# Patent

## Title
Mutated humanized 12G4 antibodies and the fragments thereof against the human anti-Mullerian hormone receptor type II

## Abstract

Novel mutated humanized 12G4 antibodies, and fragments thereof, directed against the anti-Müllerian hormone type II receptor.

## Background
The invention relates to novel mutated humanized 12G4 antibodies, and fragments thereof, directed against the anti-Müllerian hormone type II receptor.
Ovarian cancer is the main cause of gynaecological cancers and is the fifth commonest cause of mortality from cancer in women, having the following three histological origins:

    
    
        the surface epithelium (epithelial tumour with various subtypes), which represents 85-90% of ovarian cancers,
        sexual cords/stroma (granulosa tumour (3% of total ovarian cancers)), representing about 10% of ovarian tumours,
        germ cells, representing 5% of ovarian cancers.
    
    


It is generally asymptomatic during the first stages, hence its nickname “silent killer” (La Marca A., Volpe A. The Anti-Mullerian hormone and ovarian cancer. Human Reproduction Update, Vol. 13, No. 3 pp. 265-273, 2007).
There are four stages and prognoses (FIGO classification: International Federation of Gynaecology and Obstetrics) for which the survival rate decreases considerably from stage 2:
Stage I: Tumour limited to the ovaries (5-year survival: 90-70%),
Stage II: Tumour in one or two ovaries and extends to pelvis (5-year survival: 70-40%),
Stage III: Tumour in one or two ovaries, extending outside the pelvis (5-year survival: 20%),
Stage IV: Distant metastases excluding peritoneal metastases (5-year survival: <10%), (Fauci, Braunwald et al. Principles of internal medicine. Harrison's 17th edition/National Cancer Institute cancer.gov/CNGOF (French National Colleges of Gynaecologists and Obstetricians).
The main strategies used for treating ovarian cancer are surgery and chemotherapy, in particular as first-line treatment, such as a mixture of carboplatin and paclitaxel.
Monoclonal antibodies have also recently been developed such as cetuximab, which is directed against the epidermal growth factor receptor (EGFR, Ozols R. F. et al., Focus on epithelial ovarian cancer, Cancer Cell. 2004, January; 5(1): 19-24).
Other monoclonal antibodies are currently in phase III, such as abagovomab directed against CA-125, Avastin directed against vascular endothelial growth factor (VEGF-A), or farletuzumab directed against folate receptor alpha (FRA).
The human anti-Müllerian hormone is a glycoprotein of 560 amino acids, a member of the TGF-β family. It is a hormone released by the Sertoli cells of the fetal testis, which causes degeneration of the Müller duct.
It is expressed in the adult in the Sertoli cells and Leydig cells (testis) and the granulosa cells (ovary).
It plays a role in the activity of the adult ovary in regulation of folliculogenesis.
The anti-Müllerian hormone type II receptor (AMHR-II) is a peptide of 573 amino acids and has serine-threonine kinase activity.
It is involved in regression of the Müller duct associated with development of the human reproductive system. It atrophies in men, where it only forms the prostatic vesicle and the sessile hydatid, but it persists in women, where it gives rise to the fallopian tubes, the uterus and most of the vagina.
This receptor is often expressed on the tumoral epithelial cells of human ovaries.
International application WO 2008/053330 describes a murine 12G4 monoclonal antibody directed against AMHR-II for treating ovarian cancers. However, it is well known to a person skilled in the art that the administration of murine monoclonal antibodies in humans causes an immune reaction.
This international application also mentions that the antibody can be chimeric or humanized, but does not describe them as such.
However, chimeric antibodies also trigger immune reactions, and humanized antibodies, slightly immunogenic, have the shortcoming that their antigen binding affinity can decrease and consequently they become less active.
It is possible according to this application to increase said affinity by mutation of the amino acids present in the humanized antibody, in particular by modifying the peptide sequence of the humanized antibody but maintaining the hydropathic index, i.e. their hydrophobicity and their charge, for example by substitution of the following amino acids: arginine-lysine or glutamate-aspartate or serine-threonine or glutamine-asparagine or valine-leucine-isoleucine substitution.
One of the aims of the invention is to provide mutated humanized 12G4 antibodies, or fragments thereof, having an affinity at least equal to that of the corresponding unmutated chimeric antibody and specificity with respect to the AMHR-II receptor, and not triggering an immune reaction.
Another aim of the invention is also to provide means for producing said specific antibodies of the AMHR-II receptor.
The invention further relates to the use of these antibodies as drugs for treating ovarian cancers.
The invention relates to a humanized 12G4 monoclonal antibody comprising or consisting of:

    
    
        a) a light chain comprising or consisting of:
        
            a variable region the amino acid sequence of which is represented by SEQ ID NO: 2 (without leader) or SEQ ID NO: 4 (with leader), and
            a constant region the amino acid sequence of which is represented by SEQ ID NO: 6 or by a sequence having at least 80% homology with SEQ ID NO: 6,
        
        
        b) a heavy chain comprising or consisting of:
        
            a variable region the amino acid sequence of which is represented by SEQ ID NO: 8 (without leader), or SEQ ID NO: 10 (with leader), and
            a constant region the amino acid sequence of which is represented by SEQ ID NO: 12 or by a sequence having at least 80% homology with SEQ ID NO: 12,
        
        
        and said humanized 12G4 monoclonal antibody is mutated, comprises at least one mutation in the light and/or heavy chain, and has a KD for the human anti-Müllerian hormone type II receptor (AMHRII) at least equal to that of the chimeric 12G4 monoclonal antibody comprising or consisting of:
        
            a variable region the amino acid sequence of which is represented by SEQ ID NO: 14 (without leader), and
            a constant region the amino acid sequence of which is represented by SEQ ID NO: 6,
        
        
        b) a heavy chain consisting of:
        
            a variable region the amino acid sequence of which is represented by SEQ ID NO: 18 (without leader) or SEQ ID NO: 10 (with leader), and
            a constant region the amino acid sequence of which is represented by SEQ ID NO: 12,
        
        
    
    


for said receptor, preferably below 10−7M, in particular below 10−8M, in particular from 10−9M to 10−11M.
The antibodies of the invention also display an affinity at least equal to a third or a half of that of the murine 12G4 antibody.
Throughout the description, the expression in parentheses “with leader” after the sequence number indicates that said sequence comprises the signal peptide or the sequence encoding for the signal peptide, i.e. the peptide that defines that the protein will be secreted.
Conversely, the expression in parentheses “without leader” indicates that said sequence does not comprise the signal peptide or the sequence encoding for the signal peptide.
The invention is based on the inventors' finding that mutated humanized 12G4 antibodies of the invention, although having at least one mutation in the CDR (three regions determining recognition of the antigen) the hydropathic index of which is not respected, i.e. in a region that is crucial for the affinity and binding to the antigen, and which as a general rule only allows substitutions of amino acids of the same hydropathic index (for example arginine-lysine, glutamate-aspartate, serine-threonine, glutamine-asparagine or valine-leucine-isoleucine), still has the following properties:

    
    
        it has for a KD for the AMHR-II receptor (determined according to Example 1) at least similar to or even less than that of the corresponding chimeric 12G4 antibody and therefore an affinity greater than or equal to that of the corresponding chimeric 12G4 antibody, or relative to the antibodies or antibody fragments of the prior art.
        it has a specificity for the AMHR-II receptor,
        it does not trigger an immune reaction or a smaller reaction than the murine antibody
    
    


By way of example, Example 1 presents the KD obtained with antibodies of the invention produced in CHO or YB2/0 cells.
Throughout the description, the term “12G4” and the term “LFB112”, which is also used, denote the same thing and represent the same antibody.
The affinity of said antibody can be determined by a BIAcore assay that is well known to a person skilled in the art.
In the invention, the term “antibody” refers to an immunoglobulin, a multimeric protein consisting of 4 chains participating in the acquired immune response.
The immunoglobulins are well known to a person skilled in the art and consist of an assembly of two dimers, each consisting of a heavy chain and a light chain. The multimeric complex assembled by the binding of a light chain and a heavy chain by a disulphide bridge between two cysteines, the two heavy chains themselves also being joined together by two disulphide bridges.
Each of the heavy chains and of the light chains consists of a constant region and a variable region. The assembly of chains making up an antibody can define a characteristic Y-shaped three-dimensional structure, where

    
    
        the base of the Y corresponds to the constant region Fc that is recognized by complement and the Fc receptors, and
        the ends of the arms of the Y correspond to the respective assembly of variable regions of the light chain and variable of the heavy chain.
    
    


More precisely, each light chain consists of a variable region (VL) and a constant region (CL). Each heavy chain consists of a variable region (VH) and a constant region consisting of three constant domains CH1, CH2 and CH3. The domains CH2 and CH3 make up the domain Fc.
The structure of an antibody is shown schematically in FIG. 1.
The variable region of the light chain consists of three regions determining recognition of the antigen (CDR) surrounded by four framework regions. The three-dimensional folding of the variable region is such that the 3 CDRs are exposed on the same side of the protein and allow the formation of a specific structure recognizing a particular antigen.
The string-of-pearls structure of a variable region of a light or heavy chain of an antibody is shown in FIG. 2.
The antibodies described in the invention have been isolated and purified, and are different from natural antibodies because they are humanized. These antibodies are mature, i.e. they have an ad hoc three-dimensional structure allowing them to recognize the antigen, and they have all the post-translational modifications essential for their antigen recognition, in particular glycosylation and the formation of intramolecular and intermolecular disulphide bridges.
They are monoclonal antibodies, i.e. they only recognize a single antigenic determinant in the AMHR-II receptor, in contrast to polyclonal antibodies, which correspond to a mixture of antibodies, and can therefore recognize several antigenic determinants in one and the same protein.
By “chimeric monoclonal antibody” is meant, in the invention, an isolated antibody, in which the sequence of each light chain and/or of each heavy chain of the antibody of which it is constituted comprises or consists of a hybrid sequence derived from at least two different animals (or human).
In particular the chimeric 12G4 antibody is a mouse/human hybrid, which indicates that a region of the sequence of the light chains and of the heavy chains is derived from the sequence of a mouse 12G4 immunoglobulin, and that the rest of the sequence of said heavy chains and of said light chains is derived from the sequence of one, or optionally several, human immunoglobulins.
FIG. 18 gives the map of the expression vector for producing the chimeric 12G4 antibody.
By “humanized 12G4 monoclonal antibody” is meant, in the invention, an isolated antibody, in which only the CDRs of each light and heavy chain of the 12G4 antibody, in particular murine, have been grafted in the light and heavy chains of a human antibody.
FIG. 19 gives the map of the expression vector for producing the humanized 12G4 antibody.
Hereinafter, the expressions “chimeric 12G4 antibody” and “unmutated chimeric 12G4 antibody” denote the same antibody.
By “mutated humanized 12G4 monoclonal antibody” is meant, in the invention, a humanized 12G4 monoclonal antibody in which at least one mutation was carried out in the variable region of the light chain and/or the constant region of the light chain and/or the variable region of the heavy chain or the constant region of the heavy chain.
Thus, the definition of the mutated humanized monoclonal antibody of the invention covers both:

    
    
        the precursor of the mutated humanized antibody, in particular the human/mouse antibody as defined above, and
        the mutated humanized antibody, in particular human/mouse, defined above.
    
    


