HK1132752B - Novel antiproliferation antibodies - Google Patents

Abstract

The present invention relates to novel isolated antibodies, or the derived compounds or functional fragments of same, capable of inhibiting the proliferation of tumor cells in vitro and/or in vivo, said antibodies having been obtained by functional screening. More particularly, the present invention relates to the 6F4 antibody, specific to the JAM-A protein, as well as its use for the treatment of cancer. Pharmaceutical compositions composed of such antibodies are also covered.

Description

Novel antiproliferative antibodies

The present invention relates to novel antibodies, in particular murine monoclonal antibodies, chimeric antibodies and humanized antibodies, capable of inhibiting tumor growth, as well as the amino acid and nucleic acid sequences encoding these antibodies. In one aspect, the present invention relates to novel antibodies, derivative compounds or functional fragments capable of inhibiting tumor cell proliferation. The invention also comprises the use of such antibodies in medicaments for the prophylactic and/or therapeutic treatment of cancer, as well as in procedures or kits relating to cancer diagnosis. Finally, the invention includes compositions comprising such antibodies in combination with other anti-cancer compounds (e.g., antibodies) or in combination with toxins, and uses thereof in the prevention and/or treatment of certain cancers.

Generally, the criteria chosen to produce monoclonal antibodies are the identification of immunogens that are identified as potential therapeutic targets. In practice, mice are immunized with recombinant proteins corresponding to the immunogen, and after recovering the monoclonal antibodies produced by the mice, they are first screened for their ability to recognize the immunogen in a specific manner. In the second phase, the antibodies thus selected are subjected to in vivo and in vitro tests to determine their activity and their characteristics and/or mechanism of action.

This "classical" approach, although it is possible to know the target of action from the outset, often produces large quantities of antibodies that do specifically recognize a particular target, but do not exhibit significant biological activity in vivo. In the cancer field, it is indeed known that although an antibody produces good results in vitro, it is not inevitable that such an antibody subsequently exhibits true anti-tumor activity in vivo.

The present invention differs from the previous way and even in contrast to the previous one, since it is based on a "functional" approach, more particularly on the basis of a functional search for antibodies on the basis of the main screening method rather than on the antigen recognized.

More particularly, the inventors of the present invention have selected as an antibody selection parameter a specific function, namely inhibition of basal proliferation of cells without inhibiting induced proliferation of cells.

The preparation methods used are described in detail in the examples below.

By this functional approach, in a surprising manner, the inventors have prepared and selected antibodies capable of inhibiting tumor cell proliferation in a significant manner in vitro and/or in vivo.

According to a first aspect, the present invention relates to an isolated antibody, or a derived compound or functional fragment thereof, capable of inhibiting tumor cell proliferation in vitro and/or in vivo; the antibody or derived compound or functional fragment thereof comprising at least one Complementarity Determining Region (CDR) selected from the group consisting of the sequences of SEQ ID nos. 1, 2, 3, 4, 5 or 6 or at least one CDR whose sequence has at least 80%, preferably 85%, 90%, 95%, 98% identity to the sequence of SEQ ID nos. 1, 2, 3, 4, 5 or 6 after optimal alignment.

"functional fragments" of an antibody mean in particular antibody fragments, such as Fv, scFv (sc ═ single chain), Fab, F (ab')₂Fab', scFv-Fc fragment or diabody or any fragment with an extended half-life. Such antibody fragments will be described in detail later in this specification.

"derivative compounds" of an antibody refer in particular to binding proteins consisting of a peptide scaffold and one of the CDRs of at least one of the original antibodies in order to retain its ability to be recognized. Such derivatised compounds, well known to those skilled in the art, will be described in more detail in the present specification below.

More preferably, the present invention comprises an antibody, antibody-derived compound or antibody functional fragment according to the present invention obtained by genetic recombination or chemical synthesis, particularly a chimeric or humanized antibody.

According to a preferred embodiment, the antibody or derived compound or functional fragment thereof according to the invention is characterized in that it consists of a monoclonal antibody.

It is understood that "monoclonal antibody" refers to an antibody produced in a population of nearly homogeneous antibodies. More particularly, the individual antibodies in the population are identical except for some mutations that may be found in a minimal proportion to occur naturally. In other words, a monoclonal antibody consists of a homogeneous antibody produced by growing a single cell clone (e.g., a hybridoma cell, a eukaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, a prokaryotic host cell transfected with a DNA molecule encoding the homogeneous antibody, etc.) and is generally characterized by heavy chains that are one and only one class or subclass, and by light chains that are only one type. Monoclonal antibodies are highly specific and directed against a single antigen. Furthermore, in contrast to the production of polyclonal antibodies, polyclonal antibodies typically include a variety of antibodies directed against various determinants or epitopes, while each monoclonal antibody is directed against a single epitope of the antigen.

It must be understood here that the invention does not relate to antibodies in their natural form, i.e. they are not taken from their natural environment but are isolated or purified from natural sources or obtained by genetic recombination or chemical synthesis, and therefore they can carry unnatural amino acids, which antibodies will be described below.

