagonism where the response of the system in the co-presence of the antagonist and agonist corresponds to the basal

(without any ligand) activity of the system.

- An antagonist can act as partial antagonist when the maximal inhibition (even when applied at high concentration, so that all the receptors should be occupied by the antagonist) elicited by the co-presence of the antagonist and agonist is above the basal activity of the system.

- Antagonism can be competitive when the binding of agonist and antagonist is mutually exclusive. This may be because the agonist and antagonist compete for the same binding site or combine with adjacent sites that overlap. A third possibility is that different sites are involved but that they influence the receptor macro molecule in such a way that agonist and antagonist molecules cannot be bound at the same time. - Noncompetitive antagonism is observed when agonist and antagonist can be bound to the receptor simultaneously; antagonist binding reduces or prevents the action of the agonist with or without any effect on the binding of the agonist.

The deletion, addition or substitution can be classically done by any method known by the skilled artisan.

Several methods can be applied by the skilled artisan to generate deletions or insertions in a given DNA sequence. Can be mentioned without limitations, partial digestion of DNA with pancreatic DNAse I, partial digestion of DNA with restriction enzymes, linker-based insertions mutants, nested sets of deletion mutants using BAL31 nuclease, DNAse I or exonuclease III. These methods are extensively described in laboratory manuals such as Molecular Cloning, A laboratory manual (Sambrook, Fritsch and Maniatis). Several PCR-based methods can also be employed to generate deletions, insertions or site-directed mutagenesis in a DNA molecule such as overlap extension PCR (Wurch et al., 1998), but not limited to this one. To perform site directed mutagenesis, several other techniques can be used, as examples, but not limited to these ones, can be mentioned oligonucleotide based mutagenesis based on either single or double-primer methods, the Kunkel method based on uracil incorporation (Kunkel, 1985). These methods are extensively described in laboratory manuals such as Molecular Cloning, A laboratory manual (Sambrook, Fritsch and Maniatis). In a preferred embodiment of the process of the invention, said modification is selected from: i) the deletion of at least one amino acid of said hinge region amino acid sequence; and/or ii) the addition of at least one disulfide bridge into said hinge region. In order to clarify the invention, the first aspect (i) will be detailed in first and the second aspect (ii) will be detailed after. It must be understood that this ordering is only due to the writing of the present application and that both of these aspects, as it will be obvious hereinafter, are of similar importance.

In a particular embodiment, a way to modify the amino acid sequence of the hinge region will consists of the deletion of at most 2, 3 or 4 amino acids of said hinge region amino acid sequence. A particular aspect of the invention is that said monoclonal antibody is a divalent antibody. Actually, as seen below, it is possible to modulate agonistic/antagonistic activity of an antibody by modifying the structure of said antibody. For the first time, inventors report an original way to modulate such agonistic/antagonistic activity while conserving a divalent form for the antibody, aiming the conservation of good properties such as long half-life or effector functions.

It can also be mentioned here that, if the modification of the hinge region of a monoclonal antibody in order to increase the effector functions has already been reported in the prior art, it has never been reported, at the contrary, that such a modification into the hinge region could be of interest in the modulation of the agonistic/antagonistic activity of a monoclonal antibody. This is clearly the subject of the present invention witch is novel and inventive regarding the existing prior art.

As an aspect in accordance with the process of the invention, the monoclonal antibody is a chimeric antibody. By "chimeric" antibody, it is intended to indicate an antibody which contains a natural variable (light chain and heavy chain) region derived from an antibody of a given species in combination with the light chain and heavy chain constant regions of an antibody of a species heterologous to said given species (e.g. mouse, horse, rabbit, 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 example, the chimeric antibody can be produced by cloning a recombinant DNA containing a promoter and a sequence coding for the variable region of a non-human, especially murine, monoclonal antibody according to the invention and a sequence coding for the constant region of human antibody. A chimeric antibody of the invention encoded by such a recombinant gene will be, for example, a mouse-man chimera, the specificity of this antibody being determined by the variable region derived from the murine DNA and its isotype determined by the constant region derived from the human DNA. For the methods of preparation 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 Patent No. 4,816,567. As another aspect in accordance with the process of the invention, the monoclonal antibody is a humanized antibody.

By "humanized antibody", it is intended to indicate an antibody which contains CDR regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or from several) human antibodies or germline sequences. 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 et 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 Bebbington 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 0 451 216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 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.

As another aspect in accordance with the process of the invention, the monoclonal antibody is a human antibody.

The term "human antibody" includes all antibodies that have one or more variable and constant region derived from human immunoglobulin sequences. In a preferred embodiment, all of the variable and constant domains (or regions) are derived from human immunoglobulin sequence (fully human antibody). In other words, it includes any antibody which has variable and constant regions (if present) 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 making human antibodies known by the man skilled 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 immunoglobulin loci and is deficient in mouse antibody production (Green at al, 1994, Nature Genetics, 7:13-21). The XENOMOUSE™ produces an adult-like human repertoire of fully human antibodies, and generate antigen-specific human monoclonal antibodies. A second generation XENOMOUSE™ contains approximately 80% of the human antibody repertoire (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 generation of human antibody according to the invention.

The process according to the invention can be used for any type of immunoglobulin comprising a hinge region, i.e. IgA, IgD and IgG.

As an example, for the IgA isotype, the hinge region of an IgAl comprises the amino acid sequence PSTPPTPSPSTPPTPSPS (SEQ ID No. 8) and the hinge region of an IgA2 comprises the amino acid sequence PPPPP (SEQ ID No. 9).

In a similar manner, the hinge region of an IgD comprises the amino acid sequence SPKAQASSVPTAQPQAEGSLAKATTAPATTRNTRGGEEKKKEKEKEE QEERETKTP (SEQ ID No. 10).