In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, comprising at least one mutation in at least one CDR of the variable region of the light chain, and having an affinity for said receptor at least equal to that of said chimeric 12G4 monoclonal antibody.
In this embodiment, when a single mutation is present, it is located in CDR1 or CDR2 or CDR3.
When more than one mutation is present, the second as well as the others can be located in CDR1 and/or CDR2 and/or CDR3 and/or any other region of the antibody.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, further comprising at least one mutation in the FR regions of the light chain (VL).
In the present invention, the inventors discovered that when at least one mutation is carried out in CDR1 or CDR2 or CDR3 of the humanized 12G4 antibody, and at least one mutation in the variable region, in particular FR of the light chain, said at least one mutation not necessarily complying with the hydropathic index of the amino acids, nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune or less important reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, comprising at least one mutation in CDR1 and at least one mutation in the FR regions of the light chain (VL).
In the present invention, the inventors discovered that when at least one mutation is carried out in CDR1 of the humanized 12G4 antibody, and at least one mutation in the variable region, in particular FR of the light chain, said at least one mutation not necessarily complying with the hydropathic index of the amino acids, nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, comprising at least one mutation in CDR2 and at least one mutation in the FR regions of the light chain (VL).
In the present invention, the inventors discovered that when at least one mutation is carried out in CDR2 of the humanized 12G4 antibody, and at least one mutation in the variable region, in particular FR of the light chain, said at least one mutation not necessarily complying with the hydropathic index of the amino acids, nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, comprising at least one mutation in CDR3 and at least one mutation in the FR regions of the light chain (VL).
In the present invention, the inventors discovered that when at least one mutation is carried out in CDR3 of the humanized 12G4 antibody, and at least one mutation in the variable region, in particular FR of the light chain, said at least one mutation not necessarily complying with the hydropathic index of the amino acids, nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, having an ADCC against cells, in particular Cov434, Asc 1 and META 2815, expressing the AMHR II receptor, in particular greater than the ADCC against the same cells of said unmutated humanized 12G4 monoclonal antibody.
ADCC (antibody-dependent cellular cytotoxicity) is a mechanism in which, when the antibody has recognized an antigen, the portion Fc of the antibody is recognized by a receptor Fcγ of a killer cell which, after binding, is capable of killing the cell bearing the antigen.
In the present invention, the inventors discovered that when at least one mutation is carried out in one or more of the CDRs of the humanized 12G4 antibody, the affinity of which is particularly reduced relative to the corresponding chimeric or murine antibody (Example 2), and although the CDR corresponds to a particularly large region of antigen recognition, said at least one mutation makes it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and they do not cause an immune reaction or a less pronounced reaction.
In the context of the present invention, the numbering used is based on the numbering of an ScFv fragment, the heavy chain being numbered from 1 to 115 and the light chain from 131 to 236, as shown in FIGS. 3A and 3B for the humanized 12G4 antibody in which the grey-shaded beads correspond to the amino acids that are absent from said sequence.
The two chains are joined together by a linker comprising the amino acids 116 to 130.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, in which at least one of said mutations in at least one CDR of the variable region of the light chain, is located in the CDR comprised in the region containing amino acid 179 to amino acid 184 of the variable region of the light chain, the amino acid sequence of which is represented by SEQ ID NO: 2.
The region containing amino acid 179 to amino acid 184 does not correspond to the complete CDR.
In this embodiment, if the antibody only has one mutation, it is located in the region of the CDR of the light chain containing amino acid 179 to amino acid 184.
It can of course have other mutations in other CDRs.
In the present invention, the inventors discovered that when at least one mutation is carried out in the region comprising amino acid 179 to 184 of the CDR of the humanized 12G4 antibody, said at least one mutation makes it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, in which at least one of said mutations located in the CDR comprised in the region containing amino acid 179 to amino acid 184 corresponds to the substitution of at least one of the following amino acids: S179P, E184K, E184G, E184D, S182F.
The notation used here corresponds to the single-letter codes that are familiar to a person skilled in the art.
The notation S179P means for example that the amino acid serine in position 179 is replaced with a proline.
In the present invention, the inventors discovered that when at least one mutation is carried out in the region comprising amino acid 179 to 184 of the CDR of the humanized 12G4 antibody, said at least one mutation not necessarily complying with the hydropathic index of the substituted amino acids, nevertheless makes it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody.
By way of example, FIG. 17 shows the affinity of binding to the AMHR-II receptor of mutated humanized antibodies according to the invention. The 6B—78 antibody only has one mutation located in the CDR of the variable region of the light chain (E184K) in which a glutamic acid is replaced with a lysine, i.e. replacement of an acidic amino acid with a basic amino acid, consequently having a totally different charge since opposite, and yet still displaying an activity but in particular an appreciably better affinity than the unmutated humanized 12G4 antibody, and equal to that of the unmutated chimeric 12G4 antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, further comprising at least one mutation in the FR regions of the light chain (VL).
In the present invention, the inventors discovered that when at least one mutation is carried out in the region comprising amino acid 179 to 184 of the CDR of the humanized 12G4 antibody, and at least one mutation in the variable region, in particular FR of the light chain, said at least one mutation not necessarily complying with the hydropathic index of amino acids nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, further comprising at least one mutation in the heavy chain.
In the present invention, the inventors discovered that when at least one mutation is carried out in the region comprising amino acid 179 to 184 of the CDR of the humanized 12G4 antibody, at least one mutation in the variable region, in particular FR of the light chain, and at least one mutation in the heavy chain, said at least one mutation not necessarily complying with the hydropathic index of the amino acids, nevertheless make it possible not only to preserve the activity of the humanized antibody but even obtain a mutated humanized antibody having an affinity at least equal to that of the unmutated chimeric antibody, and do not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, in which at least one of said mutations in the FR regions of the light chain (VL) is located in the FR region adjacent to the region containing amino acid 179 to amino acid 184.
By way of example, FIG. 17 and Tables I and VII (fixation to the target AMHRII-Fc determined by ELISA) present the affinity of binding to the AMHR-II receptor of the mutated humanized antibody according to the invention.
Thus, the 3C—23 antibody has three mutations:

    
    
        a mutation in the CDR of the variable region of the light chain (S179P) in which a serine is replaced with a proline, i.e. replacement of a hydrophilic amino acid with a hydrophobic amino acid,
        a mutation in the variable region, in particular FR of the light chain (1177T), i.e. replacement of a hydrophobic amino acid with a hydrophilic amino acid, moreover having an entirely different value of hydropathic index, according to international application WO 2008/053330 (+4.5 for isoleucine and −0.7 for threonine), and
        a mutation in the heavy chain (Q3R), i.e. replacement of a glutamine with an arginine for which the value of the hydropathic index, according to international application WO 2008/053330, varies from −3.5 for glutamine to −4.5 for arginine,
    
    


and yet having an appreciably better affinity than that of the unmutated humanized 12G4 antibody, and greater than that of the unmutated chimeric 12G4 antibody.
Moreover, the 3C—23K antibody, which apart from the mutations of the 3C—23 antibody also has a second mutation in the CDR of the variable region of the light chain (E184K) in which a glutamic acid is replaced with a lysine, i.e. replacement of an acidic amino acid with a basic amino acid, consequently having a totally different charge since it is of opposite sign, nevertheless still displays an activity, but especially an affinity that is appreciably better than that of the unmutated humanized 12G4 antibody, and greater than that of the unmutated chimeric 12G4 antibody, and does not cause an immune reaction.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, in which at least one of said mutations in the FR regions of the light chain (VL) corresponds to the substitution of at least one of the following amino acids: I132T, A143T, T150A, S158P, L175Q, I177T, Y178H, V187A, S192T, G197D, F212S.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, in which at least one of said mutations in the heavy chain corresponds to the substitution of at least one of the following amino acids: Q1E, Q3E, Q3R, Q6E, A9T, V11A, K12R, K13R, K19E, V20A, A24G, A24V, A24T, Q39E, A40V, S31G, L45P, D56N, A76T, A79T, R87G, T58A, Q62R, V67M, I70N, T74A, S77P, A79T, S88P, E89D, F102S, A103T, L110P, S114T.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, having a light chain and a heavy chain selected from the following:

    
    
        a) a light chain comprising or consisting of a variable region the amino acid sequence of which is represented by SEQ ID NO: 2 in which at least one following substitution of amino acids located in one of the CDRs has been carried out: S179P, E184K, E184G, E184D, S182F, or
        
            a light chain comprising or consisting of a variable region the amino acid sequence of which is represented by SEQ ID NO: 2 in which at least one following substitution of amino acids located in one of the CDRs has been carried out: S179P, E184K, E184G, E184D, S182F, and at least one following substitution of amino acids located in regions FR has been carried out: I132T, A143T, T150A, S158P, L175Q, Y178H, V187A, S192T, G197D, F212S,
        
        
        and of a constant region the amino acid sequence of which is represented by SEQ ID NO: 6,
        b) a heavy chain the amino acid sequence of which is represented by SEQ ID NO: 58 in which a substitution of at least one of the following amino acids: Q1E, Q3E, Q3R, Q6E, A9T, V11A, K12R, K13R, K19E, V20A, A24G, A24V, A24T, Q39E, A40V, S31G, L45P, D56N, A76T, A79T, R87G, T58A, Q62R, V67M, I70N, T74A, S77P, A79T, S88P, E89D, F102S, A103T, L110P, S114T has been carried out.
    
    


Table VII of Example 3 presents the various clones obtained and their substitution. It also shows that the hydropathic index varies considerably as a function of the mutations, but without leading to a loss of activity and/or of affinity for the antigen and even makes it possible, for certain clones, to obtain an increase in affinity relative to the corresponding chimeric antibody (ratio Ab of the invention/chimeric antibody greater than or equal to 1).
In this embodiment, it is possible to constitute an antibody that is derived from the combination of two antibodies obtained previously and further increase the activity and the affinity for the AMHR-II receptor relative to the unmutated chimeric 12G4 antibody.
In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, having a light chain and a heavy chain selected from the following:

    
    
        a) a light chain comprising or consisting of a variable region the amino acid sequence of which is represented by:
        
            SEQ ID NO: 22 (without leader) or SEQ ID NO: 24 (with leader), or
            SEQ ID NO: 30 (without leader) or SEQ ID NO: 32 (with leader), or
            SEQ ID NO: 34 (without leader) or SEQ ID NO: 36 (with leader), or
            SEQ ID NO: 46 (without leader) or SEQ ID NO: 48 (with leader),
            and of a constant region the amino acid sequence of which is represented by SEQ ID NO: 6,
        
        
        b) a heavy chain comprising or consisting of a variable region the amino acid sequence of which is represented by:
        
            SEQ ID NO: 38 (without leader), or SEQ ID NO: 40 (with leader),
            SEQ ID NO: 26 (without leader), or SEQ ID NO: 28 (with leader),
            SEQ ID NO: 8 (without leader), or SEQ ID NO: 10 (with leader),
            SEQ ID NO: 42 (without leader), or SEQ ID NO: 44 (with leader),
            SEQ ID NO: 50 (without leader), or SEQ ID NO: 52 (with leader), and of a constant region the amino acid sequence of which is represented by SEQ ID NO: 12.
        
        
    
    


In an advantageous embodiment, the present invention relates to a mutated humanized 12G4 monoclonal antibody as defined above, having:

    
    
        a) a light chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 70 (without leader) or SEQ ID NO: 72 (with leader), and
        
        
        b) a heavy chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 74 (without leader), or SEQ ID NO: 76 (with leader), (3C—23 antibody)
        
        
    
    


or,

    
    
        a) a light chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 78 (without leader) or SEQ ID NO: 80 (with leader), and
        
        
        b) a heavy chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 58 (without leader), or SEQ ID NO: 60 (with leader), (6B—78 antibody)

or,

        
        
        a) a light chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 82 (without leader) or SEQ ID NO: 84 (with leader), and
        
        
        b) a heavy chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 86 (without leader), or SEQ ID NO: 88 (with leader), (3C—23K antibody)

or,

        
        
        a) a light chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 78 (without leader) or SEQ ID NO: 80 (with leader), and
        
        
        b) a heavy chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 90 (without leader), or SEQ ID NO: 92 (with leader), (4C—35 antibody)

or,

        
        
        a) a light chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 94 (without leader) or SEQ ID NO: 96 (with leader), and
        
        
        b) a heavy chain consisting of the amino acid sequence represented by:
        
            SEQ ID NO: 98 (without leader), or SEQ ID NO: 100 (with leader), (5B—42 antibody)
        
        
    
    


According to another aspect, the invention relates to a fragment of a mutated humanized 12G4 monoclonal antibody as defined above, selected from the group of fragments consisting of: Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, Sc(Fv)2, “diabodies”.
According to another aspect, the invention relates to a nucleic acid comprising or consisting of a sequence encoding for the light chain of a monoclonal antibody defined above and/or comprising or consisting of a sequence encoding for the heavy chain of the monoclonal antibody defined above.
In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the light chain comprises or consists of the following sequences:

    
    
        a) a sequence encoding for the variable region of the light chain represented by SEQ ID NO: 53 in which a substitution of at least one codon permitting the substitution, in one of the CDRs, of one or more of the following amino acids: S179P, E184K, E184G, E184D, S182F has been carried out,
        or,
        b) a sequence encoding for the variable region of the light chain represented by SEQ ID NO: 53 in which:
        
            at least one substitution of a codon permitting the substitution, in one of the CDRs, of one or more of the following amino acids: S179P, E184K, E184G, E184D, S182F has been carried out, and
            at least one substitution of at least one codon permitting the substitution, in one of the FRs, of one or more of the following amino acids: I132T, A143T, T150A, S158P, L175Q, Y178H, V187A, S192T, G197D, F212S, has been carried out,
        
        
        and a sequence encoding for the constant region represented by SEQ ID NO: 5.
    
    


In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the heavy chain comprises or consists of the following sequences:

    
    
        a) SEQ ID NO: 57 in which a substitution of at least one codon permitting the substitution of one or more of the following amino acids: Q1E, Q3E, Q3R, Q6E, A9T, V11A, K12R, K13R, K19E, V20A, A24G, A24V, A24T, Q39E, A40V, S31G, L45P, D56N, A76T, A79T, R87G, T58A, Q62R, V67M, I70N, T74A, S77P, A79T, S88P, E89D, F102S, A103T, L110P, S114T has been carried out.
    
    


In an advantageous embodiment, the invention relates to a nucleic acid defined above, comprising a light chain defined above and a heavy chain defined above.
In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the light chain comprises or consists of a sequence encoding for a variable region and a sequence encoding for a constant region selected from the following:

    
    
        a) variable region:
        
            SEQ ID NO: 21 (without leader) or SEQ ID NO: 23 (with leader), or
            SEQ ID NO: 29 (without leader) or SEQ ID NO: 31 (with leader), or
            SEQ ID NO: 33 (without leader) or SEQ ID NO: 35 (with leader), or
            SEQ ID NO: 45 (without leader) or SEQ ID NO: 47 (with leader), or
        
        
        b) constant region
        
            SEQ ID NO: 5.
        
        
    
    


In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the heavy chain comprises or consists of a sequence encoding for a variable region and a sequence encoding for a constant region selected from the following:

    
    
        a) variable region:
        
            SEQ ID NO: 25 (without leader) or SEQ ID NO: 27 (with leader), or
            SEQ ID NO: 7 (without leader) or SEQ ID NO: 9 (with leader), or
            SEQ ID NO: 37 (without leader) or SEQ ID NO: 39 (with leader), or
            SEQ ID NO: 41 (without leader) or SEQ ID NO: 43 (with leader), or
            SEQ ID NO: 49 (without leader) or SEQ ID NO: 51 (with leader),
        
        
        b) constant region
        
            SEQ ID NO: 11.
        
        
    
    


In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the light chain is selected from the following sequences:

    
    
        SEQ ID NO: 69 (without leader) or SEQ ID NO: 71 (with leader), or
        SEQ ID NO: 77 (without leader) or SEQ ID NO: 79 (with leader), or
        SEQ ID NO: 81 (without leader) or SEQ ID NO: 83 (with leader), or
        SEQ ID NO: 93 (without leader) or SEQ ID NO: 95 (with leader),
    
    


and the sequence encoding for the heavy chain is selected from the following sequences:

    
    
        SEQ ID NO: 73 (without leader) or SEQ ID NO: 75 (with leader), or
        SEQ ID NO: 57 (without leader) or SEQ ID NO: 59 (with leader), or
        SEQ ID NO: 85 (without leader) or SEQ ID NO: 87 (with leader), or
        SEQ ID NO: 89 (without leader) or SEQ ID NO: 91 (with leader), or
        SEQ ID NO: 97 (without leader) or SEQ ID NO: 99 (with leader).
    