More particularly, according to a preferred embodiment of the invention, the antibody or derived compound or functional fragment thereof is characterized in that it comprises a light chain comprising at least one CDR whose amino acid sequence is selected from SEQ ID No.1, 3 or 5, or at least one CDR whose sequence, after optimal alignment, has at least 80%, preferably 85%, 90%, 95% and 98%, identity with the sequence SEQ ID No.1, 3 or 5; or it comprises a heavy chain comprising at least one CDR whose amino acid sequence is selected from the group consisting of SEQ ID Nos. 2, 4 or 6, or at least one CDR whose sequence, after optimal alignment, is at least 80%, preferably 85%, 90%, 95% and 98%, identical to the sequence SEQ ID Nos. 2, 4 or 6.

More preferably, the antibody or derived compound or functional fragment thereof according to the invention is characterized in that it comprises a heavy chain comprising at least one of the three CDRs of sequences SEQ ID nos. 2, 4 and 6 or at least one sequence having a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with sequences SEQ ID nos. 2, 4 or 6.

Even more preferably, the antibody of the invention, or one of its derivative compounds or functional fragments, is characterized in that it comprises a heavy chain comprising the following 3 CDRs (CDR-H1, CDR-H2 and CDR-H3, respectively), wherein:

-CDR-H1 comprises the sequence SEQ ID No.2, 7 or 9 or a sequence which, after optimal alignment, is at least 80% identical to the sequence SEQ ID No.2, 7 or 9;

-CDR-H2 comprises the sequence SEQ ID No.4 or 11 or a sequence which, after optimal alignment, is at least 80% identical to the sequence SEQ ID No.4 or 11;

-CDR-H3 comprises the sequence SEQ ID No.6 or 12 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No.6 or 12;

according to a particular embodiment, the antibody or one of its derived compounds or functional fragments is characterized in that it comprises a heavy chain comprising the CDR-H1 having the sequence SEQ ID No.7, the CDR-H2 having the sequence SEQ ID No.4 and the CDR-H3 having the sequence SEQ ID No. 12.

According to another particular embodiment, the antibody or one of its derived compounds or functional fragments is characterized in that it comprises a heavy chain comprising the CDR-H1 having the sequence SEQ ID No.9, the CDR-H2 having the sequence SEQ ID No.11 and the CDR-H3 having the sequence SEQ ID No. 6.

According to another embodiment, the antibody according to the invention or one of its derived compounds or functional fragments is characterized in that it comprises a light chain comprising at least one of the three CDRs whose sequences are SEQ ID nos. 1, 3 and 5 or at least one sequence whose sequence, after optimal alignment, is at least 80%, preferably 85%, 90%, 95% and 98%, identical to the sequences SEQ ID nos. 1, 3 and 5.

In a preferred manner, the antibody according to the invention or one of its derivative compounds or functional fragments is characterized in that it comprises a light chain comprising the following three CDRs (CDR-L1, CDR-L2 and CDR-L3, respectively), wherein

-CDR-L1 comprises the sequence SEQ ID No.1 or 8 or a sequence which, after optimal alignment, is at least 80% identical to the sequence SEQ ID No.1 or 8;

-CDR-L2 comprises the sequence SEQ ID No.3 or 10 or a sequence which, after optimal alignment, is at least 80% identical to the sequence SEQ ID No.3 or 10;

-CDR-L3 comprises the sequence SEQ ID No.5 or a sequence having at least 80% identity after optimal alignment with the sequence SEQ ID No. 5;

according to a particular embodiment, the antibody or one of its derived compounds or functional fragments is characterized in that it comprises a light chain comprising the CDR-L1 having the sequence SEQ ID No.1, the CDR-L2 having the sequence SEQ ID No.3 and the CDR-L3 having the sequence SEQ ID No. 5.

According to another particular embodiment, the antibody or one of its derived compounds or functional fragments is characterized in that it comprises a light chain comprising the CDR-L1 having the sequence SEQ ID No.8, the CDR-L2 having the sequence SEQ ID No.10 and the CDR-L3 having the sequence SEQ ID No. 5.

In the present specification, the terms "polypeptide", "polypeptide sequence", "peptide" and "protein linked to an antibody compound or its sequence" are interchangeable.

It must be understood that the invention does not relate to antibodies in their natural form, i.e. they are not taken from their natural environment, but are isolated or purified from natural sources, or obtained by genetic recombination or chemical synthesis, and therefore they can carry unnatural amino acids, which antibodies will be described below.

In a first embodiment, complementarity determining regions or CDRs mean the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest, 5th Ed., US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to the antigen or epitope it recognizes, depending on the circumstances.

In a second embodiment, the CDR regions or CDRs refer to the highly variable regions of the heavy and light chains of an immunoglobulin as defined by IMGT.