As a particular embodiment of the invention, it is preferred to use of an IgG including, for example, IgGl , IgG2, IgG3 or IgG4.

The respective amino acid sequences corresponding to the different isotypes of IgG hinge regions are:

PKSCDKTHTCPPCP (SEQ ID NO. 11) for an IgGl, RKCCVECPPCP (SEQ ID NO. 7) for an IgG2, LKTPLFTGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKS

CDTPPPCPRCP (SEQ ID NO. 12) for an IgG3, and

SKYGPPCPSCP (SEQ ID NO. 13) for an IgG4. Still more particularly, it is preferred to use an IgGl. Actually, in the field of therapeutical antibodies, and more particularly in the treatment of cancers, it is preferred to generate IgGl to get effector functions such as ADCC and CDC in addition to functions linked to the specific binding to the targeted antigen. The process of the invention is characterized in that said monoclonal antibody is an IgGl.

"Target molecule", within the meaning of the invention, relates to any molecule to witch the monoclonal antibody is able to bind specifically or to modulate the activity. Generally, such target molecule can be named "antigen". As non limitative example of target molecule witch can be targeted by a monoclonal antibody, can be mentioned soluble ligands, receptors such as transmembrane receptors, membrane tumoral markers, etc.

In a preferred embodiment, said target molecule is a transmembrane receptor.

The expression "transmembrane receptor" relates to a protein that spans the plasma membrane of a cell, with the extracellular domain of the protein having the ability to bind to a ligand and the intracellular domain having an activity (such as a protein kinase) that can be altered (either increased or decreased) upon ligand binding.

In other words, Transmembrane receptors are integral membrane proteins, which reside and operate typically within a cell's plasma membrane, but also in the membranes of some subcellular compartments and organelles. Binding to a signalling molecule or sometimes to a pair of such molecules on one side of the membrane, transmembrane receptors initiate a response on the other side. In this way they play a unique and important role in cellular communications and signal transduction.

Many transmembrane receptors are composed of two or more protein subunits which operate collectively and may dissociate when ligands bind, fall off, or at another stage of their "activation" cycles. They are often classified based on their molecular structure, or because the structure is unknown in any detail for all but a few receptors, based on their hypothesized (and sometimes experimentally verified) membrane topology. The polypeptide chains of the simplest are predicted to cross the lipid bilayer only once, while others cross as many as seven times (the so-called G-protein coupled receptors or GPCRs) or more. Like any integral membrane protein, a transmembrane receptor may be subdivided into three parts or domains, an extracellular domain, a transmembrane domain and an intracellular domain.

The extracellular domain is the part of the receptor that sticks out of the membrane on the outside of the cell or organelle. If the polypeptide chain of the receptor crosses the bilayer several times, the external domain can comprise several "loops" sticking out of the membrane. By definition, a receptor's main function is to recognize and respond to a specific ligand, for example, a neurotransmitter or hormone (although certain receptors respond also to changes in transmembrane potential), and in many receptors these ligands bind to the extracellular domain.

In the majority of receptors for which structural evidence exists, transmembrane alpha helices make up most of the transmembrane domain. In certain receptors, such as the nicotinic acetylcholine receptor, the transmembrane domain forms a protein- lined pore through the membrane, or ion channel. Upon activation of an extracellular domain by binding of the appropriate ligand, the pore becomes accessible to ions, which then pass through. In other receptors, the transmembrane domains are presumed to undergo a conformational change upon binding, which exerts an effect intracellularly. In some receptors, such as members of the 7TM superfamily, the transmembrane domain may contain the ligand binding pocket. The intracellular (or cytoplasmic) domain of the receptor interacts with the interior of the cell or organelle, relaying the signal. There are two fundamentally different ways for this interaction a) The intracellular domain communicates via specific protein-protein- interactions with effector proteins, which in turn send the signal along a signal chain to its destination and b) with enzyme-linked receptors, the intracellular domain has enzymatic activity. Often, this is a tyrosine kinase activity. The enzymatic activity can also be located on an enzyme associated with the intracellular domain.

There are several ways for the cell to regulate the activity of a transmembrane receptor. Most of them work through the intracellular domain. The most important ways are phosphorylation and internalization (see ubiquitin) or activation of second messenger cascades such as cAMP, IP, Ca²⁺ or cGMP.

All membrane proteins showing enzymatic activities can also be targeted by antibodies with the modification described in this invention. Can be mentioned as examples, but without limitations, the matrix metalloprotease (MMP) family, the 'a disintegrin and a metalloprotease domain protease' (ADAM) family, adenylate cyclases, ...

All membrane proteins acting as ion channels, pores and transporters can also be targeted by antibodies with the modification described in this invention. Can be mentioned as examples, but without limitations, the sodium channel family, the potassium channel family, the nicotinic acetylcholine receptor family, the sigma receptors, the monoamine transporter family.

More broadly, all membrane proteins identified as specific markers for a given disease can also be targeted by an antibody treatment, which antibody can as well be improved by the modifications described in this invention.

In a preferred embodiment of the invention, said transmembrane receptor is selected from the group consisting of the tyrosine kinase receptor and GPCRs.

In a more preferred embodiment, said transmembrane receptor is a tyrosine kinase receptor selected preferentially in the group consisting of IGF-IR, c-Met, RON, AxI, VEGF, VEGFR, Her-2neu, homodimers and heterodimers of the ErbB family, etc.

In the present application, and more particularly in the following specification, sequences will be defined in reference to 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., Pommie, 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 amino acids always have the same position, for instance cysteine 23 (lst-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides a standardized delimitation of the framework regions (FRl-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 regions: CDRl-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 representations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M. -P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M. -P., Current Bioinformatics, 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)].

For the man skilled in the art, it will be obvious to transpose the invention described according the IMGT system into any other numbering system such as, for example, the Kabat numbering system.