    


In an advantageous embodiment, the invention relates to a nucleic acid defined above, in which the sequence encoding for the light chain and the sequence encoding for the heavy chain are as follows:

    
    
        a) sequence encoding for the light chain SEQ ID NO: 69 (without leader) or SEQ ID NO: 71 (with leader), and
        b) sequence encoding for the heavy chain SEQ ID NO: 73 (without leader) or SEQ ID NO: 75 (with leader),
        (3C—23 antibody)
    
    


or,

    
    
        a) sequence encoding for the light chain SEQ ID NO: 77 (without leader) or SEQ ID NO: 79 (with leader), and
        b) sequence encoding for the heavy chain SEQ ID NO: 57 (without leader) or SEQ ID NO: 59 (with leader),
        (6B—78 antibody)
    
    


or,

    
    
        a) sequence encoding for the light chain SEQ ID NO: 81 (without leader) or SEQ ID NO: 83 (with leader), and
        b) sequence encoding for the heavy chain SEQ ID NO: 85 (without leader) or SEQ ID NO: 87 (with leader),
        (3C—23K antibody)
    
    


or,

    
    
        a) sequence encoding for the light chain SEQ ID NO: 77 (without leader) or SEQ ID NO: 79 (with leader), and
        b) sequence encoding for the heavy chain SEQ ID NO: 89 (without leader) or SEQ ID NO: 91 (with leader),
        (4C—35 antibody)
    
    


or,

    
    
        a) sequence encoding for the light chain SEQ ID NO: 93 (without leader) or SEQ ID NO: 95 (with leader), and
        b) sequence encoding for the heavy chain SEQ ID NO: 97 (without leader) or SEQ ID NO: 99 (with leader),
        (5B—42 antibody).
    
    


According to another aspect, the invention relates to an expression vector comprising at least one nucleic acid defined above, said nucleic acid being under the control of the elements permitting its expression.
“Expression vector” defines, in the invention, a DNA molecule that has elements permitting its replication (duplication) in at least one living organism. These elements permitting replication are in particular origins of replication in yeast or bacteria, or elements controlling the replication of a virus.
The vectors according to the invention are in particular plasmids, phages, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), modified genomes of replicative viruses or of integrative viruses etc.
These vectors are called “expression vectors” as they have nucleotide sequences that permit the expression, i.e. the transcription to RNA, of the nucleotide sequences that they control.
In the invention, said nucleic acid sequence contained in said vector is placed “under the control of the elements permitting its expression”. This means that said expression vector has at least one transcription initiation sequence such as a promoter of a virus, for instance the early promoter of the simian virus SV40, or of the Cytomegalovirus (CMV) or the promoter sequences of the Rous sarcoma virus (RSV), and in particular a sequence or promoter comprising a TATAA box. Moreover, said vector also has at least one transcription termination sequence, and in particular a polyadenylation sequence, derived from a mammalian, in particular human, gene.
To these sequences, which are indispensable for expression of the nucleotide sequence contained in said vector, other sequences may be added for regulating or modulating the expression of said sequence. A non-limitative list comprises: introns of mammalian, and in particular human, genes, sequences for regulating transcription of the enhancer type or sequences of mammalian, and in particular human, genes that have been transcribed but not translated.
An advantageous embodiment of the invention relates to an expression vector as defined above, comprising at least one nucleic acid selected from the nucleic acids comprising the following sequences SEQ ID NO 59, 71, 75, 79, 83, 87, 91, 95 or 99.
In another advantageous embodiment, the invention relates to an expression vector as defined above, comprising

    
    
        a first nucleic acid selected from the nucleic acids with the following sequences: SEQ ID NO 71, 79, 83 or 95, said first nucleic acid being under the control of the elements permitting its expression, and
        a second nucleic acid selected from the nucleic acids with the following sequences:
    
    


SEQ ID NO 59, 75, 87, 91 or 99, said second nucleic acid being under the control of the elements permitting its expression.
This expression vector therefore comprises two aforementioned sequences of nucleic acids, and more particularly comprises a nucleic acid sequence encoding for the light chain of the monoclonal antibody defined above, and a nucleic acid sequence encoding for the heavy chain of the monoclonal antibody defined above.
Preferably said expression vector contains a first element permitting expression of the nucleic acid sequence encoding for the light chain of the monoclonal antibody defined above and a second element permitting expression of the nucleic acid sequence encoding for the heavy chain of the monoclonal antibody defined above, said first element and said second element, permitting expression of said sequences of nucleic acids, being identical or different, and preferably identical. These control elements are in particular the long terminal repeat (LTR) sequences of the virus RSV.
Another embodiment of the invention relates to an expression vector defined above, comprising at least one antibiotic resistance gene.
By “at least one resistance gene” is meant, in the invention, that said expression vector can contain 1 or 2, or 3 or 4 or 5 or 6 antibiotic resistance genes.
“Antibiotic resistance gene” defines, in the invention, a gene the expression product of which exerts a cytostatic effect (inhibition of growth) or cytolytic effect (cellular death) on cells. The antibiotics to which the invention relates in particular have an effect on prokaryotic cells, but can also have an effect on eukaryotic cells, whether of yeasts, plants, insects, amphibians or mammals.
More particularly, the aforementioned expression vector has an antibiotic resistance gene specific to prokaryotic cells and at least one, preferably 2, antibiotic resistance genes specific to eukaryotic cells.
As antibiotics specific to prokaryotic cells: ampicillin, tetracycline and derivatives thereof, hygromycin, kanamycin etc. may be mentioned. As antibiotics specific to eukaryotic cells: G418, Geneticin (salts of G418), puromycin, methotrexate, blasticidin etc. may be mentioned.
The transcription units (TUs) of interest coding for the heavy chain and the light chain are cloned in the form of cDNA and under the dependence of the RSV promoter. This promoter corresponds to the LTR (long terminal repeat) of the Rous sarcoma virus, which contains an enhancer element in its 5′ region.
An artificial intron optimized for alternative splicing and composed of a donor sequence at 5′ isolated from human beta-globin and at 3′ of an acceptor sequence derived from the gene of the variable of the heavy chain of immunoglobulin is cloned immediately at 3′ of the promoter. The TUs of interest are terminated with polyadenylation sequences derived from the growth hormone (GH) gene of human origin (hGH) for the heavy chain and bovine origin (bGH) for the light chain. This difference of origin in the choice of the polyAs has the aim of limiting recombinations between the genes of interest. This combination of LTRRSV promoter, chimeric intron, cDNA and polyA sequence was selected as it confers high transcriptional and translational activity in the YB2/0 cell line.
The expression vector contains in addition to the TUs of interest, several TUs for resistances to chemical molecules:
Bla gene: This gene (called Amp in the restriction maps of the vectors) expresses the enzyme beta-lactamase in the bacterium (prokaryotic promoter) and confers resistance to ampicillin.
Neo gene: This gene codes for the enzyme npt II (neomycin-phosphotransferase II) under the control of the promoter SV40 and confers resistance to various antibiotics such as neomycin, kanamycin or G418 on transfected mammalian cells expressing this gene.
Dhfr gene: This gene codes for the enzyme DHFR (DiHydroFolate Reductase) under the control of the promoter SV40 and confers resistance to methotrexate (MTX). This method can be used for gene amplification by increasing the concentration of MTX thus resulting from an increase in antibody production by the transfected cells.
FIGS. 18 to 22 give the maps of the expression vectors used for producing clones 3C—23, 6B—78, 3C—23K, 4C—35 and 5B—42.
In another aspect, the present invention relates to a host cell or cell line transformed by a nucleic acid defined above and/or an expression vector defined above.
In particular, said cell or cell line is characterized in that it


    
    
        displays apoptosis of less than 25%,
        is stable during cell divisions, and
        secretes at least 14 μg/ml of monoclonal antibody defined above.
    
    


The concept of cellular stability implies that the cells resulting from cloning of the cloned cells derived from the cells containing at least one vector permitting expression of a monoclonal antibody according to the invention are capable, during the various divisions, of conserving their properties of antibiotic resistances and of producing monoclonal antibodies.
In yet another aspect, the invention relates to a pharmaceutical composition, and in particular a vaccine composition, comprising at least

    
    
        a monoclonal antibody defined above, or
        a nucleic acid defined above, or
        a vector defined above, or
        a fragment of said monoclonal antibody defined above,
    
    


together with a pharmaceutically acceptable vehicle.
Advantageously, the invention relates to a pharmaceutical composition, and in particular a vaccine composition, comprising at least one monoclonal antibody defined above, together with a pharmaceutically acceptable vehicle.
The dosage of the active ingredient depends in particular on the method of administration, and is easily determined by a person skilled in the art.
“A pharmaceutically acceptable vehicle” refers to a non-toxic material that is compatible with a biological system such as a cell, a cell culture, a tissue or an organism.
A therapeutically effective amount (unit dose) can vary from 0.01 mg/kg to 500 mg/kg, preferably from 0.1 mg/kg to 500 mg/kg, preferably from 0.1 mg/kg to 100 mg/kg, preferably from 0.1 mg/kg to 20 mg/kg, preferably from 0.1 mg/kg to 10 mg/kg, and more preferably from 1 mg/kg to 10 mg/kg, in one or more weekly administrations, for several weeks or months.
Moreover, a therapeutically effective amount (unit dose) can vary from 0.2 mg/m2 to 10 g/m2, preferably from 0.2 mg/m2 to 1 g/m2, preferably from 2 mg/m2 to 1 g/m2, preferably from 20 mg/m2 to 1 g/m2, and more preferably from 20 mg/m2 to 0.5 g/m2, in one or more weekly administrations, for several weeks or months.
The pharmaceutical composition of the invention can in particular be administered intravenously, in particular by injection or by gradual perfusion, subcutaneously, systemically, locally by infiltrations, per os, or by the respiratory or pulmonary route by means of an aerosol.
The preparations for parenteral administration can include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters such as ethyl oleate. Aqueous vehicles comprise water, alcohol/water solutions, emulsions or suspensions.
The advantageous pharmaceutical form of the pharmaceutical composition of the invention can be administered by the oral route and comprises

    
    
        a monoclonal antibody defined above, or
        a nucleic acid defined above, or
        a expression vector defined above, or
        a fragment of said monoclonal antibody defined above,

together with an excipient, in the presence or absence of a propellant.

    
    


In one embodiment of the invention, the aerosol is in the form of a liquid containing the mutated humanized antibody and an excipient. The excipients are most often alcohols, but any other excipient known to a person skilled in the art can be used in the context of the invention. The aerosol in liquid form can be combined with a propellant gas such as chlorofluorocarbons (CFCs) or hydrofluorocarbons (HFAs).
The aerosol in liquid form can also consist of lipid microparticles and an excipient. In this case, the excipients can be selected from synthetic dipalmitoylphosphatidylcholine (DPPC), lactose or hydroxyethyl starch (HES). The microparticles are then administered by means of an insufflator.
In another embodiment of the invention, the aerosol is in the form of powder. The powder is composed of particles with a size between 1 and 10 μm and preferably less than 9 μm, or preferably less than 5 μm. As a non-limitative example, the following methods can be used for obtaining a dry powder: spraying accompanied by freeze-drying or crystallization by ultrasound, directed precipitation.
The aerosol will be administered, depending on whether it is in liquid or solid form, by means of a nebulizer which can be pneumatic, ultrasonic or of the sieve type or by means of a metering aerosol (of pressurized liquid, mechanical, electrohydrodynamic, thermal) for the liquid formulations or by means of an inhaler for the solid formulations. (Reychler G., Dessanges J F and Vecellio L, Rev. Mal. Respir, 2007; 24: 1013-1023).
According to another aspect, the invention relates to a product comprising a first pharmaceutical preparation comprising a monoclonal antibody defined above, and a second pharmaceutical preparation comprising a conventional anticancer compound, in particular paclitaxel or a platinum salt, in particular oxaliplatin, cisplatin or carboplatin, as a combined preparation for simultaneous, separate or sequential use in the treatment of patients with diseases associated with the human anti-Müllerian hormone type II receptor, in particular
ovarian cancer, in particular metastatic ovarian cancer, serous cancer, hypernephroma,
endometrioid, colloidal epithelium,
prostate cancer,
germ cell cancer,
endometrial cancer,
mixed Müllerian malignant tumour of the uterus,
leiomyosarcoma,
endometrial stromal sarcoma.
According to another aspect, the invention relates to the use of at least:
a monoclonal antibody defined above, or
a fragment of said monoclonal antibody defined above, or
a nucleic acid defined above, or
a vector defined above, or
a cell defined above,

    
    
        for preparing a drug intended for treating or preventing a disease associated with the human anti-Müllerian hormone type II receptor, in particular:
    
    


ovarian cancer, in particular metastatic ovarian cancer, serous cancer, hypernephroma,
endometrioid, colloidal epithelium,
prostate cancer,
germ cell cancer,
endometrial cancer,
mixed Müllerian malignant tumour of the uterus,
leiomyosarcoma,
endometrial stromal sarcoma.
By “treatment” is meant the manner of treating a pathology that has developed, the symptoms of which are visible. By “prevention” is meant the manner of preventing said pathology from developing.
In an advantageous embodiment, the invention relates to the use of an antibody defined above, or of a fragment of the latter defined above, for diagnosing and/or monitoring ovarian cancer.
In an advantageous embodiment, the invention relates to the use of an antibody defined above, or of a fragment of the latter defined above, additionally comprising a conventional anticancer drug, in particular paclitaxel or a platinum salt, in particular oxaliplatin, cisplatin or carboplatin.
According to another aspect, the invention relates to:

    
    
        a monoclonal antibody as defined above, or
        a fragment of said monoclonal antibody as defined above, or
        a nucleic acid as defined above, or
        a vector as defined above, or
        a cell as defined above,
        for use in the treatment or prevention of a pathology associated with the human anti-Müllerian hormone type II receptor, in particular:
    
    


ovarian cancer, in particular metastatic ovarian cancer, serous cancer, hypernephroma,
endometrioid, colloidal epithelium,
prostate cancer,
germ cell cancer,
endometrial cancer,
mixed Müllerian malignant tumour of the uterus,
leiomyosarcoma,
endometrial stromal sarcoma.
In an advantageous embodiment, the monoclonal antibody defined above or a fragment of the latter defined above is used for diagnosing and/or monitoring a cancer associated with the human anti-Müllerian hormone type II receptor, in particular:
ovarian cancer, in particular metastatic ovarian cancer, serous cancer, hypernephroma,
endometrioid, colloidal epithelium,
prostate cancer,
germ cell cancer,
endometrial cancer,
mixed Müllerian malignant tumour of the uterus,
leiomyosarcoma,
endometrial stromal sarcoma.
In an advantageous embodiment, the monoclonal antibody defined above or a fragment of the latter defined above, or the nucleic acid defined above or the vector defined above or the cell defined above, additionally comprises a conventional anticancer drug, in particular paclitaxel or a platinum salt, in particular oxaliplatin, cisplatin or carboplatin.
According to another aspect, the invention relates to a kit comprising at least:

    
    
        a monoclonal antibody as defined above, or
        a fragment of said monoclonal antibody as defined above, or
        a nucleic acid as defined above, or
        a vector as defined above, or
        a cell as defined above,
        for use in diagnosing a pathology associated with the human anti-Müllerian hormone type II receptor, in particular ovarian cancer.
    