The unique numbering of IMGT is defined as compared to the variable domain regardless of the type or species of antigen receptor or chain [ Lefranc m. -p ], Immunology Today 18, 509(1997)/Lefranc m. -p ], the immunologies, 7, 132-flaponic 136(1999)/Lefranc, m. -p, Pommi e, c, Ruiz, m. In the unique numbering of IMGT, the positions of conserved amino acids are always the same, such as cysteine 23 (1 st-CYS), tryptophan 41 (conserved-TRP), hydrophobic amino acid 89, cysteine 104 (2 nd-CYS), phenylalanine or tryptophan 118(J-PHE or J-TRP). The IMGT is uniquely numbered with respect to the framework regions (FR 1-IMGT: positions 1 to 26, FR 2-IMGT: 39 to 55, FR 3-IMGT: 66 to 104 and FR 4-IMGT: 118 to 128) and the complementarity determining region CDR 1-IMGT: 27 to 38, CDR 2-IMGT: 56 to 65 and CDR 3-IMGT: 105 to 117 provide a standardized delimitation. The gap (gap) represents an unoccupied position, and the CDR-IMGT length (indicated in parenthesis, separated by dots, e.g. [8.8.13]) becomes the key information. The unique number of IMGT used in the 2D representative graphs is designated IMGT colloids de trees [ Ruiz, m, and Lefranc, m. — p., Immunogenetics, 53, 857-.

There are three heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs as used herein is intended to mean a region comprising one or more, or even all, of the major amino acid residues, as the case may be, that contribute to the binding affinity of an antibody to its recognized antigen or epitope.

For clarity, it must be understood that in the following description and more particularly in tables 2 and 3, the CDRs are defined by IMGT numbering, kabat numbering and general numbering.

Common numbering regroups some of the residues of each CDR that are identical for IMGT and the CDRs defined by the Kabat numbering system.

The IMGT numbering system defines CDRs according to the IMGT system described above, while the Kabat numbering system defines CDRs according to the Kabat system described above.

More particularly, CDR-L1 consists of SEQ ID No.1(QDINNY) of the common numbering and IMGT numbering system and SEQ ID No.8(KASQDINNYIA) of the kabat numbering system.

CDR-L2 consists of SEQ ID No.3(YTS) of the common numbering and IMGT numbering system and SEQ ID No.10(YTSTLQA) of the kabat numbering system.

CDR-L3 consists of each of SEQ ID No.5(LQYDNLWT) in the three numbering systems.

For the heavy chain, CDR-H1 consists of SEQ ID No.2 of the common numbering system (TDYS), SEQ ID No.7 of the IMGT numbering system (GYSFTDYS), and SEQ ID No.9 of the kabat numbering system (TDYSMY).

CDR-H2 consists of SEQ ID No.4(IDPYNGGT) of the common numbering system and the IMGT numbering system and SEQ ID No.11(YIDPYNGGTRYNQKFKG) of the kabat numbering system.

Finally, CDR-H3 consists of SEQ ID No.6(QTDYFDY) in the common and kabat numbering system, whereas it consists of SEQ ID No.12(ARQTDYFDY) in the IMGT numbering system.

In the sense of the present invention, a "percentage of identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues obtained after an optimized alignment between the two sequences being compared, this percentage being purely statistical, and the differences between the two sequences being randomly distributed along their length. Comparisons between two nucleic acid or amino acid sequences are typically made by comparing the two optimally aligned sequences, either in segments or with an "alignment interface". Optimized alignments for comparison, in addition to manual comparisons, can be determined by the local homology algorithm of Smith and Waterman (1981) [ ad. 482], local homology algorithm by Neddleman and Wunsch (1970) [ j.mol.biol.48: 443], similarity finding method by Pearson and Lipman (1988) [ proc.natl.acad.sci.usa 85: 2444] or by Computer Software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis, or by the composition Software BLAST NR or BLAST P).

The percent identity between two nucleic acid or amino acid sequences is determined by comparing two optimally aligned sequences, wherein the nucleic acid or amino acid sequences being compared may have additions or deletions compared to the reference sequence which optimizes the alignment between the two sequences. Percent identity is determined by determining the number of positions of the identical amino acid nucleotide or residue between the two sequences, dividing the number of identical positions by the total number of positions in the alignment interface, and multiplying the result by 100 to obtain the percent identity between the two sequences.

For example, the BLAST program "BLAST 2 sequence" (Tatusova et al, "BLAST 2 sequences-a new tool for composing protein and nucleotide sequences", FEMS Microbiol, 1999, Lett.174: 247-http://www.ncbi.nlm.nih.gov/gorf/bl2.htmlThe program is available using default parameters (in particular the parameters "open gap penalty": 5 and "extended gap penalty": 2; the matrix of choices proposed by the program, for example the "BLOSUM 62" matrix). The percent identity between two compared sequences was calculated directly by the program.