The IMGT unique numbering for all IG and TR V-REGIONs of all species relies on the high conservation of the structure of the variable region. This numbering, set up after aligning more than 5 000 sequences, takes into account and combines the definition of the framework (FR) and complementarity determining regions (CDR), structural data from X-ray diffraction studies, and the characterization of the hypervariable loops. The delimitations of the FR-IMGT and CDR-IMGT regions have been defined. Similarly, the IMGT unique numbering has been applied for the C-

DOMAIN, and allows for precise delimitation of Ig-like domains. The C-DOMAIN corresponds to the complete C-REGION, to most of the C-REGION or to only part of the C-REGION, depending on the immunoglobulin (IG) type.

The IMGT numbering for C-DOMAIN (IG and TR) is derived as the IMGT unique numbering for V-DOMAIN, from the princeps IMGT unique numbering for V- REGION, up to position 104. Amino acid positions can therefore be easily compared between the C-DOMAIN and the V-DOMAIN.

To precisely locate the Hinge regions, the IMGT numbering of C-DOMAIN was applied to precisely locate CHl and CH2 domains. The Hinge region includes all amino acid residues in between the last residue of IMGT-CHl and the first residue of IMGT-

CH2.

All other immunoglobulin numbering schemes such as Kabat or A. Honegger, covering the same Hinge domain are included in the present invention.

As a preferred example of the invention, based on the IMGT numbering system as above described, the amino acids sequence of the hinge region of an IgGl comprises the residues Hl to H14 with the segment Hl to H9 corresponding to the Upper Hinge and the segment HlO to H14 corresponding to the Core Hinge. More particular, the human IgGl hinge region comprises the amino acid sequence PKSCDKTHTCPPCP (SEQ ID No. 11) and the murine IgGl hinge region comprises the amino acid sequence PRDCGCKPCICT (SEQ ID NO. 14).

Table 1

In a preferred embodiment of the invention, it is considered a modification aiming at reducing the length of the protein sequence coding for the hinge region of a divalent antibody. More particularly, the process according to the invention comprises a step of deletion of at least one amino acid in the hinge region.

As previously mentioned, it is preferred the deletion of at most 2 amino acids of said hinge region.

As previously mentioned, it is preferred the deletion of at most 3 amino acids of said hinge region.

As previously mentioned, it is preferred the deletion of at most 4 amino acids of said hinge region. In a particular use of the process of the invention, the modification consists of at least a deletion of an amino acid selected from the amino acid in position Hl, H5, H6, H9 or H12.

More particularly, inventors have demonstrated the implication of particular residue and a particular inventive aspect of the invention consists of the selection of certain residues.

In the preferred case of an IgGl, the amino acid in position Hl consists of a Proline; the amino acid in position H5 consists of an aspartate in the human version and of a Glycine in the murine version; the amino acid in position H6 consists of a Lysine; the amino acid in position H9 consists of a Threonine in the human version and of a Proline in the murine version; and the amino acid in position H12 consists of a Pro line in the human version.

According to a preferred embodiment, this deletion has to be done in the "upper hinge" region. In a more preferred embodiment, this deletion is part of the "upper hinge" constituted, for an IgGl for example, of the amino acids Hl to H9 by comparison to the "Core hinge" constituted of the amino acids HlO to H 14.

More particularly, said modification consists of the deletion of the amino acids H6 and H9. In the present application, the amino acid numbering is done regarding the

IMGT system as previously described. It is obvious that any other numbering system, with a modification of the numbering but not the nature of residue implicated in the hinge region, must be considered as equivalent. As an example, renumbering the identified amino acid part of the invention (according to the IMGT system) in the Kabat system must be considered as equivalent.

Another aspect of the invention is based on the addition of at least one disulfide bridge into the hinge region.

More particularly, the process of the invention is characterized in that the modification consists of the introduction of at least one Cysteine into the "upper hinge" region.

According to the inventors, a plausible explanation is based on a possible "rigidifcation" of the hinge resulting from either the reduction of the length and/or the introduction of another disulfide bridge. Such a "rigidifϊcation" will allow to maintain a appropriate spatial conformation of the antibody with, as a consequence, an improved antagonistic activity.

It is clear that any method aiming at rigidifying the hinge region must be considered as an equivalent method to the process according to the present invention.

The introduction of a Cysteine can be done by addition of such an amino acid, said addition being done by any method known by the man skilled in the art.

Another preferred way to introduce a Cysteine into the hinge region consists of a substitution of at least an amino acid. More particularly, a preferred way to introduce a Cysteine into the hinge region consists of a substitution of at least an amino acid selected from Hl to H9. Such a substitution can be done by any method known by the skilled artisan.

More particularly, the process of the invention comprises the substitution of the Threonine in position H7 into the "upper hinge" region by a Cysteine. In another embodiment, the process of the invention comprises the substitution of the Lysine in position H6 into the "upper hinge" region by a Cysteine.

According to another embodiment, it is also possible to reduce the length of the hinge region and/or to add a disulfide bridge by changing the whole of the amino acid sequence encoding for the hinge region. As a preferred example, the modification of the process of the invention consists of a replacement of the amino acids Hl to H14 of the IgGl hinge region by the amino acid Hlto H14 of an IgG2 hinge region, preferably when said monoclonal antibody which is desired to improve its antagonistic activity is an IgGl antibody.

In another application, the invention relates to a process of screening for an antagonist monoclonal antibody directed against a specific target molecule, or a divalent functional fragment or derivative thereof, said antibody being capable of inhibiting one or more of the biological activities of said target molecule, wherein said process comprises the steps of:

(a) selecting an initial antibody with an initial level of inhibition of said one or more biological activity of said target molecule,

(b) modifying the amino acid sequence of the hinge region of said initial antibody by the process of the invention, (c) evaluating the modified antibody of step (b) for its ability to inhibit said one or more biological activity of said target molecule, and

(d) selecting, as a positive result, the antibody of step (c) with an inhibition level of said one or more biological activity of said target molecule higher than the initial level of said inhibition.