    


According to another aspect, the invention relates to a method of diagnosing a pathology associated with the human anti-Müllerian hormone type II receptor, in particular ovarian cancer, on a human biological sample, comprising the following steps:

    
    
        a. labelling a biopsy previously obtained from a patient,
        b. determining the presence of a human anti-Müllerian hormone type II receptor.
    
    


According to another aspect, the invention relates to a method of diagnosing a pathology associated with the human anti-Müllerian hormone type II receptor, in particular ovarian cancer, on a human biological sample, comprising the following steps:

    
    
        a. obtaining a biopsy from a patient,
        b. labelling the biopsy,
        c. determining the presence of a human anti-Müllerian hormone type II receptor.
    
    


Labelling of the biopsy is carried out according to techniques that are well known to a person skilled in the art.
The presence of the receptor can be determined by techniques that are well known to a person skilled in the art, such as immunoassay, binding etc.
According to another aspect, the invention relates to a method of treating a pathology associated with the human anti-Müllerian hormone type II receptor, in particular ovarian cancer, on a human biological sample, comprising the following steps:

    
    
        a. obtaining a biopsy from a patient,
        b. labelling the biopsy,
        c. determining the presence of a human anti-Müllerian hormone type II receptor,
        d. if the presence of a human anti-Müllerian hormone type II receptor is determined, treating the patient with:
        
            i. a monoclonal antibody as defined above, or
            ii. a fragment of said monoclonal antibody as defined above, or
            iii. a nucleic acid as defined above, or
            iv. a vector as defined above, or
            v. a cell as defined above.
        
        
    
    





DESCRIPTION OF THE FIGURES
FIG. 1 corresponds to a diagrammatic representation of an antibody. The black parts correspond to the constant parts of the heavy chains, the parts in dark grey correspond to the constant part of the light chain, the parts in light grey correspond to the variable part of the heavy chain, and the white parts correspond to the variable part of the light chain. —S—S— represents the disulphide bridges established between two cysteines. The CDR and framework regions are indicated by arrows. The Fab and Fc fragments are also shown.
FIG. 2 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of a variable part of a light chain or of a heavy chain of immunoglobulin. The black beads correspond to the amino acids forming the framework regions, and the grey beads correspond to the amino acids representing the CDRs.
FIG. 3 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain (FIG. 3A: amino acids 1-115, SEQ ID NO: 8) and of the light chain (FIG. 3B: amino acids 131-236, SEQ ID NO: 2) of the humanized 12G4 antibody with the numbering adopted for defining the position of the mutations. The grey-shaded beads correspond to amino acids that are not present in the sequence and which therefore are not counted in the numbering.
FIG. 4 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the chimeric 12G4 antibody (SEQ ID NO: 66).
FIG. 5 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the chimeric 12G4 antibody (SEQ ID NO: 62).
FIG. 6 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the unmutated humanized 12G4 antibody (SEQ ID NO: 58) and of the mutated humanized 12G4 antibody (6B—78; SEQ ID NO: 58).
FIG. 7 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the unmutated humanized 12G4 antibody (SEQ ID NO: 54).
FIG. 8 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the mutated humanized 12G4 antibody (3C—23; SEQ ID NO: 74).
FIG. 9 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the mutated humanized 12G4 antibody (3C—23; SEQ ID NO: 70).
FIG. 10 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the mutated humanized 12G4 antibody (6B—78; SEQ ID NO: 78).
FIG. 11 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the mutated humanized 12G4 antibody (3C—23K; SEQ ID NO: 86).
FIG. 12 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the mutated humanized 12G4 antibody (3C—23K; SEQ ID NO: 82).
FIG. 13 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the mutated humanized 12G4 antibody (4C—35; SEQ ID NO: 90).
FIG. 14 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the mutated humanized 12G4 antibody (4C—35; SEQ ID NO: 78).
FIG. 15 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the heavy chain of the mutated humanized 12G4 antibody (5B—42; SEQ ID NO: 98).
FIG. 16 corresponds to a diagrammatic string-of-pearls representation of the amino acid sequence of the variable part of the light chain of the mutated humanized 12G4 antibody (5B—42; SEQ ID NO: 94).
FIG. 17 presents determination of the binding affinity of the antibody to the AMHR-II receptor in a conventional ELISA assay obtained with mutated antibodies (Fab) according to the invention.
The X-axis shows the concentration of (Fab) in μg/ml and the Y-axis shows the OD at 450 nm.
The dotted curve with empty white circles represents the binding of the unmutated humanized 12G4 antibody.
The curve with filled black triangles represents the binding of the mutated humanized 12G4 antibody, having a mutation in the CDR (E184K) of the variable region of the light chain (6B—78 antibody).
The curve with empty white triangles represents the binding of the mutated humanized 12G4 antibody, having a mutation in the CDR (S179P) of the variable region of the light chain, a mutation in the FR region (1177T) of the variable region of the light chain and a mutation in the variable region of the heavy chain (Q3R) (3C—23 antibody).
The curve with empty white circles represents the binding of the mutated humanized 12G4 antibody, having a mutation in the CDR (E184K) of the variable region of the light chain, a mutation in the CDR (S179P) of the variable region of the light chain, a mutation in the FR region (I177T) of the variable region of the light chain and a mutation in the variable region of the heavy chain (Q3R) (3C—23K antibody).
The curve with filled black circles represents the binding of the unmutated chimeric 12G4 antibody.
FIG. 18 corresponds to the diagrammatic representation of the H622-14 cloning vector for the chimeric 12G4 antibody containing the heavy chain where the leader VH AMHR-II is fused to the variable region of the heavy chain (VH AMHR-II), itself fused to the constant region of human immunoglobulin (CH T125), and the light chain where the leader VK AMHR-II is fused to the variable region of the light chain (VK AMHR-II), itself fused to the constant region of human immunoglobulin (CK T125).
The various regulatory elements (promoters, chimeric introns, polyadenylation sites, etc.) as well as the antibiotic resistance genes and the origins of replication are also shown.
FIG. 19 corresponds to the diagrammatic representation of the H622-18 cloning vector for the unmutated humanized 12G4 antibody containing the heavy chain where the humanized leader VH AMHR-II is fused to the variable region of the heavy chain (humanized VH AMHR-II), itself fused to the constant region of human immunoglobulin (CH T125), and the light chain where the leader VK AMHR-II is fused to the variable region of the light chain (humanized VK AMHR-II), itself fused to the constant region of human immunoglobulin (CK T125).
The various regulatory elements (promoters, chimeric introns, polyadenylation sites, etc.) as well as the antibiotic resistance genes and the origins of replication are also shown.
FIG. 20 corresponds to the diagrammatic representation of the H622-18 cloning vector MAO 3C23 for the mutated humanized 12G4 antibody 3C—23 containing the heavy chain where the leader VH 3C—23 is fused to the variable region of the heavy chain (VH 3C—23), itself fused to the constant region of human immunoglobulin (CH T125), and the light chain where the leader VK 3C—23 is fused to the variable region of the light chain (VK 3C—23), itself fused to the constant region of human immunoglobulin (CK T125).
The various regulatory elements (promoters, chimeric introns, polyadenylation sites, etc.) as well as the antibiotic resistance genes and the origins of replication are also shown.
FIG. 21 corresponds to the diagrammatic representation of the H622-18 cloning vector MAO 6B—78 for the mutated humanized 12G4 antibody 6B—78 containing the heavy chain where the humanized leader VH AMHR-II is fused to the variable region of the heavy chain (humanized VH AMHR-II), itself fused to the constant region of human immunoglobulin (CH T125), and the light chain where the leader VK 6B—78 is fused to the variable region of the light chain (VK 6B—78), itself fused to the constant region of human immunoglobulin (CK T125).
The various regulatory elements (promoters, chimeric introns, polyadenylation sites, etc.) as well as the antibiotic resistance genes and the origins of replication are also shown.
FIG. 22 corresponds to the diagrammatic representation of the H622-18 cloning vector MAO 3C—23K for the mutated humanized 12G4 antibody 3C—23K containing the heavy chain where the leader VH 3C—23K is fused to the variable region of the heavy chain (VH 3C—23K), itself fused to the constant region of human immunoglobulin (CH T125), and the light chain where the leader VK 3C—23K is fused to the variable region of the light chain (VK 3C—23K), itself fused to the constant region of human immunoglobulin (CK T125).
The various regulatory elements (promoters, chimeric introns, polyadenylation sites, etc.) as well as the antibiotic resistance genes and the origins of replication are also shown.
FIG. 23 presents the diagrammatic protocols for construction of the scFv fragments.
The black arrow under region 2⁄3 of VH indicates the sequence encoding for the N-terminal 2⁄3 of the peptide bond.
The black arrow under region 2/3 of VL indicates the sequence encoding for the C-terminal 2/3 of the peptide bond.
FIGS. 24A and 24B show the subcloning of the nucleotide sequences of the light chains VL-CL and heavy chains VH-CH1 of the mLFB112 and huLFB112 antibodies into the pMG62-Fab expression vectors.
FIG. 24A: mLFB112
FIG. 24B: huLFB112
FIG. 25 presents the ADCC activity of the humanized anti-AMHRII antibodies of the invention compared to that of the unmutated humanized 12G4 antibody. The results are expressed as percentage lysis of the ASC1 cell (Y-axis) as a function of the amount of antibody added in ng/ml (X-axis). Mean±SEM.
The curve with diamonds represents the anti-AMHRII antibody 3C—23 (R901 3C—23), the curve with triangles with the point upwards represents the anti-AMHRII antibody 6B—78 (R901 6B—78), the curve with triangles with the point downwards represents the anti-AMHRII antibody 3C—23K (R901 3C—23K), the curve with circles represents the unmutated humanized anti-AMHRII antibody 12G4.
FIG. 26 shows the map of the pIRES-neo plasmid expression vector used for generating the cov434-AMHRII line.
FIGS. 27A and 27B present the ADCC activity of chimeric and humanized anti-AMHRII antibodies produced in the YB2/0 cells (FIG. 27A) and the CHO cells (FIG. 27B) on the COV434-AMHRII line.
The results are expressed as percentage lysis of the COV434-AMHRII cells (Y-axis) as a function of the amount of antibody added in ng/ml (X-axis). Mean of 3 assays±SEM.
FIG. 27A: the curve with diamonds represents the unmutated chimeric 12G4 anti-AMHRII antibody, the curve with filled squares represents the anti-AMHRII antibody YB2/0 3C—23 (R901 3C—23), the curve with triangles with the point downwards represents the anti-AMHRII antibody YB2/0 6B—78 (R901 6B—78), the curve with triangles with the point upwards represents the anti-AMHRII antibody YB2/0 3C—23K (R901 3C—23K) and the curve with empty rectangles represents the anti-CD20 antibody used as negative control.
FIG. 27B: the curve with diamonds represents the unmutated chimeric 12G4 anti-AMHRII antibody, the curve with triangles with the point upwards represents the anti-AMHRII antibody CHO 3C—23 (R901 3C—23), the curve with triangles with the point downwards represents the anti-AMHRII antibody CHO 3C—23K (R901 3C—23K), the curve with circles represents the anti-AMHRII antibody CHO 6B—78 (R901 6B—78) and the curve with empty rectangles represents the anti-CD20 antibody used as negative control.
FIG. 28 shows the ADCC activity of the humanized anti-AMHRII antibodies produced in YB2/0 and CHO cells on the Asc 1 line. The results are expressed as percentage lysis of the Asc 1 cells (Y-axis) as a function of the amount of antibody added in ng/ml (X-axis). Mean of 3 tests±SEM.
The curve with diamonds represents the anti-AMHRII antibody YB2/0 3C—23K, the curve with triangles with the point upwards represents the anti-AMHRII antibody CHO 3C—23K.
FIG. 29 shows the ADCC activity of the humanized anti-AMHRII antibodies produced in YB2/0 and CHO on the META 2815 line. The results are expressed as percentage lysis of the META 2815 cells (Y-axis) as a function of the amount of antibody added in ng/ml (X-axis). Mean of 3 tests±SEM.
The curve with diamonds represents the anti-AMHRII antibody YB2/0 3C—23K, the curve with triangles with the point upwards represents the anti-AMHRII antibody CHO 3C—23K, the curve with circles represents the anti-CD20 antibody used as negative control (anti-CD20 A/R603 09/045).
FIG. 30 shows the effect of the anti-AMHRII antibody 3C—23K on proliferation of the COV434-AMHRII cells. The value 100% corresponds to the proliferation of the COV434-AMHRII cells observed without antibody (mean of 3 tests±SD).
From left to right, the histograms show:
The control without antibody, an antiP24 antibody, the anti-AMHRII antibody YB2/0 3C—23K, the anti-AMHRII antibody CHO 3C—23K, an antiP24 antibody in the presence of a cross-linking agent (CK), the anti-AMHRII antibody YB2/0 3C—23K in the presence of CK, the anti-AMHRII antibody CHO 3C—23K in the presence of CK, colchicine at 1 μg/ml.
FIG. 31 shows the effect of the anti-AMHRII antibody 3C—23K on the proliferation of META 2815 cells. The value 100% corresponds to the proliferation of the META 2815 cells observed without antibody (mean of 3 tests±SD).
From left to right, the histograms show:
The control without antibody, an antiP24 antibody, the anti-AMHRII antibody YB2/0 3C—23K, the anti-AMHRII antibody CHO 3C—23K, a cross-linking agent alone, an antiP24 antibody in the presence of a cross-linking agent (CK), the anti-AMHRII antibody YB2/0 3C—23K in the presence of CK, the anti-AMHRII antibody CHO 3C—23K in the presence of CK, colchicine at 1 μg/ml.
FIGS. 32A and 32B show the variation of tumour volumes (FIG. 32A) and the survival curves (FIG. 32B) under the effect of the treatment with 3C23K-YB2/0 with intraperitoneal injections of antibody performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections (black arrows) in the cov434-AMHRII model.
FIG. 32A:
Y-axis: tumour volumes in mm3,
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.
FIG. 32B:
Y-axis: percentage survival
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.
FIGS. 33A and 33B show the variation of the tumour volumes (FIG. 33A) and the survival curves (FIG. 33B) under the effect of the treatment with 3C—23K-YB2/0, intraperitoneal injections of antibody performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections (black arrows) in an Asc1a5 model.
FIG. 33A:
Y-axis: tumour volumes in mm3,
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.
FIG. 33B:
Y-axis: percentage survival
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.
FIGS. 34A and 34B show the variation of the tumour volumes (FIG. 34A) and survival curves (FIG. 34B) under the effect of the treatment with 3C—23K-YB2/0, intraperitoneal injections of antibody performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections (black arrows) in the META 2815 model.
FIG. 34A:
Y-axis: tumour volumes in mm3,
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.
FIG. 34B:
Y-axis: percentage survival
X-axis: days after injection of the tumour cells.
Curve with diamonds: vehicle
Curve with rectangles: anti-AMHRII antibody YB2/0 3C—23K.