Preferred embodiments include those containing embodiments of the reference sequence, certain modifications, in particular deletions, additions or substitutions, truncations or extensions of at least one amino acid, for amino acid sequences having at least 80%, preferably 85%, 90%, 95% and 98% identity compared to the reference amino acid sequence. In the case of substitution of one or more consecutive or non-consecutive amino acids, such substitution is preferred: i.e., the substituted amino acid is replaced with an "equivalent" amino acid. The term "equivalent amino acid" as used herein refers to an amino acid that may be substituted with one of the structural amino acids, but which does not alter the biological activity of its corresponding antibody and amino acids as defined in the specific examples below.

Equivalent amino acids can be determined based on their structural homology to the substituted amino acid or based on comparative testing of biological activity between the various antibodies that may be produced.

As a non-limiting example, table 1 below summarizes possible substitution sites that may be substituted without significantly altering the biological activity of the corresponding modified antibody; the reverse substitution may occur naturally under the same conditions.

TABLE 1

Original residues

Substituent group

Ala(A)	Val、Gly、Pro
		Arg(R)	Lys、His
Asn(N)	Gln
		Asp(D)	Glu
Cys(C)	Ser
		Gln(Q)	Asn
Glu(G)	Asp
		Gly(G)	Ala
His(H)	Arg
		Ile(I)	Leu
Leu(L)	Ile、Val、Met
		Lys(K)	Arg
Met(M)	Leu
		Phe(F)	Tyr
Pro(P)	Ala
		Ser(S)	Thr、Cys
Thr(T)	Ser
		Trp(W)	Tyr
Tyr(Y)	Phe、Trp
		Val(V)	Leu、Ala

It is known to those skilled in the art that, in the current state of the art, the largest variation (length and composition) among the six CDRs is found among the three heavy chain CDRs, more particularly CDR-H3 of this heavy chain. As a result, it is clear that: preferred characteristic CDRs of the antibody according to the invention or of a derived compound or functional fragment thereof are the three CDRs of the heavy chain, i.e. the CDRs encoded by the sequences SEQ ID nos. 2, 4 and 6, respectively, even more preferred is the CDR corresponding to CDR-H3 encoded by the sequence SEQ ID No. 6.

In a particular embodiment, the invention relates to a murine antibody or a derivative compound or a functional fragment thereof.

In another embodiment of the invention, an antibody or derivative compound or functional fragment thereof is disclosed, comprising a light chain of the following three CDRs:

the sequence is CDR-L1 of SEQ ID No.1 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 1;

the sequence is CDR-L2 of SEQ ID No.3 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 3;

and

the sequence is CDR-L3 of SEQ ID No.5 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 5;

and

a heavy chain comprising the following three CDRs:

the sequence is CDR-H1 of SEQ ID No.7 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 7;

the sequence is CDR-H2 of SEQ ID No.4 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 4;

and

the sequence is CDR-H3 of SEQ ID No.12 or a sequence which after optimal alignment is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID No. 12.

In another embodiment of the invention, an antibody or derivative compound or functional fragment thereof is disclosed, comprising a light chain of the following three CDRs:

-the sequence is CDR-L1 of SEQ ID No.8, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with the sequence SEQ ID No. 8;

-the sequence is CDR-L2 of SEQ ID No.10, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence SEQ ID No. 10; and

-the sequence is CDR-L3 of SEQ ID No.5, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with the sequence SEQ ID No. 5; and

a heavy chain comprising the following three CDRs:

-the sequence is CDR-H1 of SEQ ID No.9, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence SEQ ID No. 9; and

-the sequence is CDR-H2 of SEQ ID No.11, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with sequence SEQ ID No. 11; and

-the sequence is CDR-H3 of SEQ ID No.6, or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98% identity with the sequence SEQ ID No. 6.

According to another embodiment, the antibody or derived compound or functional fragment thereof according to the invention is characterized in that it comprises a light chain sequence comprising a sequence whose amino acid sequence is SEQ ID No.13 or which, after optimal alignment, has at least 80%, preferably 85%, 90%, 95% and 98%, identity with the sequence SEQ ID No. 13; and in that it comprises a heavy chain sequence comprising the amino acid sequence SEQ ID No.14 or a sequence which after optimal alignment has at least 80%, preferably 85%, 90%, 95% and 98%, identity with the sequence SEQ ID No. 14.

Also disclosed is a humanized antibody or a derived compound or functional fragment thereof, characterized in that it comprises a light chain sequence comprising a sequence whose amino acid sequence is SEQ ID No.17 or which after optimal alignment has at least 80% identity with the sequence SEQ ID No.17, and wherein it comprises a heavy chain sequence comprising an amino acid sequence of SEQ ID No.18 or 19 or which after optimal alignment has at least 80% identity with the sequence SEQ ID No.18 or 19.

As above, the invention also relates to any compound derived from an antibody according to the invention.

More particularly, the antibody or derivative compound or functional fragment thereof according to the invention is characterized in that said derivative compound consists of a binding protein comprising a peptide scaffold onto which at least one CDR is grafted in such a way as to retain all or part of the antibody binding site recognition properties of the original antibody.