Initial antibodies can be selected amongst the existing antibodies such as, without limitation, antibodies antagonists to IGF-IR, c-Met, RON, AxI, VEGF,

VEGFR, Her-2neu, homodimers and heterodimers of the ErbB family. As non limitative preferred example, said initial antibodies can consist of Herceptin, Pertuzumab, Cetuximab, anti- VEGFR or anti-IGF-lR antibodies.

"Inhibition level", within the meaning of the invention, illustrates the antagonistic activity of an antibody. Such inhibition level can be determined by any method known by the skilled artisan such as, without limitation, such as a) direct cell counting or use of 3[H]Thymidine, tetrazoline salts or any other fluorescent mean to evaluate proliferation, b) western blotting, phospho-ELISA or alpha-screen assays to monitor signal transduction, c) BRET or FRET analysis for dimerization assay, d) microscopy or fluorescent methods to monitor migration, invasion, angiogenesis or morphogenesis and e) calliper measurement of tumors for in vivo evaluations.

This screening process can be used for the improvement of validated antibodies or as a selection stage for research or pre-clinical antibodies.

Another aspect in accordance with the invention relates to a monoclonal antibody directed against a specific target molecule, or divalent functional fragments or derivatives thereof, obtainable by the process of the invention, said antibody being characterized in that it comprises a hinge region amino acid sequence selected from the group consisting of SEQ ID No. 1 (PRDCGCKPCICT), SEQ ID No. 2

(PKSCGCKPCICT), SEQ ID NO. 3 (PKSCGCKPCICP), SEQ ID No. 4 (PRDCGCKPCPPCP), SEQ ID NO. 5 (PRDCGCHTCPPCP), SEQ ID No. 6 (PKSCDCHCPPCP) or SEQ ID No. 7 (RKCC VECPPCP).

A preferred monoclonal antibody obtained by the implementation of the process of the invention can be characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID No. 1 (PRDCGCKPCICT), SEQ ID No. 2 (PKSCGCKPCICT), SEQ ID No. 3 (PKSCGCKPCICP), SEQ ID No. 4 (PRDCGCKPCPPCP), SEQ ID NO. 5 (PRDCGCHTCPPCP), SEQ ID No. 6 (PKSCDCHCPPCP) or SEQ ID No. 7 (RKCC VECPPCP).

In a preferred embodiment, said monoclonal antibody is a human antibody, more preferred is an IgGl antibody. The invention also relates to an isolated nucleic acid encoding for a monoclonal antibody as previously described, i.e. comprising a hinge region amino acid sequence selected from the group consisting of SEQ ID No. 1 (PRDCGCKPCICT), SEQ ID No. 2 (PKSCGCKPCICT), SEQ ID NO. 3 (PKSCGCKPCICP), SEQ ID No. 4 (PRDCGCKPCPPCP), SEQ ID No. 5 (PRDCGCHTCPPCP), SEQ ID No. 6 (PKSCDCHCPPCP) or SEQ ID No. 7 (RKCC VECPPCP).

The invention comprises an isolated nucleic acid comprising a nucleic sequence selected from the group consisting of SEQ ID No. 15 to SEQ ID No. 21.

Also part of the invention is an expression vector or a transformed host cell comprising an isolated nucleic acid as previously described and, more particularly, an isolated nucleic acid comprising a nucleic sequence selected from the group consisting of SEQ ID No. 15 to SEQ ID No. 21.

Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures wherein:

Figures IA and IB: Effect a series of murine and corresponding chimeric anti-c- Met Mabs produced as a human IgGl /kappa isotype on c-Met receptor phosphorylation on A549 cells.

Figure IA: agonist effect calculated as percentage versus maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml]. Figure IB: antagonist effect calculated as percentage of inhibition of the maximal stimulation of c-Met phosphorylation by HGF [100 ng/ml] .

Figures 2 A and 2B: Comparison between murine 224Gl 1 Mab and chimeric 224Gl 1 Mabs containing various engineered hinge regions, on c-Met receptor phosphorylation on A549 cells. Figure 2 A: agonist effect calculated as percentage 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 3 A and 3B, 4 and 5: c-Met dimerization and activation BRET models.

Figures 6 A and 6B: c-Met recognition by chimeric and humanized 224Gl 1 forms.

Figures 7 A, 7B and 7C: Effect of murine and chimeric antibodies on HGF- induced proliferation of NCI-H441 cells in vitro. NCI-H441 cells were plated in serum- free medium. Twenty four hours after plating (Figure 7A) ml IEl and [HEl] chim, (Figure 7B) m227Hl and [227Hl ] chim or (Figure 7C) m224Gl l and [224Gl 1] chim were added either in absence or in presence of HGF. Black arrows indicate the wells plated with cells alone either in absence lέ or in presence ^l of HGF. A murine IgGl (mlgGl) was introduced as an isotype control.

Figure 8: In vivo comparison of murine and chimeric 224Gl 1 Mabs on the NCI- H441 xenograft model.

Figures 9 A and 9B: Effect of the murine 224Gl 1 Mab and of various chimeric and humanized versions of this antibody on HGF-induced proliferation 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 presence of HGF. In panel (Figure 9A), the murine m224Gl l, chimeric IgGl [224Gl 1] chim, humanized IgGl [224Gl 1] [HzI], [224Gl 1] [Hz2], [224Gl 1] [Hz3] versions were shown. In panel (Figure 9B), the murine m224Gl l and various chimeric IgGl forms ([224GI l] chim, [224Gl 1] [MH chim], [224Gl 1] [MUP9H chim], [224Gl 1] [MMCH chim], [224Gl 1] [TH7 chim]) were presented. Black arrows indicate the wells plated with cells alone either in absence lέ or in presence ^l of HGF. A murine IgGl was introduced as a negative control for agonist activity. The m5D5 was used as a dose-dependent full agonist control. Figure 10: Effect of the murine 224Gl 1 Mab and of various chimeric and humanized versions of this antibody on HGF-induced proliferation 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 presence of HGF. The murine m224Gl l, [224Gl 1] chim, [224Gl 1] [TH7 chim]) IgGl chimeric forms and [224Gl 1] [TH7 HzI], [224Gl 1] [TH7 Hz3]), were presented. Black arrows indicate the wells plated with cells alone either in absence lέ or in presence ^l of HGF. A murine IgGl was introduced as a negative control for agonist activity. The m5D5 was used as a dose-dependent full agonist control.