EXAMPLES
Example 1
Determination of the Affinity of the Anti-AMHR-II Antibodies
The affinity of the antibodies for their antigen, AMHR-II, is determined by the SPR (Surface Plasmon Resonance) technique on BIACore X100 (BIACore, GE Healthcare). The AMHR-II recombinant receptor, expressed in the form of fusion protein with a region Fc, is immobilized by covalent coupling between its amine functions and the carboxyl groups of dextran activated in succinimide esters, present on the surface of the type CM5 sensor chip. The COOH groups of the sensor chip are activated for 7 minutes with EDC/NHS mixture (0.1 M of N-hydroxysuccinimide and 0.1 M of 3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide) at a flow of 10 μl/min then the AMHR-II/Fc fusion protein, diluted to 5 μg/ml in 10 mM sodium acetate buffer, pH 4.0, is injected at 5 μl/min on track 2 of the sensor chip so as to reach 300 RU. The ester groups that have not reacted with the amines of the fusion protein are deactivated by injection of a solution of ethanolamine-HCl 1M, pH 8.5 for 7 min at a flow of 10 μl:min. Track 1, serving as negative control, was activated and deactivated like track 2.
All the measurements are carried out at 25° C. The antibodies to be analysed are diluted in HBS-EP running buffer (BIACore, GE Healthcare) at concentrations from 6.25 to 3333 nM and injected on the sensor chip for 2 min at a flow of 30 μl/min. The dissociation step is monitored for 10 min and then the surface is regenerated by injection of 10 mM glycine buffer, pH 1.5 for 30 s at 10 μl/min.
The sensorgrams obtained are analysed using the 1:2 kinetic model of the BIAevaluation 3.1 software.
Results
The antibodies were produced in CHO or YB 2/0 cells (Table I)












TABLE I







ka


KDchimeric/


Antibody
Mutations
(M−1s−1)
kd (s−1)
KD (nM)
KDmutated




















12G4-
NA
3.5 × 103
7.4 × 10−4
212
—


Chimeric


6B_78
VL-E184K
1.6 × 104
1.3 × 10−3
82
2.6


YB2/0


3C_23
VH-Q3R
3.6 × 104
3.3 × 10−3
92
2.3


YB2/0
VL-I177T/



S179P


3C_23K
VH-Q3R
  4 × 104
8.6 × 10−4
21
10


YB2/0
VL-I177T/



S179P/



E184K


6B_78
VL-E184K
1.5 × 104
1.2 × 10−3
81
2.6


CHO


3C_23
VH-Q3R
  5 × 104
4.3 × 10−3
86
2.5


CHO
VL-I177T/



S179P


3C_23K
VH-Q3R
4.1 × 104
  1 × 10−3
25
8.5


CHO
VL-I177T/



S179P/



E184K









The mutations introduced in the 6B—78 and 3C—23 antibodies induce an increase in affinity for the AMHR-II antigen by a factor of 2.3 to 2.6 relative to the chimeric antibody (12G4—chimeric).
The mutations of the two antibodies 6B—78 and 3C—23 have a synergistic effect; introduction of the mutation of the 6B—78 antibody into the 3C—23 antibody causes an increase in affinity by a factor of 10.
Example 2
Determination of the Affinity of Chimeric or Humanized Murine 12G4 Antibody on cov434-AMHR-II Cells (Epitope Peptide of Sequence: GGGGNLTQDRAQVEMQGSR (SEQ ID NO: 101) and GGGGNLTQARGQVEMQGSR (SEQ ID NO: 102) for the Negative Control Peptide)

















Affinity




Dissociation
constant at



Association constant
constant
equilibrium



















humanized 12G4
1.83 × 103 M−1 · s−1
9.62 × 10−3 s−1
5.26 × 10−6M


chimeric 12G4
6.49 × 103 M−1 · s−1
1.53 × 10−3 s−1
2.35 × 10−7M


murine 12G4

1.47 × 10−3 s−1
of the order





of 10−7M





The affinity of the chimeric antibody determined on the AMHR-II human receptor is about 10−8M.






Example 3
Preparation of Mutated Humanized 12G4 Antibodies
The murine antibody is substantially equivalent to the chimeric antibody and displays strong affinity.
The humanized 12G4 antibody (huLFB112) was obtained by grafting hypervariable CDR loops of the murine 12G4 antibody (mLFB112) on a protein framework of a human nature (“CDR grafting”).
The humanized antibody has an appreciable loss of affinity compared with the murine antibody.
The final objective is therefore to increase the affinity of the humanized antibody so as to restore the initial binding characteristics of the murine antibody. This optimization will be performed by means of a cycle of molecular evolution by the MutaGen technology owned by the Millegen company.
3.1 Construction and Validation of the Molecular Tools
3.1.1. Construction of the scFv Fragments
The nucleotide sequences coding for the variable regions of the light chains (VL) and of the heavy chains (VH) of the murine and humanized antibodies were amplified by PCR using suitable primers. The amplified sequences were then combined together so as to generate a recombinant antibody fragment of the scFv type. Several constructions were performed in this way: VH-VL or VL-VH orientation and use of two different peptide bonds (peptide bond of 15 or of 18 amino acids). A total of 8 constructions were performed, 4 for the murine antibody and 4 for the humanized antibody. The principle for construction of the scFv fragments is illustrated below (scheme I). The sequences coding for these scFv were then subcloned into the MilleGen phagemid expression vector (pMG58). This vector makes it possible to express antibody fragments of the scFv type and display them on the surface of a type M13 bacteriophage (phage-scFv).
The nucleotide sequences of the VH and VL domains of the murine and humanized antibodies were verified by DNA sequencing.
The protocol is summarized in FIG. 23.
3.1.2. Expression of the scFv on the Surface of the Phages and Characterization by ELISA
The amount of target supplied (80 μg) did not allow us to test all 8 constructions made. The murine antibody mLFB112 expressed in the form of a scFv is called mVH-VL hereinafter whereas the humanized antibody huLFB112 is called huVH-VL.
3.1.2.1. Production of the Phages-scFv
The XL1-Blue bacteria transformed by the pMG58 vectors containing the DNA coding for the scFv mVH-VL on the one hand and the scFv huVH-VL on the other hand are cultured at 30° C. to an OD600 nm of 0.5-0.6. After adding IPTG and infecting the bacteria with auxiliary phages (M13KO7, New England Biolabs), the cultures are cultured at 26° C. overnight. The next day, the phage particles (phages-scFv) are recovered from the culture supernatant, precipitated by means of a PEG/NaCl solution, concentrated (100×) and quantified.
In this case, a concentration of the order of 8×1011 phages/ml is obtained for the two scFv.
3.1.2.2. ELISA-Phages Assay
The functionality of the scFv mVH-VL and huVH-VL produced on the surface of the phages was verified by direct ELISA assays.
Protocol:

    
    
        1) Immobilization of the target: 100 μl/well of the recombinant target diluted to 5 μg/ml in PBS1X i.e. 500 ng/well, overnight at 4°. Use of Nunc-Immuno Plate Maxisorp microtitre plates,
        2) Saturation: 200 μl/well of PBS1X-Skimmed milk 4%, incubation 2 h at 37° C.,
        3) Binding: 100 μl/well of the solutions of murine and humanized phages-scFv diluted in PBS1X-Milk 2%-Tween20 0.05% (twofold dilution series), incubation 2 h at 37° C.,
        4) Detection: 100 μl/well of the anti-phages M13 antibody coupled to peroxidase (dilution 1/10000, GE Healthcare), incubation 2 h at 37° C.
        5) Detection: 100 μl/well of TMB
        6) Neutralization: 100 μl/well of H2SO4 
        7) Measurement of OD at 450 nm

Results (Table II):

    
    












TABLE II







Number
mVH-VL
huVH-VL
Ratio














of phages

Without


Without

mVH-VL/


per well
+covering
covering
Diff
+covering
covering
Diff
huVH- VL





3.00E+10
2.805
0.158
2.647
1.247
0.150
1.097
2.4


3.00E+10
2.193
0.085
2.107
0.689
0.096
0.593
3.6


3.00E+09
1.570
0.064
1.506
0.395
0.072
0.323
4.7


3.00E+09
0.946
0.059
0.887
0.226
0.056
0.170
5.2


3.00E+09
0.495
0.049
0.446
0.135
0.049
0.086
5.2





Diff: specific binding (difference between the wells with covering and without covering


The ratio mVH-VL/huVH-VL was calculated with the value of specific binding (“Diff”)






3.1.3: Construction of the Fab Fragments
The nucleotide sequences of the light chains VL-CL and heavy chains VH-CH1 of the antibodies mLFB112 and huLFB112 were subcloned into the pMG62-Fab expression vectors (FIGS. 24A and 24B).
pMG62-Fab Expression Vectors
A) The two chains VL-CL and VH-CH1 are expressed starting from the pLac promoter upstream of the light chain, the heavy chain VH-CH1 is fused to a tag for detection (peptide V5) and a tag for purification with IMAC (6×His).
B) Each of the light and heavy chains is dependent on a promoter. RBS: Ribosome Binding Site.
3.2 Construction and Validation of the Molecular Tools
Construction of the Library by MutaGenTM
The objective defined for this step was to obtain a database of 5×106 variants with 1 to 2 amino acid mutations per scFv.
Mutations were introduced within the domains VL and VH of the humanized antibody huLFB112 by means of MutaGenTM technology. Finally, a large database composed of about 5×107 mutated clones with 1 to 5 mutations of amino acids per scFv, i.e. 10 times the diversity initially envisaged was obtained.
For this, several sub-databases were constructed according to different experimental conditions: conditions U, M, US and UE defined by different nucleotide primers, mutase enzymes used and number of replications. These sub-databases are 4 in number and are designated R20U, 45M, R20US and R20UE. For all of these sub-databases, a total of 295 sequences was carried out for accurately defining the different characteristics of mutagenesis. Table III below gives an idea of the principal data obtained from analysis of the sequencing operations.







TABLE III







Analysis of the mutations of the different sub-databases










Name of database














R20U
45M
R20US
R20UEta
TOTAL
















Size of
2.0E+07
6.0E+06
9.9E+07
5.0E+05
1.3E+08


database


Condition
U
M
US
UE


Number of
87
67
85
56
295


sequences







Analysis of the nucleotide sequences












Frequency
2.76
3.66
3.31
4.9



of mutations


per kb


% of
10%
20%
 6%
 5%


deletions


% of
 2%
—
0.8% 
—


additions


% of
72%
80%
93%
95%


substitutions


Frequency
2.0
2.45
3.10
3.68


of mutations


per kb


(without


deletions)







Analysis of the amino acid sequences












% of
63%
46%
44%
41%



sequences


(by weight)


(+ silent


mutation)


% of
13%
28%
13%
27%


sequences


with reading


frame shift


(+ stop


codon)


% of
24%
25%
43%
32%


sequences


with mutated


amino acids


Number of
4.8E+06
1.5E+06
4.3E+07
1.6E+05
4.9E+07


scFv clones


with mutated


amino acids


Number of
1.3
1.2
1.8
2.11


mutations


of amino


acids per


scFv


scFv clones
65%
49%
50%
33%
2.5E+07


with 1


mutated


amino acid


scFv clones
25%
34%
33%
39%
1.6E+07


with 2


mutated


amino acids


scFv clones
10%
13%
 8%
 6%
4.2E+06


with 3


mutated


amino acids


scFv clones
—
 4%
 6%
22%
2.5E+06


with 4


mutated


amino acids


scFv clones
—
—
 3%
—
1.2E+06


with 5


mutated


amino acids









3.2. Elaboration of the Selection Conditions
In order to evaluate different selection strategies, an artificial mixture was prepared between the murine and humanized phages-scFv (1/200 mixture, mLFB112/huLFB112), the objective being to simulate screening of the database. This simulation of screening must make it possible to validate different selection conditions which have the aim of rapidly amplifying the most affine clone within this artificial mixture (i.e. the murine clone mLFB112 in this case).
The various strategies evaluated:
i) Use of a constant amount of immobilized target in the course of the selection cycles (Cond 1)
ii) Decrease of the amount of immobilized target in the course of the selection cycles (Cond 2)
ii) Test for a “koff” condition: long incubation time of the phages-scFv with the target (Cond 3)
These various conditions were performed in 3 selection cycles. Sequencing was performed on the clones retained after each selection cycle. The results are shown in Table IV below.