One or more of the six CDR sequences of the invention may also be present on various immunoglobulin scaffolds. In this case, the protein sequence makes it possible to reconstruct a peptide backbone which facilitates the folding of the grafted CDRs, so that it retains its antibody-binding site antigen-recognition properties.

In general, the skilled person knows how to determine the type of protein scaffold on which to implant at least one CDR from the original antibody.

More particularly, it is known that the maximum number of conditions (Skerra A., J.mol.Recogn., 2000, 13: 167-:

good phylogenetic conservation;

known three-dimensional structures (e.g. by crystallography, NMR spectroscopy or any other technique known to the person skilled in the art);

-small in volume;

-little or no post-transcriptional modification; and/or

Easy to produce, express and purify.

The origin of such a protein scaffold may be, but is not limited to, a structure selected from the group consisting of: fibronectin (preferably fibronectin type III domain 10), lipocalin, anticalin (Skerra a., j.biotechnol., 2001, 74 (4): 257-75), protein Z from domain B of Staphylococcus aureus (staphyloccocus aureus) protein a, thioredoxin a, or proteins with a repeat motif like "ankyrin repeat" (Kohl et al, PNAS, 2003, vol.100, No.4, 1700-1705), "armadillo (armadillo) repeat", "leucine-rich repeat" and "tetratricopeptide (tetratricopeptide) repeat".

Scaffolds derived from toxins (e.g. scorpions, insects, plants, molluscs, etc.) and neuronal NO synthase (PIN) should be mentioned.

An example of a non-limiting way of such a hybrid construct is to insert the CDR-H1 (heavy chain) of the anti-CD 4 antibody, namely named 13B8.2, in one of the loop regions of PIN, so that the new binding protein obtained retains the same binding properties as the original antibody (Bes et al, biochem. Biophys. Res. Commun., 2006, 343(1), 334-344). On a purely illustrative basis, it may also be mentioned that the CDR-H3 (heavy chain) of an anti-lysozyme VHH antibody is grafted onto a loop region of a novel oncostatin (neocarzinostatin) (Nicaise et al, Protein Science, 2004, 13 (7): 1882-1891).

Finally, as described above, such a peptide scaffold may comprise one to six CDRs from the original antibody. Preferably, but not necessarily, the skilled person will select at least one heavy chain CDR, the latter known to be the main cause of antibody specificity. It will be apparent to those skilled in the art that one or more relevant CDRs may be selected by those skilled in the art, and those skilled in the art will select known techniques thereafter (Bes et al, FEBS letters 508, 2001, 67-74).

A particular aspect of the invention relates to a method for selecting an antibody derivative compound according to the invention, said derivative compound being capable of inhibiting the growth of tumor cells in vivo and/or in vitro and comprising a peptide scaffold onto which at least one antibody CDR is grafted, characterized in that it comprises the following steps:

a) contacting a compound consisting of a peptide scaffold onto which at least one antibody CDR is grafted, in vitro and under conditions such that cells are grown, with a biological sample comprising tumor cells capable of growth; and

b) said compound is selected if it is capable of inhibiting the growth of these tumor cells and is characterized in that said at least one grafted CDR is selected from the group consisting of:

-the sequence is a CDR of SEQ ID No.1, 8 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No.1, 8;

-the sequence is a CDR of SEQ ID No.3, 10 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No.3, 10;

-the sequence is the CDR of SEQ ID No.5 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No. 5;

-the sequence is a CDR of SEQ ID No.2, 7, 9 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No.2, 7, 9;

-the sequence is a CDR of SEQ ID No.4, 11 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No.4, 11; and

-the sequence is a CDR of SEQ ID No.6, 12 or a sequence having at least 80%, preferably 85%, 90%, 95% and 98% identity after optimized alignment with the sequence SEQ ID No.6, 12.

According to a preferred mode, the method may comprise, in step a), contacting in vitro a compound comprising a peptide scaffold onto which at least two or three CDRs of the antibody are grafted.

According to an even more preferred mode of the method, the peptide scaffold is selected from the scaffolds or binding proteins whose structure is mentioned above.

It will be apparent that these examples are in no way limiting and that any other structure known or obvious to a person skilled in the art shall be considered to be covered within the protection of the present patent application.

The invention therefore relates to an antibody or a derived compound or functional fragment thereof, characterized in that the peptide scaffold is selected from the following proteins: a) phylogenetically well-preserved proteins, b) structurally robust proteins, c) proteins with well-known 3-D molecular composition, D) proteins that are small in size and/or e) proteins that contain modifications that can be deleted and/or inserted without altering the stability properties.

According to a preferred embodiment, the antibody, or derived compound or functional fragment thereof, according to the invention is characterized in that said peptide scaffold is selected from i) a scaffold from fibronectin, preferably a scaffold of fibronectin type III domain 10, lipocalin, anticalin, protein Z from domain B of protein a from staphylococcus aureus, thioredoxin a or a protein with a repeating motif such as "ankyrin repeat" (Kohl et al, PNAS, 2003, vol.100, No.4, 1700-1705), "armadillo repeat", "leucine-rich repeat" and "tetratricopeptide repeat" or III) neuronal synthase NO (PIN).