Example 1: Construction of chimeric Mabs and functional evaluation of c- Met receptor phosphorylation status

Several mouse Mabs, targeting a prototypical tyrosine kinase receptor (c-Met receptor) were reformatted as chimeric Mabs carrying mouse variable domains and human constant domains. Their intrinsic activities were analyzed based on a functional assay monitoring inhibition of ligand (HGF)-dependent c-Met receptor phosphorylation. Upon PCR-cloning of mouse variable domain sequences (VH, VL), chimeric

Mabs were constructed upon ligation of a {Nhel-Bcll} restriction fragment carrying either mouse VH or VL sequences into a pCEP4 vector (InVitrogen, US) carrying the entire coding sequence of the constant domain of either a human light chain Ckappa or a human heavy chain [CH1-Hinge-CH2-CH3] of an IgGl immunoglobulin. All cloning steps were performed according to conventional molecular biology techniques as described in the Laboratory manual (Sambrook and Russel, 2001) or according to the supplier's instructions. Each genetic construct was fully validated by nucleotide sequencing using Big Dye terminator cycle sequencing kit (Applied Biosystems, US) and analyzed using a 3100 Genetic Analyzer (Applied Biosystems, US). Production of the corresponding chimeric Mabs was performed using suspension-adapted HEK293 EBNA cells (InVitrogen, US) grown in serum-free medium Excell 293 (SAFC Biosciences) supplemented with 6 mM glutamine. Transient transfection was performed using linear 25 kDa polyethyleneimine (PEI) (Polysciences). Cultivation process was monitored on the basis of cell viability and Mab production. Mabs were purified using a conventional chromatography approach on a Protein A resin (GE Healthcare, US).

All different forms of Mabs were produced at levels suitable with functional evaluations. Productivity levels are typically ranging between 15 and 30 mg/1 of purified Mabs.

Functional evaluations were performed on A549 human lung cancer cells. c-Met receptor phosphorylation status was monitored on cell lysates using a specific capture

ELISA assay. A goat anti-c-Met Mab (R&D, ref AF276) was used as capture antibody whereas the detection antibody corresponded to an anti-phospho-c-Met Mab (Biosource ref KHO0281). Luminescence readings were recorded on a Mithras LB920 multimode plate reader (Berthold).

All three murine Mabs HEl, 224Gl 1 and 227Hl yielded comparable intrinsic activities on c-Met receptor phosphorylation: almost no agonist activity by their own (less than 5 % of HGF effect, Figure IA), and a strong inhibition of HGF [100 ng/ml]- induced c-Met receptor phosphorylation (> 70 % inhibition of HGF effect, Figure IB).

Very surprisingly, by modifying exclusively the Mab's constant domain to switch from a mouse IgGl /kappa to a human IgGl kappa, a complete modification of the intrinsic activities of the resulting chimeric Mabs was observed (Figures IA- IB). Indeed, strong agonism (reaching 20 % of HGF effect for cl lEl, Figure IA) was observed associated with an important decrease in the antagonist efficacy (only remaining 60 % of inhibition of HGF effect for c224Gl l, Figure IB). This effect was independent of the variable domain of the antibody since the same phenomenon was observed for the three investigated Mabs.

Example 2: Design, cloning and production of engineered hinge versions Based on the observation made above, it is hypothesized that the impaired pharmacological profile observed upon reformatting of mouse IgGl into human IgGl Mabs was due to the human IgGl domain. On one hand, it was known in the literature that activation of the c-Met receptor was associated with its dimerization, and that inhibition of c-Met receptor dimerization may inhibit c-Met receptor phosphorylation and downstream signaling.

On the other hand, Mabs are by essence divalent molecules due to their inherent structural basis and thus, they may act as inducers of c-Met receptor dimerization. Therefore, it is hypothesized that by restricting the conformational flexibility of chimeric Mabs, such as rotation, bending or wagging (see Roux et al., 1997), it could be possible to regain the intrinsic activities of interest (strong antagonism and weak agonism) of the parental murine Mabs. This hypothesis is reinforced by analysis of the respective sequences of mouse and human IgGl hinge regions (also referred as IgGl H- region):

Mouse IgGl H-region PRDCGCKPCICT (SEQ ID No. 1)

Human IgGl H-region PKSCDKTHTCPPCP (SEQ ID No. 11) Human IgG2 H-region RKCCVECPPCP (SEQ ID No. 7)

This alignment shows that the mouse IgGl H-region is shorter and contains one additional disulfide bridge (Cys) as compared to the human IgGl H-region. It also shows that the human IgG2 H-region resembles that of mouse IgGl H-region, both in its length (11 AA) and number of disulfide bridges (4).

Therefore, it is speculated that increased rigidity of the human IgGl H-region could be obtained by introducing stabilizing mutations such as Cys residues and/or by shortening of this particular segment. This putative increased rigidity of the H-region may be associated with improved functional properties of the engineered human IgGl Mab.

A series of 7 engineered versions have been designed (Table 2) by making chimeric H-regions upon exchange of either N-terminal or C-terminal hinge portions between mouse and human sequences. Construction of an IgG2 equivalent was as well performed.