TABLE IV







Evaluation of the three screening strategies











Bp1
Bp2
Bp3














Conditions 1





Covering
500 ng
500 ng
500 ng


Number of scFv phages used for the
1.6E+11
1.2E+11
2.0E+11


selection cycle


Number of scFv phages recovered
3.3E+5
2.4E+5
2.7E+6


at the end of the selection cycle


% of mLFB112/huLFB112
0/100
1/99
90/10


Conditions 2


Covering
500 ng
100 ng
100 ng


Number of scFv phages used for the
1.6E+11
1.2E+11
1.8E+11


selection cycle


Number of scFv phages recovered
3.3E+5
2.4E+5
2.7E+6


at the end of the selection cycle


% of mLFB112/huLFB112
0/100
1/99
30/70


Conditions 3


Covering
500 ng
100 ng koff
100 ng koff


Number of scFv phages used for the
1.6E+11
1.8E+11
1.6E+11


selection cycle


Number of scFv phages recovered
3.3E+5
6.7E+5
2.0E+6


at the end of the selection cycle


% of mLFB112/huLFB112
0/100
1/99
30/70









It appears from these results that condition 1 (fixed amount of target) gives best performance for amplifying the clone with better affinity, mLFB112 (9 clones out of 10). Using a smaller amount of target (100 ng/well) seems less suitable; only 3 clones out of 10 after 3 selection cycles correspond to the clone with better affinity. The same applies to condition 3 based on a long incubation time (“koff selection”) which does not allow sufficient amplification of clone mLFB112. Moreover, the number of phages recovered for this last-mentioned condition after 3 cycles is not high (2×104 phages). It therefore seemed to us to be inadvisable to use conditions 2 and 3 for a more diversified mixture of clones, as is the case for the database constructed in the context of this project.
3.3. Primary Screening (Selection Cycles)
After elaborating the screening conditions, it was therefore decided to use 2 screening conditions:
Cond A: 1 μg/well of target for 4 selection cycles then 2 cycles with 0.5 μg/well
Cond B: 0.5 μg/well for 6 selection cycles.
The results obtained are presented in Table V below.







TABLE V







Results of the selection cycles














Selection
Selection
Selection
Selection
Selection
Selection



cycle 1
cycle 2
cycle 3
cycle 4
cycle 5
cycle 6

















Conditions A








Covering
  1 μg
  1 μg
  1 μg
  1 μg
0.5 μg
0.5 μg


Number of scFv phages
4.8E+11
8.0E+11
2.8E+11
8.0E+11
4.5E+11
6.0E+11


used for the selection


cycle


Number of scFv phages
2.0E+5 
1.3E+5 
2.4E+5 
7.5E+5 
7.3E+5 
1.4E+5 


recovered at the end of the


selection cycle


Conditions B


Covering
0.5 μg
0.5 μg
0.5 μg
0.5 μg
0.5 μg
0.5 μg


Number of scFv phages
4.8E+11
8.0E+11
3.2E+11
6.4E+11
7.5E+10
4.2E+11


used for the selection


cycle


Number of scFv phages
2.0E+5 
8.6E+5 
5.4E+5 
1.3E+5 
2.6E+5 
6.5E+5 


recovered at the end of the


selection cycle









After these selections, the clones obtained were sequenced starting from the 3rd selection cycle. The results obtained were compared with those obtained for the starting database (Table VI).







TABLE VI







Results of the sequencing operations














Selection
Selection
Selection
Selection



Database
cycle 3
cycle 4
cycle 5
cycle 6
















Number of
295
87
168
114
98


sequences


% of
39%
26%
26%
35%
20%


sequences


with mutated


amino acids


% of
14%
13%
27%
32%
49%


sequences


with reading


frame shift


% of
47%
61%
48%
34%
32%


sequences


(by weight)


(+ silent


mutation)





Database: initial database resulting from mixing the 4 databases R20U, 45M, R20US, R20Ueta.


Analysis of these sequencing operations revealed the presence of redundant clones. In total, from all of the clones sequenced, 113 unique mutated clones were obtained.






3.4. Secondary Screening (ELISA-Phages)
The secondary screening consists of analysing the clones selected at the end of the primary screening individually. For this, the 113 unique mutated clones were transferred to a culture plate (96 well-1.2 ml).
After production of the phage particles, the culture supernatants containing the phages-scFv were used for carrying out an ELISA binding assay. The binding of the mutated clones was evaluated at two dilutions (1⁄2 and 1⁄4 of the supernatants containing the scFv-phages). The murine clones (mLFB112) and humanized clones (huLFB112) constructed under the scFv-phages format were used as references on each of the assay plates. Each of the mutated clones was tested at least twice.
The results are expressed as a ratio, i.e. the differences in binding (OD405 nm) between the mutated clones and the references huLFB112 and mLFB112.

    
    
        Ratio relative to the humanized scFv, huLFB112 (Ratio/huLFB112)
        Ratio relative to the murine scFv, mLFB112 (Ratio/mLFB112)

Out of the 113 clones tested, only the best clones are presented below.

    
    


Table VII below presents the various clones obtained and the mutations present (position and substitution of amino acids) in the light and/or heavy chain, as well as the binding affinity of clones determined by ELISA.
The values shown after the substitutions correspond to the changes in the values of the hydropathic index as a function of the various substitutions.
The values of binding affinity correspond to the ratio of the binding affinity of the antibody of the invention for the AMHRR-II receptor to the binding affinity of the unmutated humanized 12G4 antibody or the binding affinity of the unmutated chimeric 12G4 antibody.
The values of binding affinity given are the mean values of at least four values and the figures in parentheses correspond to the standard deviation.
Table VII shows that with the substitutions that were carried out, although the latter lead to a large change in the hydropathic index, the binding affinity of the antibody for the receptor is much greater than that of the unmutated humanized 12G4 antibody and at least equal to that of the unmutated chimeric 12G4 antibody: ratio AB invention/chimeric 12G4 greater than or equal to 1.
The mutated humanized antibody displays an affinity that is restored or even greater than that of the chimeric or murine antibody.









TABLE VII









FIXATION TO THE TARGET



AMHRII-Fc DETERMINED BY



ELISA












Numbering
Numbering
Ratio
Ratio



VH
VL
AB invention/humanized
AB invention/chimeric


clone
1-115
131-236
12G4
12G4





4C_35
L45P +3.8_−1.6
E184K −3.5_−3.9
4.3 (0.5)
1.9 0.4)


5B_81
L45P +3.8_−1.6
—
3.5 (0.9)
1.6 (0.3)


6B_78
—
E184K −3.5_−3.9
NT
NT


3C_23
Q3R −3.5_−4.5
I177T +4.5_−0.7
2.6 (1.1)
1.2 (0.3)




S179P −0.8_−1.6


3C_23K
Q3R −3.5_−4.5
I177T +4.5_−0.7
NT
NT




S179P −0.8_−1.6




E184K −3.5_−3.9


5B_42
T74A −0.7_1.8
S179P −0.8_−1.6
NT
NT


4F_196
Q3E −3.5_−3.5
—
3.0 (1.0)
1.5 (0.3)



Q62R −3.5_−4.5



E89D −3.5_−3.5


6B_87
Q1E −3.5_−3.5
—
2.1 (0.2)
1.0 (0.1)



A24V +1.8_+4.2


4F_169
Q6E −3.5_−3.5
—
2.0 (0.7)
1.0 (0.2)



T58A −0.7_+1.8


3D_74
—
S158P −0.8_−1.6
  2 (0.7)
0.8 (0.2)


4C_44
R87G −3.9_−0.4

NT
NT


5A_66
V67M +4.2_+1.9
F212S +2.8_−0.8
2.2 (0.4)
1.1 (0.1)


6B_14
S31G −0.8_−0.4
—
2.6 0.3)
1.1 (0.2)



Q39E −3.5_−3.5


4C_47
Q3E −3.5_−3.5
—
NT
NT



S88P −0.8_−1.6


4E_153
D56N −3.5_−3.5
—
2.0 (0.4)
1.0 (0.1)



I70N +4.5_−3.5



F102S +2.8_−0.8


3C_24
—
E184G −3.5_−0.4
NT
NT


5B_18
Q3E −3.5_−3.5
—
1.8 (0.2)
0.9 (0.2)



A9T +1.8_−0.7



A103T +1.8_−0.7


5B_84
Q1E −3.5_−3.5
—
NT
NT



A24G +1.8_−0.4


6B_86
Q3E −3.5_−3.5
G179D −0.4_−3.5
NT
NT


4D_91
Q1E −3.5_−3.5
—
1.7 (0.1)
0.8 (0.2)



V11A +4.2_+1.8


6B_76
A40V +1.8_+4.2
S179P −0.8_−1.6
1.7 (0.1)
0.8 (0.2)


5A_28
—
Y178H −1.3_−3.2
1.6 (0.6)
0.8 (0.2)




S179P −0.8_−1.6


3D_57
A76T +1.8_−0.7
—
NT
NT



A79T +1.8_−0.7


6A_80
A24V +1.8_+4.2
—
1.5 (0.2)
1.3 (0.1)



Q62E −3.5_−3.5


5B_67
K12R −3.9_−4.5
—
NT
NT


5B_86
S31G −0.8_−0.4
I132T +4.5_−0.7
NT
NT



Q39E −3.5_−3.5
A143T +1.8_−0.7


5A_73
A24V +1.8_+4.2
—
NT
NT


5B_33
A76T +1.8_−0.7
—
NT
NT


3B_71
S114T −0.8_−0.7
S179P −0.8_−1.6
NT
NT


3B_87
—
L175Q +3.8_−3.5
NT
NT


3D_68
—
T150A −0.7_1.8
NT
NT


4E_112
L110P +3.8_−1.6
V187A +4.2_+1.8
NT
NT




S192T −0.8_−0.7


5B_54
A24T +1.8_−0.7
—
NT
NT


6A_18
K13R −3.9_−4.5
—
NT
NT


3C_40
—
P224A −1.6_+1.8
NT
NT


5A_83
Q62E −3.5_−3.5
—
NT
NT



S179P −0.8_−1.6



A79T +1.8_−0.7


5A_19
Q3E −3.5_−3.5
S182F −0.8_+2.8
NT
NT


3A_29
V20A +4.2_+1.8
—
NT
NT





(Q1E, Q3E, Q6E, K19E, Q39E and Q62E: TAG codon suppressed, translated to E in the E. coli bacteria XL1-blue used)


NT: not tested






Example 4
Comparison of Clones Having an Improvement of Affinity
The positive clones 3C—23K, 3C—23 and 6B—78 were compared with one another by determining the binding affinity of the antibody to the AMHR-II receptor in a conventional ELISA assay obtained with soluble mutated antibodies (Fab) according to the invention.
The results obtained are presented in FIG. 17.
Example 5
Establishment of the Transfected Line AMHRII cov434-AMHRII
The cov434-AMHRII line was generated by transfection of a plasmid expressing the cDNA coding for AMHRII in the granulosa tumour line cov434 (van den Berg-Bakker, C., et al., 1993. Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumour cell line: Growth features and cytogenetics. International Journal of Cancer 53: 613; Zhang, H. et al., 2000. Characterization of an immortalized human granulosa cell line (COV434). Molecular Human Reproduction 6: 146) not expressing AMHRII.
Briefly, the cDNA of AMHRII was cloned into the commercial plasmid pIRES-neo (Clontech—Takara Bio Europe, France; references 6060-1). Owing to the IRES sequence, AMHRII and neo are expressed under the control of one and the same promoter CMV (FIG. 26).
This construction was stably transfected in the granulosa cancer line cov434 (transfection agent Fugene, Roche). The transfectants obtained were then screened by cytometry and by Western blotting for expression of the AMHRII receptor. After subcloning, the cellular clone cov434-AMHRII-1F3, containing a vector of the pIRES-neo type, was retained for the in vitro and in vivo studies. This line is designated cov434-AMHRII hereinafter.
Example 6
Establishment of Primary Lines from Ascitic Fluid or Biopsy from Patients with Ovarian Epithelial Carcinoma
The main steps in establishing the lines derived from samples of ascitic fluid (Asc 1 line) are as follows:

    
    
        D0: Receiving the ascitic fluid and starting culture of the sample immediately. The sample is centrifuged for 5 min at 1000 rev/min and the pellet is taken up in 2 mL of medium for seeding a T25 flask (RPMI 10% FCS medium).
        D2-D46: Culture with regular microscopic observation. Fresh medium is added regularly during this period. Washing with PBS is also carried out every other day in order to remove the contaminating erythrocytes and fibroblasts.
        Around D13, all the contaminating cells have disappeared and a still heterogeneous confluent lawn of cells is observed (two different types).
        D46: Transfer: on D46 a homogeneous lawn of tumour cells is observed and the cells are then washed and transferred by scraping to be re-seeded in two T75 flasks.
        D61: Evaluation of AMHRII expression by FACS: after washing with PBS, the cells are detached from the scraper and analysed by flow cytometry (labelling AcM 12G4 10 μg/mL, AcII anti-mouse FITC). The databases are constructed at this stage. The positive AMHRII line is cultured for 10 more days in order to confirm expression of the AMHRII receptor.
        D71: Confirmation of AMHRII expression by FACS.
    