Another aspect of the invention relates to functional fragments of the above antibodies.

More particularly, the object of the invention is an antibody or a derivative compound or a functional fragment thereof, characterized in that said functional fragment is selected from the group consisting of fragments Fv, Fab, (Fab')₂Fab', scFv-Fc or diabodies, or any fragment with an extended half-life, such as a PEGylated fragment.

Such a functional fragment of the antibody of the present invention is composed of, for example, fragments Fv, scFv (single chain sc), Fab, and F (ab')₂Fab ', scFv-Fc or diabodies or any fragment whose half-life is extended by chemical modification, e.g.addition of polyalkylene glycol such as polyethylene glycol (PEGylation) (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F (ab')₂-PEG and Fab' -PEG), or incorporated into liposomes, microspheres, or PLGA; the fragment comprises at least one CDR characteristic of the invention. The CDR can exert in particular the activity of the general meaning of the antibody from which it is derived, even partial activity.

Preferably, the functional fragment comprises or includes a partial sequence of the variable heavy or light chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity as the antibody from which it is derived, and sufficient affinity, preferably at least 1/100 equal to the affinity of the antibody from which it is derived, more preferably at least 1/10 equal to the affinity of the antibody from which it is derived.

Such a functional fragment will comprise at least five amino acids, preferably 6, 7, 8, 10, 15, 25, 50 or 100 consecutive amino acids of the sequence of the antibody from which it is derived.

Preferably, these functional fragments will be Fv, scFv, Fab, F (ab')₂F (ab'), scFv-Fc or diabody types, these functional fragments generally having the same binding specificity as the antibody from which they are derived. According to the invention, the antibody fragment of the invention is usefulObtained by methods such as enzymatic digestion (including pepsin or papain) and/or cleavage of disulfide bonds by chemical reduction. Antibody fragments can also be obtained by recombinant genetic techniques (also known to those skilled in the art) or by peptide synthesis techniques, e.g., peptide synthesis can be synthesized using automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

The object of the present invention is also the original murine antibody, i.e. the antibody according to the invention or a derivative compound or functional fragment thereof, characterized in that said antibody is a murine antibody comprising the light chain of the amino acid sequence SEQ ID No.15 or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No.15 and the heavy chain of the amino acid sequence SEQ ID No.16 or a sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequence SEQ ID No. 16.

For clarity, table 2 below summarizes the various amino acid sequences corresponding to the antibodies of the present invention.

Table 2 (wherein Mu. is rat and Hu. is humanized)

Another particular aspect of the invention relates to a chimeric antibody or a derived compound or functional fragment thereof, characterized in that said antibody further comprises light and heavy chain constant regions derived from an antibody of a species heterologous to mouse, in particular to human.

Another particular aspect of the invention also relates to a humanized antibody or a derived compound or functional fragment thereof, characterized in that the constant regions of the light and heavy chains derived from a human antibody are the λ or κ region and the γ 1, γ 2 or γ 4 region, respectively.

According to another aspect, the invention relates to a murine hybridoma capable of secreting a monoclonal antibody according to the invention, in particular a murine hybridoma deposited at 6.7.2006 under accession number I-3646 to the center for cultures of french microorganisms (CNCM, Pasteur Institute, Paris, France). The hybridoma was obtained by fusing Balb/C immunized mouse spleen cells with the myeloma Sp2/O-Ag14 cell line.

Monoclonal antibody, herein designated 6F4, or a derivative compound or functional fragment thereof, characterized in that said antibody is secreted by a hybridoma deposited on 4/7/2006 under accession number I-3646 to CNCM, obviously forming part of the present invention.

The antibodies of the invention also comprise chimeric or humanized antibodies.

Chimeric antibodies are antibodies derived from a given species and comprising the natural variable regions (light and heavy chains) in combination with the constant regions of the light and heavy chains of an antibody of a species heterologous to the given species.

Antibodies or chimeric fragments of antibodies can be prepared by using recombinant genetic techniques. For example, chimeric antibodies may be produced by cloning recombinant DNA comprising a promoter and sequences encoding the variable regions of a non-human monoclonal antibody (particularly a murine monoclonal antibody) as described herein and sequences encoding the constant regions of a human antibody. The chimeric antibody encoded by such a recombinant gene according to the present invention may be, for example, a mouse-human chimera, the specificity of which is determined by the variable region derived from murine DNA and the isotype of which is determined by the constant region derived from human DNA. Methods for making chimeric antibodies are described in Verhoeyn et al (BioEssays, 8: 74, 1988).

"humanized antibody" refers to an antibody comprising the CDR regions of an antibody of non-human origin, the remainder of the antibody molecule being derived from one (or several) human antibody(s). In addition, some of the framework segment residues (referred to as FR) may be modified to retain binding affinity (Jones et al, Nature, 321: 522-525, 1986; Verhoeyen et al, Science, 239: 1534-1536, 1988; Riechmann et al, Nature, 332: 323-327, 1988).