Table 2

WT-IgG2 WT-IgG₁ Variants

Hu-IgG₂ Hu-IgG₁ Mu-IgGi MH MMCH MMH MUP9H MUC7H TH7CΔ6,9

-IgG₁ -IgG₁ -IgG₁ -IgG₁ -IgG₁ -IgG₁

- P P P P P P P P

R K R R K K R R K

K S D D S S D D S

C C C C C C C C C

_ D G G G G G G D

_ K - - - - - - -

C T C C C C C C C

V H K K K K K H H

E T P P P P P T -

C C C C C C C C C

P P I I I I P P P

P P - - - - P P P

C C C C C C C C C

P P T T T P P P P

As an example of hinge engineering, the variable domain (heavy and light chains) of a mouse anti-c-Met Mab - called 224Gl 1, was chosen. These mouse sequences were fused in a first instance to human constant domains [Ckappa] for the light chain and [CH1-Hinge-CH2-CH3] for the human IgGl heavy chain. Modification of the hinge region was performed by exchanging a {Nhel-Bcll} restriction fragment by the equivalent portion carrying the desired modifications, each respective {Nhel- Bcll} fragment being synthesized by global gene synthesis (Genecust, LU). All cloning steps were performed according to conventional molecular biology techniques as described in the Laboratory manual (Sambrook and Russel, 2001) or according to the supplier's instructions. Each genetic construct was fully validated by nucleotide sequencing using Big Dye 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- free 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 μg/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). All different forms of Mabs were produced at levels suitable with functional evaluations. Productivity levels are typically ranging between 15 and 30 mg/1 of purified Mabs.

Example 3: Evaluation of the engineered Mabs in a Phospho-c-Met-specific ELISA assay

A549 cells were seeded in a 12 multiwell (MW) plate in complete 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 μg/ml 15 minutes prior to ligand stimulation. Ice-cold lysis buffer was added 15 minutes after the addition of HGF to stop the phosphorylation reaction. Cells were scaped mechanically and cell lysates were collected by centrifugation at 13000 rpm for 10 min. at 4°C and correspond to the supernatant phase. Protein content was quantified using a BCA kit (Pierce) and stored at -20 ⁰C until use. The phosphorylation status of c- Met was quantified by ELISA. A goat anti-c-Met Mab (R&D, ref AF276) was used as a capture antibody (overnight coating at 4°C) and after a saturation step with a TBS-BSA 5% buffer (1 hour at room temperature (RT)), 25 μg of protein lysates were added to each well of the coated 96MW plate. After a 90 minutes incubation at RT, plates were washed four time and the detection 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 detection was performed by adding Luminol. Luminescence readings were on a Mithras LB920 multimode plate reader (Berthold).

A series of engineered versions of the heavy chain hinge domain was constructed and assayed in the c-Met receptor phosphorylation assay. As shown in Figure 2 A, compared to 224GI l[IgGl-CMm], an important reduction of the agonist effect associated with the hlgGl/kappa isotype was observed for both the IgG2 -based construct and for some engineered IgGl/kappa constructs [MH, MUP9H and TH7, Figure 2A]. Weakest and comparable agonism activities were obtained with 224GI l[MH-IgGl], containing a fully murine IgGl hinge region and 224G11[TH7], containing the most human engineered IgGl hinge region. A concomitant increase in antagonist efficacy was as well obtained [Figure 2B]. Thus, both IgG2 -based and engineered hlgGl /kappa-based TH7 hinge mutant associated with murine 224Gl 1 variable domain, yielded functional activities almost similar to that of the mouse 224Gl 1 Mab. However, the comparison of agonistic/antagonistic activities of 224G 11 [MMCH-IgG 1-chim] with 224Gl l[IgGl-chim] demonstrated that an increased antagonistic activity could be obtain by antibody engineering irrespectively of the intrinsic agonistic properties of such antibody.

Example 4: BRET analysis

In a first set of experiments, it was controlled that irrelevant mouse IgGl, human IgGl and human IgG2 had no effect of HGF induced BRET signal in both BRET models (Figure 3). Those Mabs were used further as controls. The effect of IgGl chimeric forms of mouse 224Gl 1 Mab ([224GI l] chim), mouse HEl Mab ([HEl] chim) and mouse 227Hl Mab ([227Hl] chim) on c-Met dimerisation and c-met activation BRET model were then evaluated.

While mouse 224Gl 1 Mab inhibited 59% of the HGF induced BRET signal on c-Met dimerisation model, [224Gl 1] chim Mab inhibited only 29% (Figure 4).

[224Gl 1] chim antibody was also less effective in inhibiting HGF induced c-Met activation since [224Gl 1] chim and m224Gl l antibodies inhibited 34.5% and 56.4% of

HGF induced BRET signal (Figure 5). Moreover, m224Gl l alone had no effect on c-

Met activation while [224Gl 1] chim had a partial agonist effect on c-Met activation corresponding to 32.9% of the HGF induced signal. This partial agonist effect of the

[224Gl 1] chim was also seen on c-Met dimerisation BRET model since [224Gl 1] chim alone induced a BRET increase corresponding to 46.6% of HGF-induced signal versus

21.3% for m224Gl l.

Example 5: c-Met recognition by chimeric and humanized 224Gl 1 forms

A direct ELISA has been set up to determine 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 μg/ml on 96-well Immunlon II plates. After an overnight incubation at 4°C, wells were saturated with a 0.5% gelatine/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 anti-mouse IgG HRP for detecting the murine antibody and a goat anti-human Kappa light chain HRP for chimeric and humanized antibody recognition. Plates were incubated for one hour and the peroxydase substrate TMB Uptima was added for 5 mn before neutralization with H₂SO₄ IM. Results presented in Figures 6 A and 6B showed that all tested forms were comparable for c-Met recognition.