    


The lines thus established are kept in RPMI 10% FCS medium, with one passage per week (1/15 dilution).
In the case of biopsies (META 2815 line), the primary tumour is first maintained on nude mice (grafts of the sample in the interscapular space, 3 successive passages on mice) then the tumour is removed and dilacerated before being taken up in culture medium. A protocol identical to that of the samples of ascitic fluid is then applied.
Example 7
Evaluation of the Affinity of the Various Candidate Humanized Antibodies
This study was conducted on the original murine 12G4 antibody as well as on the candidate humanized antibodies 3C23, 6B78 and 3C23K (of sequence 3C—23, 6B—78 and 3C—23K respectively) produced in YB2/0. The affinity of these antibodies was evaluated on the cov434-AMHRII cells.
Briefly, KD was determined by the saturation method, by adding increasing doses of radiolabelled antibody to a constant number of cov434-AMHRII cells. The cells (1×106 in 50 μl PBS/BSA 0.5%) were incubated (final volume 150 μl) for 1 h at 4° C. in the presence of increasing doses of antibody labelled beforehand with iodine 125 (125I). For each antibody, fourfold dilutions were carried out in PBS/BSA 0.5% from the solutions of labelled and unlabelled antibody (84.4 μg/ml). The nonspecific fixation was evaluated by incubating the cells in the presence of a 100 times molar excess of unlabelled antibody.
After incubation, the samples were frozen in liquid nitrogen and then analysed in a gamma counter. The specific fixation was determined by subtracting the fixation obtained in the presence of an excess of unlabelled antibody.
Scatchard analysis, performed on PRISM software, made it possible to determine the affinity constants presented in Table VIII.







TABLE VIII







Dissociation constant (KD) of the anti-AMHRII antibodies












murine 12G4
3C23
6B78
3C23K















KD
15.41 +/− 0.97
7.33 +/− 0.44
6.68 +/− 0.21
5.30 +/− 0.38


(nM)









According to this study, it appears that the humanized anti-AMHRII antibody 3C23K has the best affinity (KD=5.3 nM) compared with the two other candidate antibodies 6B78 and 3C23. The 3C23K antibody also has an affinity about three times greater than that of the original murine 12G4 antibody (KD=15.4 nM).
Example 8
Comparison of the ADCC Activity of the Antibodies of the Invention Versus the ADCC Activity of the Unmutated Humanized 12G4 Antibody
1 Material and Methods
1.1 Principle of the Methods
ADCC
The ASC1 target cells obtained from patients are adherent and are prepared on the day before the assay. They are detached with trypsin and incubated in EMS+5% FCS in a flat-bottomed plate at a rate of 50 μl per well at a concentration of 6×105 cells/ml. The plates are incubated overnight at 37° C., 7% CO2.
The next day, the cells have adhered to the bottom of the well. The supernatant is aspirated and the necessary volume of buffer per well is added for carrying out the assay in the presence of NK and antibody.
The killer cells (NK cells) are purified beforehand by the negative depletion technique developed by the company Miltenyi (Miltenyi Biotec—NK cell isolation kit human ref 130-092-657), from peripheral blood of healthy donors. The ADCC technique consists of incubating the NK cells with ASC1 target cells in the presence of different concentrations of the humanized anti-AMHRII antibody (0.005 to 5000 ng/ml) with E/T ratio of 10/1. After 4 hours of incubation, the cytotoxic activity induced by the anti-AMHRII antibodies is measured by colorimetry by determining, in the supernatants, an intracellular enzyme called lactate dehydrogenase (LDH) released by the lysed target cells (Roche Diagnostics—Cytotoxicity Detection Kit LDH ref 11644793001).
1.2 Elements Studied
Anti-AMHRII antibodies:

    
    
        829 10 054, humanized YB2/0, R901 3C23K
        829 10 050, humanized YB2/0, R901 3C23
        829 10 051, humanized YB2/0, R901 6B78
        632 07 107, unmutated humanized anti-AMHRII 12G4
    
    


ASC1 cells culture dossier 871 10 063
The results are presented in FIG. 25.
Table IX presents the raw data corresponding to FIG. 25.









TABLE IX









% of lysis (ADC 1193 11 081)
















829
632 07 107



Ac


10 054
Anti



ng/ml
829 10 050
829 10 051
3C23K
AMHRII


Ac ng/ml
(Log)
3C23 YB20
6B78 YB20
YB20
hum 1st















0.001
−3.000
0
0
0
0


0.005
−2.301
8
14
8
18


0.05
−1.301
9
5
10
2


0.5
−0.301
13
3
26
0


5
0.699
35
28
51
7


50
1.699
46
42
67
24


500
2.699
52
48
79
50


5000
3.699
63
53
75
50









Table X presents the Emax and EC50 obtained with the various antibodies.













TABLE X










632 07 107





829 10 054
Anti



829 10 050
829 10 051
3C23K
AMHRII



3C23 YB20
6B78 YB20
YB20
hum 1st




















Emax
65.55
51.89
79.02
52.40


(% of lysis)


EC50 (ng/ml)
6.298
5.383
1.704
52.35









Example 9
Comparison of the ADCC Activity of the Antibodies of the Invention Versus the Chimeric 12G4 Antibody
The ADCC activity of the humanized candidate antibody 3C—23K (hal2G4 of sequence 3C—23K: mutations VHQ3R (SEQ ID NO: 82 (without leader) or SEQ ID NO: 84 (with leader)), and VLI177T/S179P/E184K (SEQ ID NO: 86 (without leader), or SEQ ID NO: 88 (with leader)) was evaluated.
Briefly, the effector cells (NK killer cells; NK: Natural Killer) are purified beforehand by the negative depletion technique developed by the company Miltenyi (Miltenyi Biotec—NK cell isolation kit human ref 130-092-657), from peripheral blood of healthy donors, after a first step of purification of the mononucleated cells on Ficoll.
The in vitro assay of ADCC activity consists of incubating the NK cells with target cells (cov434-AMHRII, Asc 1 and META 2815 lines), in the presence of different concentrations of anti-AMHRII antibodies (chimeric antibody ch12G4, humanized 3C—23K-YB2/0 antibody, produced in YB2/0, and 3C—23K-CHO, produced in CHO). The effector/target ratio applied is 15/1. The antibodies are diluted in culture medium at concentrations ranging from 0.005 to 5000 ng/ml.
After 4 hours of incubation, the cytotoxic activity induced by the anti-AMHRII antibodies is measured by colorimetry by determining, in the supernatants, an intracellular enzyme called lactate dehydrogenase (LDH) released by the lysed target cells (Roche Diagnostics—Cytotoxicity Detection Kit LDH ref 11644793001).
The percentage lysis is calculated from the following formula:

% of lysis=[(ER−SR)/(100−SR)]−[(NC−SR)/(100−SR)

with: ER=release of LDH in the presence of antibodies and of NK cells

    
    
        SR=spontaneous release of LDH from the target cell alone
        NC=release of LDH in the presence of NK cells and absence of antibodies.
    
    


The results are expressed in percentage lysis as a function of the amount of antibody. The Emax and EC50 values are calculated with the PRISM software.
The results obtained on the cov434-AMHRII line are presented in FIG. 27. The low activity of the 3C—23K-CHO antibody does not allow a plateau to be obtained under the assay conditions. In order to compare the efficacy of the antibodies, calculation of 50% relative is carried out in this case, which represents the amount of 3C—23K-CHO antibody required to reach 50% of the plateau of the chimeric antibody (50% relative=1). According to this evaluation, the humanized antibody having the best ADCC activity on the COV434-AMRHII line is the 3C—23K antibody (50% relative: 0.84) produced in YB2/0. The humanized antibodies of sequence 3C—23 and 6B—78 have a value of 50% relative, equal to 6.41 and 36.92, respectively.
The results obtained on the Asc 1 line are presented in FIG. 28. The 3C—23K-YB2/0 antibody has a dose-dependent cytotoxic activity on the Asc 1 cells with an EC50 estimated at 2.24 ng/ml. The low activity of the 3C—23K-CHO antibody does not allow a plateau to be obtained under the assay conditions. In order to compare the efficacy of the two antibodies, calculation of 50% relative is carried out in this case, which represents the amount of 3C—23K-CHO antibody required to reach 50% of the plateau of the 3C—23K-YB2/0 antibody (50% relative=1). According to this evaluation, the cytotoxic activity of the 3C—23K-YB2/0 antibody is about 40 times greater than that of the antibody produced in CHO (50% relative=39.4).
Similarly, the results presented in FIG. 29 show that the antibodies 3C—23K-YB2/0 and 3C—23K-CHO induce dose-dependent lysis on the META 2815 cells. The cytotoxic activity of the 3C—23K-YB2/0 antibody (EC50=30.5 ng/ml) is about 146 times greater than that of the 3C—23K-CHO antibody (EC50=466.9 ng/ml).
Taken together, these results indicate that the 3C—23K anti-AMHRII antibodies produced in YB2/0 have the capacity to induce lysis of the cells expressing the AMHRII antigen. The difference in EC50 between the anti-AMHRII-YB2/0 and anti-AMHRII-CHO antibodies suggests a particular advantage for the anti-AMHRII-YB2/0 antibody under conditions of low antigenic expression, or of low penetrance of the antibody to the tumour.
Example 10
Studies of Cellular Proliferation
Inhibition of cellular proliferation was demonstrated by measuring cell growth over time in the presence or absence of the anti-AMHRII antibodies tested.
Briefly, the target cells (cov434-AMHRII, Asc 1, META2815) are cultured in P6 plates (1×105 cells/well) for 72 h at 37° C., in the presence of the anti-AMHRII antibodies (10 μg/ml) expressed in CHO or YB2/0, with or without cross-linking agent (AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Fcγ Fragment Specific ref: 109-006-008, Jackson Immunoresearch, France). The cells are treated with trypsin for 5 minutes and then counted in the CEDEX, an automatic cell counter based on cellular viability (trypan blue). A positive control of inhibition of proliferation is established in the presence of 1 μg/ml of colchicine (Ref: C3915, Sigma-Aldrich, France). A negative control is established in the presence of a non-relevant antibody (anti-P24). All the dilutions are prepared in culture medium (RPMI, 10% FCS). The results are expressed as percentage proliferation, the value 100% corresponding to the proliferation of the cells observed in the absence of antibody.
The results obtained with the cov434-AMHRII line are presented in FIG. 30.
According to these observations the antibodies 3C—23K-YB2/0 and 3C—23K-CHO induce about 40% inhibition of cellular proliferation of the cov434-AMHRII cells, in the presence of a cross-linking agent (CK). This cytostatic effect is not observed in the presence of a non-relevant antibody (antibody p24) whereas the colchicine positive control (10 μg/ml) induces 88% inhibition.
Similarly, the results presented in FIG. 31 show that the antibodies 3C—23K-YB2/0 and 3C—23K-CHO induce about 40% inhibition of cellular proliferation on the META 2815 line in the presence of a cross-linking agent (CK).
This inhibition of cellular proliferation might be the consequence of cellular signalling induced by the anti-AMHRII antibodies on the cov434-AMHRII and META 2815 lines.
Example 11
Effect In Vivo of the 3C—23K-YB2/0 Antibody on COV434-AMHRII Tumours
The antitumour efficacy of the 3C—23K-YB2/0 antibody was evaluated in late treatment on female Swiss nude mice after subcutaneous injection (s.c.) of COV434-AMHRII tumour cells. The intraperitoneal (i.p.) injections (inj) of antibody were performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections. The group treated with the 3C—23K-YB2/0 antibody was compared with the group treated with the vehicle (PBS).
Material and Methods
Female Swiss nude mice were used (Harlan). On day 0 of the experiment, the mice were given a subcutaneous injection of 7.106 COV434-AMHRII tumour cells mixed with Matrigel (ratio 1:1). The animals were then treated by i.p. injection of PBS or 3C—23K-YB2/0 with 10 mg/kg/inj starting from day 16 (tumour volume between 84 and 270 mm3, 3 injections per week for 6 weeks (total 18 injections).
Tumour volume was measured 2 to 3 times per week. Tumour volume (TV) was calculated using the following formula: TV (mm3)=(length×width×height)/2, in which the length corresponds to the largest diameter of the tumour and the width corresponds to the smallest diameter of the tumour.
The curves of tumour growth were plotted using the mean of the tumour volumes (MTV). The animals were euthanased when the individual tumour volume had reached 2000 mm3. In each of the groups, the curves were stopped when 30% of the animals in the group had been euthanased.
The inhibition of tumour growth (T/C), defined as the ratio of the median tumour volume of the treated groups relative to the control group treated with the vehicle, was calculated as follows: T/C=(median TV of the treated group/median TV of the vehicle group)×100

    
    
        T/C above 42%, the product is considered to be ineffective.
        T/C between 42% and 10%, the product has an anti-tumour effect.
        T/C below 10%, the product is truly effective.
    
    


The statistical differences between the different groups were obtained with the Kruskal-Wallis test, using the ANOVA comparison (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05. A logrank test, for comparing the survival parameters of the study, was also performed via ANOVA (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05.
Results
The 3C—23K-YB2/0 antibody shows anti-tumour activity, since a delay is observed in COV434-AMHRII (FIGS. 32A and 32B).
Statistical comparison of the tumour volumes at each measurement point, once the treatment has been started, shows that the 3C—23K-YB2/0 antibody delays tumour growth (Kruskal-Wallis, via ANOVA). The T/C ratio calculated between the groups treated with 3C—23K-YB2/0 and vehicle shows a significant difference at all the measurement points, also once the treatment has been started. The logrank test also shows that in terms of survival, the group treated with 3C—23K-YB2/0 is statistically different from the group treated with the vehicle.
Table XI below shows the evolution of the tumour volumes (treated/control ratio, T/C in %) under the effect of the treatment with 3C—23K-YB2/0 in the cov434-AMHRII model.









TABLE XI









Measurement day













15
21
25
30
32


















T/C %
108
60
42
22
13










Table XII below presents the statistical analyses obtained in the cov434-AMHRII model.










TABLE XII









ANOVA
Kruskall-Wallis













Measurement day
F-ratio
P-value
Sig.
Test
P-value
Sig.
