The humanized antibodies or functional fragments thereof described herein can be prepared by techniques known to those skilled in the art (such as, for example, those described in Singer et al, J.Immun., 150: 2844-.

Furthermore, the present invention relates to a humanized antibody derived from the above-mentioned murine antibody.

More specifically, the humanization methods of the 6F4 antibody are detailed in examples 2 and 3 for the light and heavy chains, respectively.

Preferably, the human antibody-derived light and heavy chain constant regions are lambda or kappa and gamma-1, gamma-2 or gamma-4 regions, respectively.

In embodiments corresponding to IgG1 of the IgG1 isotype, the antibodies are additionally characterized as exhibiting effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and/or complementarity-dependent cytotoxicity (CDC).

In another aspect of the invention, applicants have also identified antigens recognized by the antibodies of the invention.

The method for accomplishing this step is described in detail below in example 4.

JAM-A is a membrane protein belonging to the immunoglobulin superfamily (IgSF) and to the family of conjugated adhesion molecules (JAM). In humans, the JAM family contains several members including JAM-A, JAM-B, JAM-C, A33 and the A34 protein. Among the JAM family members, JAM-A has the highest homology to JAM-B and JAM-C, about 35% amino acid sequence identity and 45% similarity to these two proteins. JAM-A protein is also known as JAM A, F11R, F11 receptor, JAM-1, JAM1, PAM-1 or CD 321.

Two subtypes of different length extracellular domains of JAM-a precursors were identified:

-subtype a: 299 amino acids (SEQ ID No.61)

-subtype b: 259 amino acids (SEQ ID No.63)

The nucleotide sequences of the two subtypes are represented by SEQ ID No.62 for subtype a and SEQ ID No.64 for subtype b.

The human cell surface expressed protein is a single polypeptide chain with an intracellular C-terminal domain, a single transmembrane domain (21 amino acids) and an N-terminal extracellular region containing two "Ig like" domains.

JAM-a has an N-glycosylation site, with an Asn residue at position 185 of subtype a and 145 of subtype b, and two disulfide bonds, one between Cys residues at positions 50 and 109 of the IgN-terminal domain, and one between Cys residues at positions 153 and 212 of the second Ig domain.

Crystallography confirmed the presence of two extracellular Ig-like domains (Kostrewa et al, 2001, EMBO J.16: 4391-4398; Prota et al, 2003, Proc. Natl. Acad. Sci. USA, 100: 5366-5371). The two domains are connected by a tripeptide linker (sequence VLV [127- & 129], subtype A). These structural studies also demonstrated the significance of JAM-a in the interaction between the cognate antigens, which comprise the extracellular region, on the cell surface; this region is produced recombinantly and is capable of forming homodimers in solution (Bazzoni et al, 2000, J.biol.chem.275: 30970-30976), and also makes it possible to identify the amino acids involved in this interaction: arg 59, Glu 61, Lys 63, Leu 72, Tyr 75, Met 110, Glu 114, Tyr 119, and Glu 121. The tripeptide RVE [59-61] is relatively conserved in the JAM family (RLE for JAM-B and RIE for JAM-C) and constitutes the minimal motif for homodimer formation (Kostrewa et al, 2001, EMBO J.16: 4391-4398).

On epidermal and endothelial cells, JAM-A was found mainly at tight junctions (Liu et al, 2000, J.CellSci., 113: 2363-. The cytoplasmic region contains at its C-terminal position a type II PDZ domain (sequence FLV [ 298-. Murine antibodies directed against the [ 111-.

JAM-A and integrin alphavbeA₃Interact with and participate in endothelial cells along vitronectin (integrin. alpha. v. beta.)₃Ligand of (3)) migration (Naik and Naik, 2005, j. cell sci.119: 490-499). anti-JAM-A antibody J3F.1 as anti- α v β₃The antibody inhibited endothelial cell migration and bFGF-induced angiogenesis in vitro in the same manner (Naik et al, 2003, Blood, 102: 2108-2114). Various signaling pathways within endothelial cells were demonstrated: MAP kinase, PI 3-kinase, and PKC (Naik et Naik, 2005, J.CelSci., 119: 490-499; Naik et al, 2003, Blood, 102: 2108-2114; Naik et al, 2003, Artherioscope, Thromb, Vase, biol, 23: 2165-2171).

Monocytes, lymphocytes, neutrophils, and platelets also express JAM-A (Williams et al, 1999, mol. Immunol, 36: 1175-1188). However, the JAM-A protein was originally identified as a receptor for the F11 antibody, and the F11 antibody was able to activate platelets and induce platelet aggregation (Naik et al, 1995, biochem. J., 310: 155-162; Sobocka et al, 2000, Blood, 95: 2600-2609). The peptides [28-60] and [97-109] belong to the epitope of the F11 antibody and are involved in the phenomena of platelet activation and aggregation and homodimerization (Babinska et al, 2002, Thromb. Haemost., 87: 712-721).