Example 6: Effect of murine and chimeric antibodies on HGF-induced proliferation 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% L- Glutamine (Invitrogen Corporation). For proliferation 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 cells/well in 200 μl of serum 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 μg/ml (final concentration in each well). In this experiment, a murine IgGl Mab was added as a murine isotype control and the tested antibodies were the following one: m224Gl l, ml IEl, m227Hl and their human IgGl chimeric forms respectively identified as [224Gl 1] chim, [HEl] chim and [227Hl ] chim. Wells plated with cells alone -/+ HGF were also included. Then cells were pulsed with 0.25 μCi of [³H]Thymidine (Amersham Biosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of [ HJThymidine 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 Figures 7A, 7B and 7C demonstrated that, as expected, the murine antibodies displayed no agonist effect when added alone to cancer cells whatever the tested dose. No significant inhibition of the HGF-induced proliferation was observed with the isotype control regarding to the high cpm variations observed for this isotype control in this experiment. When added alone, neither murine m224Gl 1 nor ml IEl or m227Hl showed any agonist effect compared to the mlgGl isotype control Mab or cells alone. Dose dependent anti-proliferative activities reaching 78%, 80% or 80% were respectively for m224Gl l, ml IEl or m227Hl Mabs (% 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 these 3 Mabs induced a significant, dose dependent agonist effect when added alone with growth stimulations close to the one observed with HGF for [HEl] chim and [227Hl ] chim respectively. For these 2 antibodies displaying particularly high intrinsic agonist activities, the antagonist effect was significantly decreased with 53% and 21% inhibitory effects compared to 80% observed for their both murine forms. The agonist effect observed with the chimeric [224Gl 1] chim was also dose dependent but it was lower than the ones observed for [HEl] chim and [227Hl ] chim. However this agonist effect had an impact on the in vitro inhibition of HGF-induced proliferation that shifted from 78% for the murine m224Gl 1 to 50% for its chimeric form. To determine whether such "lower" in vitro intrinsic agonist activity was compatible with an unchanged in vzVo effect, both m224Gl 1 and [224Gl 1] chim were produced for in vivo testing. As, in previous studies, the 30 μg/mice dose had demonstrated a significant in vivo activity, that dose was selected for in vivo evaluation.

Example 7: In vivo comparison of murine and chimeric 224Gl 1 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 athymic mice were housed in sterilized filter-topped cages, maintained in sterile conditions and manipulated according to French and European guidelines. Mice were injected subcutaneously with 9 x 10⁶ cells. Then, six days after cell implantation, tumors were measurable (approximately 100 mm³), animals were divided into groups of 6 mice with comparable tumor size and treated first with a loading dose of 60 μg of antibody/mice and then twice a week with 1 mg/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 8 demonstrate that the murine Mab devoided of agonist activity in vivo behave, as expected, 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 8: Effect of the murine 224G11 Mab and of various chimeric and humanized versions of this antibody on HGF-induced proliferation 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% L- Glutamine (Invitrogen Corporation). For proliferation 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 3.75 x 10⁴ cells/well in 200 μl of serum 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 μg/ml (final concentration in each well). In this experiment, murine IgGl Mab was added as a murine isotype control and as an agonist negative control. The tested antibodies were the following one: i) m224Gl l, ii) its human IgGl chimeric forms respectively identified as [224Gl 1] chim, [224Gl 1] [MH chim], [224Gl 1] [MUP9H chim], [224Gl 1] [MMCH chim], [224Gl 1] [TH7 chim] iii) its humanized IgGl forms respectively described as [224Gl 1] [HzI], [224Gl 1] [Hz2], [224Gl 1] [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 full agonist positive control and thereafter called m5D5. Then cells were pulsed with 0.25 μCi of [³H]Thymidine (Amersham Biosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of [³H]Thymidine 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 9A demonstrated that as expected neither the isotype control nor the m224Gl 1 displayed any agonist activity on NCI-H441 proliferation. The isotype control was without effect on HGF-induced cell proliferation whereas m224Gl 1 showed a 66% inhibition when added at the final concentration of 10 μg/ml. The m5D5 used as an agonist control showed, as expected, a full dose dependent agonist effect when added alone to the cells. As already observed, the [224Gl 1] chim Mab displayed a significant dose- dependent 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 dependent agonist effects compared to the m224Gl l form. [224Gl 1] [HzI], [224Gl 1] [Hz2] and [224Gl 1] [Hz3] had comparable antagonist activities about 46, 30 and 35%. These activities are significantly lower than the one observed for m224Gl l. In Figure 9B, various IgGl chimeric forms were tested. Compared to [224Gl 1] chim form which displayed a dose- dependent agonist effect when added alone to NCI-H441 cells, the [224Gl 1] [MH chim], [224Gl 1] [MUP9H chim], [224Gl 1] [MMCH chim], [224Gl 1] [TH7 chim] forms were without significant intrinsic agonist effect. Their antagonist activity was higher than the one observed for the m224Gl 1 Mab (57%) with inhibitions reaching 79, 78, 84 and 93% respectively for [224Gl 1] [MH chim], [224Gl 1] [MUP9H chim], [224Gl 1] [MMCH chim] and [224Gl 1] [TH7 chim].

Example 9: In vitro effect of various IgGl chimeric and humanized form of the 224Gl 1 Mab NCI-H441 cells from ATCC were routinely cultured in RPMI 1640 medium

(Invitrogen Corporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1% L- Glutamine (Invitrogen Corporation). For proliferation 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 3.75 x 10⁴ cells/well in 200 μl of serum 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 μg/ml (final concentration in each well). In this experiment, murine IgGl Mab was added as a background negative control for agonist activity and the tested antibodies were the following one: i) m224Gl l, ii) its human IgGl chimeric forms respectively identified as [224Gl 1] chim, [224Gl 1] [TH7 chim] iii) its humanized IgGl forms respectively described as [224Gl 1] [TH7 HzI], [224Gl 1] [TH7 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 full agonist positive control and thereafter called m5D5. Then cells were pulsed with 0.25 μCi of [³H]Thymidine (Amersham Biosciences AB, Uppsala, Sweden) for 7 hours and 30 minutes. The magnitude of [³H]Thymidine 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.