15
0.03
0.8628

0.00577
0.93945



21
8.34
0.0098
*
5.67427
0.01721
*


5
12.95
0.0021
*
10.56570
0.00115
*


30
34.94
0.0000
*
13.73030
0.00021
*


32
39.04
0.0000
*
12.90670
0.00033
*









Example 12
Effect In Vivo of the 3C—23K-YB2/0 Antibody on Asc1A5 Tumours
The antitumour efficacy of the 3C—23K-YB2/0 antibody was evaluated in late treatment on female Swiss nude mice after subcutaneous injection (s.c.) of Asc1A5 tumour cells (clone of the original Asc 1 line). The intraperitoneal (i.p.) injections (inj) of the antibody were performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections. The group treated with the 3C—23K-YB2/0 antibody was compared with the group treated with the vehicle (PBS).
Material and Methods
Female Swiss nude mice were used (Harlan). On day 0 of the experiment, the mice were given a subcutaneous injection of 7.106 Asc 1A5 tumour cells mixed with Matrigel (1:1 ratio). The animals were then treated by i.p. injection of PBS or 3C—23K-YB2/0 with 10 mg/kg/inj starting from day 12 (tumour volume between 40 and 160 mm3, 3 injections per week for 6 weeks (total 18 injections)).
Tumour volume was measured 2 to 3 times per week. Tumour volume (TV) was calculated using the following formula: TV (mm3)=(length×width×height)/2, in which length corresponds to the largest diameter of the tumour and width corresponds to the smallest diameter of the tumour.
The curves of tumour growth were plotted using the mean of the tumour volumes (MTV). The animals were euthanased when the individual tumour volume had reached 2000 mm3. In each of the groups, the curves were stopped when 30% of the animals in the group had been euthanased.
The inhibition of tumour growth (T/C), defined as the ratio of the median tumour volume of the treated groups to the control group treated with the vehicle, was calculated as follows: T/C=(median TV of treated group/median TV of vehicle group)×100

    
    
        T/C above 42%, the product is considered to be ineffective.
        T/C between 42% and 10%, the product has an anti-tumour effect.
        T/C below 10%, the product is truly effective.
    
    


The statistical differences between the different groups were obtained with the Kruskal-Wallis test, using the ANOVA comparison (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05. A logrank test, for comparing the survival parameters of the study, was also performed via ANOVA (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05.
Results
The 3C—23K-YB2/0 antibody shows anti-tumour activity, since a delay is observed in tumour growth compared to the group treated with the vehicle in the Asc1A5 model (FIGS. 33A and 33B).
Statistical comparison of the tumour volumes at each measurement point, once the treatment has been started, shows that the 3C—23K-YB2/0 antibody delays tumour growth (Kruskal-Wallis, via ANOVA). The T/C ratio calculated between the groups treated with 3C—23K-YB2/0 and vehicle shows a significant difference at all the measurement points, also once the treatment has been started. The logrank test also shows that in terms of survival, the group treated with 3C—23K-YB2/0 is statistically different from the group treated with the vehicle.
Table XIII below presents the evolution of the tumour volumes (treated/control ratio, T/C in %) under the effect of the treatment with 3C—23K-YB2/0 obtained in the Asc1a5 model.










TABLE XIII









Measurement day















12
17
24
27
31
35



















T/C %
101
33
8
8
7
6










Table XIV below presents the statistical analyses obtained in the Asc1a5 model.











TABLE XIV









ANOVA
Kruskall-Wallis













Measurement day
F-ratio
P-value
Sig.
Test
P-value
Sig.
















12
0.02
0.8817

0.0995
0.7523



17
29.19
0.0001
*
11.3108
0.0007
*


24
103.56
0
*
11.2941
0.0007
*


27
32.08
0.0001
*
11.3108
0.0007
*


31
33.37
0
*
11.3274
0.0007
*


35
57.97
0
*
10.5788
0.0011
*









Example 13
Effect In Vivo of the 3C—23K-YB2/0 Antibody on Meta2815 Tumours
The antitumour efficacy of the 3C—23K-YB2/0 antibody was evaluated in late treatment on female Swiss nude mice after subcutaneous injection (s.c.) of Meta 2815 tumour cells. The intraperitoneal (i.p.) injections (inj) of antibody were performed at intervals of 2-3 days at a dose of 10 mg/kg/inj for a total of 18 injections. The group treated with the 3C—23K-YB2/0 antibody was compared with the group treated with the vehicle (PBS).
Material and Methods
Female Swiss nude mice were used (Harlan). On day 0 of the experiment, the mice were given a subcutaneous injection of 8.106 Meta2815 tumour cells. The animals were then treated by i.p. injection of PBS or 3C—23K-YB2/0 with 10 mg/kg/inj starting from day 33 (tumour volume between 45 and 240 mm3, 3 injections per week for 6 weeks (total 18 injections).
Tumour volume was measured 2 to 3 times per week. Tumour volume (TV) was calculated using the following formula: TV (mm3)=(length×width×height)/2, in which length corresponds to the largest diameter of the tumour and width corresponds to the smallest diameter of the tumour.
The curves of tumour growth were plotted using the mean of the tumour volumes (MTV). The animals were euthanased when the individual tumour volume had reached 2000 mm3. In each of the groups, the curves were stopped when 30% of the animals in the group had been euthanased.
The inhibition of tumour growth (T/C), defined as the ratio of the median tumour volume of the treated groups to the control group treated with the vehicle, was calculated as follows: T/C=(median TV of treated group/median TV of vehicle group)×100

    
    
        T/C above 42%, the product is considered to be ineffective.
        T/C between 42% and 10%, the product has an anti-tumour effect.
        T/C below 10%, the product is truly effective.
    
    


The statistical differences between the different groups were obtained with the Kruskal-Wallis test, using the ANOVA comparison (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05. A logrank test, for comparing the survival parameters of the study, was also performed via ANOVA (Statgraphics centurion XV software). The differences were regarded as significant if P<0.05.
Results
The 3C—23K-YB2/0 antibody shows anti-tumour activity, since a delay is observed in tumour growth compared to the group treated with the vehicle in the Meta2815 model (FIGS. 34A and 34B).
Statistical comparison of the tumour volumes at each measurement point, once the treatment has been started, shows that the 3C—23K-YB2/0 antibody delays tumour growth (Kruskal-Wallis, via ANOVA). The T/C ratio calculated between the groups treated with 3C—23K-YB2/0 and vehicle shows a significant difference at all the measurement points, also once the treatment has been started. The logrank test also shows that in terms of survival, the group treated with 3C—23K-YB2/0 is statistically different from the group treated with the vehicle.
Table XV below presents the evolution of the tumour volumes (treated/control ratio, T/C in %) under the effect of the treatment with 3C—23K-YB2/0 in the META 2815 model.









TABLE XV









Measurement day













33
38
42
47
52


















T/C %
92
38
26
22
21










Table XVI presents the statistical analyses obtained in the META 2815 model.











TABLE XVI









ANOVA
Kruskall-Wallis













Measurement day
F-ratio
P-value
Sig.
Test
P-value
Sig.
















33
0
0.9899

0
1



38
11.21
0.0007
*
8.30769
0.0039
*


42
9.51
0.0116
*
7.41026
0.0064
*


47
12.7
0.0052
*
8.33684
0.0038
*


52
16.14
0.0024
*
8.30769
0.0039
*

## Claims

1. A mutated humanized 12G4 monoclonal antibody, comprising or consisting of:
a) a light chain comprising or consisting of:
a variable region (VL) according to amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 4, said VL consisting of three regions determining antigen recognition (CDR) surrounded by four framework regions (FR), and a constant region according to amino acid sequence SEQ ID NO: 6 or a sequence having at least 80% homology with SEQ ID NO: 6, and

b) a heavy chain comprising or consisting of:
a variable region (VH) according to amino acid sequence SEQ ID NO: 8, or SEQ ID NO: 10, and
a constant region according to amino acid sequence of SEQ ID NO: 12 or a sequence having at least 80% homology with SEQ ID NO: 12,

wherein said mutated humanized 12G4 monoclonal antibody comprises at least one mutation selected from the group consisting of:
a mutation located in one of the CDR regions of the VL that is a substitution of at least one amino acid selected from the group consisting of S179P, one of E184K, E184G, or E184D, and S182F,
a mutation located in one of the FR regions of VL that is a substitution of at least one amino acid selected from the group consisting of: I177T, I132T, A1431, 1150A, S158P, L175Q, Y178H, V187A, 51921, G197D, and F212S, and
a mutation located in the heavy chain that is a substitution of at least one amino acid selected from the group consisting of: Q1E, Q3E or Q3R, Q6E, A9T, V11A, K12R, K13R, K19E, V20A, one of A24G, A24V or A24T, Q39E, A40V, S31G, L45P, D56N, A76T, A79T, R87G, T58A, Q62R, V67M, I70N, T74A, S77P, A79T, S88P, E89D, F102S, A1031, L110P, and 51141, and
said mutated humanized 12 G4 monoclonal antibody has a KD for the human anti-Müllerian hormone type II receptor (AMHRII) at least equal to that of the chimeric 12G4 monoclonal antibody, comprising or consisting of:
a) a light chain consisting of:
a variable region according to amino acid sequence SEQ ID NO: 14, and
a constant region according to amino acid sequence SEQ ID NO: 6,

b) a heavy chain consisting of:
a variable region according to amino acid sequence SEQ ID NO: 18, or SEQ ID NO: 10, and
a constant region according to amino acid sequence SEQ ID NO: 12,

for said receptor below 10−7M.

2. The mutated humanized 12G4 monoclonal antibody according to claim 1, wherein said at least one mutation is said substitution in said CDR region.

3. The mutated humanized 12G4 monoclonal antibody according to claim 1, wherein said at least one mutation is said substitution in said FR regions of VL.

4. The mutated humanized 12G4 monoclonal antibody according to claim 1, wherein said at least one mutation is said substitution in the heavy chain.

5. The mutated humanized 12G4 monoclonal antibody according to claim 1, wherein:
a) the light chain comprises or consists of a variable region according to amino acid sequence of SEQ ID NO: 2 in which said at least one substitution in said CDR region is selected from the group consisting of: S179P, one of E184K, E184G, or E184D, and S182F, or the light chain comprises or consists of a variable region according to amino acid sequence SEQ ID NO: 2 in which said at least one substitution in said CDR region is selected from the group consisting of: S179P, one of E184K, E184G, or E184D, and S182F, and said at least one substitution in the FR regions of VL is a substitution of at least one amino acid selected from the group consisting of: of I177T, I132T, A143T, T150A, S158P, L175Q, Y178H, V187A, S192T, G197D, or F212S, and a constant region according to amino acid sequence SEQ ID NO: 6, and
b) the heavy chain comprises or consists of a variable region according to amino acid sequence SEQ ID NO: 8 in which said at least one substitution is selected from the group consisting of: Q1E, Q3E or Q3R, Q6E, A9T, V11A, K12R, K13R, K19E, V20A, one of A24G, A24V or A24T, Q39E, A40V, S31G, L45P, D56N, A76T, A79T, R87G, T58A, Q62R, V67M, I70N, T74A, S77P, A79T, S88P, E89D, F102S, A103T, L110P, and 5114T.

6. The mutated humanized 12G4 monoclonal antibody according to claim 1, wherein
a) the light chain comprises or consists of a variable region according to an amino acid sequence selected from the group consisting of: SEQ ID NO: 22 or SEQ ID NO: 24, SEQ ID NO: 30 or SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36, and SEQ ID NO: 46 or SEQ ID NO: 48, and of a constant region according to amino acid sequence SEQ ID NO: 6, and
b) the heavy chain comprises or consists of a variable region according to an amino acid sequence selected from the group consisting of: SEQ ID NO: 38 or SEQ ID NO: 40, SEQ ID NO: 26 or SEQ ID NO: 28, SEQ ID NO: 8 or SEQ ID NO: 10, SEQ ID NO: 42 or SEQ ID NO: 44, and SEQ ID NO: 50 or SEQ ID NO: 52, and of a constant region according to amino acid sequence SEQ ID NO: 12.

7. The mutated humanized 12G4 monoclonal antibody according to claim 5, wherein the combination of the light chain and the heavy chain is selected from the group consisting of:
a) the light chain consists of SEQ ID NO: 70 or SEQ ID NO: 72, and
b) the heavy chain consists of SEQ ID NO: 74 or SEQ ID NO: 76;
a) the light chain consists of SEQ ID NO: 78 or SEQ ID NO: 80, and
b) the heavy chain consists of SEQ ID NO: 58 or SEQ ID NO: 60;
a) the light chain consists of SEQ ID NO: 82 or SEQ ID NO: 84, and
b) the heavy chain consists of SEQ ID NO: 86 or SEQ ID NO: 88;
a) the light chain consists of SEQ ID NO: 78 or SEQ ID NO: 80, and
b) the heavy chain consists of SEQ ID NO: 90 or SEQ ID NO: 92; and
a) the light chain consists of SEQ ID NO: 94 or SEQ ID NO: 96, and
b) the heavy chain consists of SEQ ID NO: 98 or SEQ ID NO: 100.

8. A fragment of the mutated humanized 12G4 monoclonal antibody according to claim 1, selected from the group consisting of: Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, Sc(Fv)2, and diabodies.

9. Pharmaceutical composition, comprising at least
a monoclonal antibody according to claim 1, or
a fragment of said monoclonal antibody selected from the group consisting of: Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, Sc(Fv)2, and diabodies,

together with a pharmaceutically acceptable vehicle.

10. Product comprising a first pharmaceutical preparation comprising a monoclonal antibody according to claim 1, and a second pharmaceutical preparation comprising a conventional anticancer compound as a combined preparation for simultaneous, separate or sequential use in the treatment of patients with ovarian cancers.

11. A kit comprising at least:
the mutated monoclonal antibody according to claim 1, or
a fragment of said mutated monoclonal antibody selected from the group of fragments consisting of: Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, Sc(Fv)2, and diabodies,

for use in diagnosing a pathology associated with the human anti-Müllerian hormone type II receptor.