Figure 10 showed that the m224Gl l Mab displayed the usual inhibitory effect (74% inhibition). The chimeric IgGl form [224Gl 1] chim had as expected a dose dependant intrinsic agonist effect and a lower antagonist effect compared to the murine form: 33% versus 74% inhibition. The [224Gl 1] [TH7 chim] had a very weak agonist activity in this experiment. In addition, 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 the [224Gl 1] [TH7 chim] with respectively 67 and 76% inhibition for [224Gl 1] [TH7 HzI] and [224Gl 1] [TH7 Hz3].

Claims

1. A process of improving the antagonistic activity of a monoclonal antibody directed against a specific target molecule, or a divalent functional fragment or derivative thereof, said antibody being capable of inhibiting one or more of the biological activities of said target molecule, wherein said process comprises a stage of reconfiguration of the hinge region consisting of a modification of the amino acid sequence of said hinge region by the deletion, the addition or the substitution of at least one amino acid.

2. The process of claim 1 wherein said modification is selected from: i) the deletion of at least one amino acid of said hinge region amino acid sequence; and/or ii) the addition of at least one disulfide bridge into said hinge region.

3. The process of claim 2 wherein the modification i) consists of the deletion of at most 2, 3 or 4 amino acids of said hinge region amino acid sequence.

4. The process of one of claims 1 to 3, wherein said monoclonal antibody is a divalent antibody.

5. The process of one of claims 1 to 3, wherein said monoclonal antibody is a chimeric antibody.

6. The process of one of claims 1 to 3, wherein said monoclonal antibody is a humanized antibody.

7. The process of one of claims 1 to 3, wherein said monoclonal antibody is a human antibody.

8. The process of one of claims 1 to 7, wherein said monoclonal antibody is an IgGl.

9. The process of one of claims 1 to 8, wherein said target molecule is a transmembrane receptor.

10. The process of claim 9, wherein said transmembrane receptor is selected from the group consisting of the tyrosine kinase receptors and GPCRs.

11. The process of one of claims 1 to 10, wherein said modification consists of at least a deletion of an amino acid selected from the amino acid in position Hl, H5, H6, H9 or H12.

12. The process of one of claims 1 to 10, wherein said modification consists of at least a deletion of an amino acid in the "upper hinge" region located between the positions Hl and H9 of said hinge region amino acid sequence.

13. The process of claim 11 or 12, wherein said modification consists of the deletion of the amino acids H6 and H9.

14. The process of one of claims 1 to 13, wherein said modification consists of the introduction of at least one Cysteine into the "upper hinge" region.

15. The process of claim 14, wherein said modification consists of the substitution of the Threonine in position H7 into the "upper hinge" region by a Cysteine.

16. The process of claim 14, wherein said modification consists of the substitution of the Lysine in position H6 into the "upper hinge" region by a Cysteine.

17. The process of claims 1 to 10, wherein said modification consists of a replacement of the amino acids Hl to Hl 4 of the IgGl hinge region by the amino acid Hl to H14 of an IgG2 hinge region when said monoclonal antibody which is desired to improve its antagonistic activity is an IgGl.

18. A process of screening for an antagonist monoclonal antibody directed against a specific target molecule, or a divalent functional fragment or derivative thereof, said antibody being capable of inhibiting one or more of the biological activities of said target molecule, wherein said process comprises the steps of:

(a) selecting an initial antibody with an initial level of inhibition of said one or more biological activity of said target molecule,

(b) modifying the amino acid sequence of the hinge region of said initial antibody by the process of one of claims 1 to 17, (c) evaluating the modified antibody of step (b) for its ability to inhibit said one or more biological activity of said target molecule, and

(d) selecting, as a positive result, the antibody of step (c) with an inhibition level of said one or more biological activity of said target molecule higher than the initial level of said inhibition.

19. A monoclonal antibody directed against a specific target molecule, or divalent functional fragments or derivatives thereof, obtainable by the process of one of claims 1 to 18, characterized in that it comprises a hinge region amino acid sequence selected from the group consisting of SEQ ID No. 1 (PRDCGCKPCICT), SEQ ID No. 2 (PKSCGCKPCICT), SEQ ID NO. 3 (PKSCGCKPCICP), SEQ ID No. 4 (PRDCGCKPCPPCP), SEQ ID NO. 5 (PRDCGCHTCPPCP), SEQ ID No. 6 (PKSCDCHCPPCP) or SEQ ID No. 7 (RKCC VECPPCP).

20. A monoclonal antibody, characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID No. 1 (PRDCGCKPCICT), SEQ ID No. 2 (PKSCGCKPCICT), SEQ ID No. 3 (PKSCGCKPCICP), SEQ ID No. 4 (PRDCGCKPCPPCP), SEQ ID No. 5 (PRDCGCHTCPPCP), SEQ ID No. 6 (PKSCDCHCPPCP) or SEQ ID No. 7 (RKCC VECPPCP).

21. A monoclonal antibody of one of claims 19 and 20, wherein said antibody is a human antibody.

22. A monoclonal antibody of one of claims 19 to 21, wherein said antibody is an IgGl antibody.

23. An isolated nucleic acid encoding for a monoclonal antibody of claim 19 to 22.

24. An isolated nucleic acid of claim 23, wherein said nucleic acid comprises a nucleic sequence is selected from the group consisting of SEQ ID No. 15 to SEQ ID No. 21.

25. An expression vector or a transformed host cell comprising an isolated nucleic acid of claim 23 or 24.