The rat antibody BV11, directed against murine JAM-A, inhibited the trans-endothelial cell migration of monocytes in vitro and in vivo (Del Maschio et al, 1999, J.exp.Med., 190: 1351-1356). Ostermann and colleagues thereof (2002, Nature Immunol., 3: 151-158) showed that JAM-A is alpha_Lβ₂Or ligands of LFA-1 (lymphocyte function-associated antigen 1) integrin, which are overexpressed by induction of certain chemochemokines during the development of anti-inflammatory responses and are required for leukocyte extravasation or migration to sites of inflammation. JAM-A, via a second Ig-like domain, contributes to the adhesion of T lymphocytes and neutrophils and to the migration across endothelial cells (Ostermann et al, 2002, Nature Immunol, 3: 151-158), and thus plays an important role in the homing of leukocytes to sites of inflammation.

The JAM-A protein is also involved in the phenomenon of viral infection. JAM-A is indeed a receptor for reovirus (reovirus), a virus that forms certain types of encephalitis by means of interaction with the adhesive protein sigma 1 (Barton et al, 2001, Cell 104: 441-. anti-JAM-A antibodies inhibit reovirus binding to JAM-A (Forrest et al, 2003, J.biol.chem., 278: 48434-48444).

To date, none of the above viruses directed against human-type JAM-a exhibit in vivo activity, with much lower antitumor activity. Such antibodies are only useful as research tools. Thus, there is a real lack of antibodies in the prior art that have anti-tumor activity both in vitro and in vivo.

According to a particular aspect, the antibody or derived compound or functional fragment thereof according to the invention is characterized in that it is capable of specifically binding to the JAM-a protein (according to the english name "Junctional addition Molecules").

According to another particular aspect, the antibody or derived compound or functional fragment thereof according to the invention is characterized in that it exhibits a K binding to JAM-A_DIs about 1nM to about 1 pM. More preferably, theK binding to JAM-A_DIs about 10pM to about 40 pM.

The term "K_D"refers to the dissociation constant of a given antibody-antigen complex. K_D＝K_off/K_onIn, K_offConsists of the "end rate" constant for dissociation of the antibody from the antibody-antigen complex, and K_onConsisting of the level of antibody binding to antigen (Chen Y. et al, 1999, J.mol.biol, 293: 865-881).

A novel aspect of the present invention relates to an isolated nucleic acid characterized in that it is selected from the following nucleic acids (including any degenerate genetic code):

a) nucleic acid, DNA or RNA encoding the antibody of the invention or a derivative compound or functional fragment thereof;

b) nucleic acids complementary to the nucleic acids defined in a);

c) a nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent conditions to at least one of the nucleic acid sequences SEQ ID nos. 20 to 31 or to at least one CDR of a sequence which, after optimal alignment, is at least 80%, preferably 85%, 90%, 95% and 98% identical to the sequence SEQ ID nos. 20 to 31; and

d) a nucleic acid of at least 18 nucleotides capable of hybridising, under high stringency conditions, to at least the light chain of nucleic acid sequence SEQ ID No.32 or 36 and/or the heavy chain of nucleic acid sequence SEQ ID No.33, 37 or 38 or to a heavy chain of sequence 32 or 36 and/or 33, 37 or 38 which is at least 80% identical after optimal alignment.

Table 3 below summarizes the various nucleotide sequences involved in the antibodies of the present invention.

TABLE 3

The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this specification to refer to the precise sequence of nucleotides (modified or unmodified), to define a segment or region of nucleic acid, which may or may not comprise non-natural nucleotides, and is double-stranded DNA, single-stranded DNA, or a transcript of such DNA.

It is also intended herein to include nucleotide sequences in which the present invention does not relate to the native chromosomal environment, i.e., in the native state. The sequences according to the invention have been isolated and/or purified, i.e.they have been sampled, directly or indirectly, for example by copying, the environment of which has been at least partially altered. Isolated nucleic acids obtained by recombinant genetics, e.g., of a host cell, or obtained by chemical synthesis, are also to be mentioned.

By "a nucleic acid sequence exhibits a percentage identity with the preferred sequence of at least 80%, preferably 85%, 90%, 95% and 98% after optimal alignment" is meant a nucleic acid sequence which exhibits certain modifications (such as in particular deletions, truncations, extensions, chimeric fusions and/or substitutions, in particular disruptions) relative to a reference nucleic acid sequence. Preferably, these sequences are sequences which encode the same amino acid as the reference sequence, are related to the degeneracy of the genetic code, or are complementary sequences which specifically hybridize (preferably under high stringency conditions) to the reference sequence, in particular the sequences defined below.

By "hybridizing under highly stringent conditions" is meant that conditions involving temperature and ionic strength are selected in such a way that hybridization between two complementary DNA fragments is maintained. On the basis of a pure explanation, the highly stringent conditions of the hybridization step for the purpose of defining the above-mentioned polynucleotide fragments are advantageously as follows.

DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42 ℃ for 3 hours in phosphate buffer containing 5 XSSC (1 XSSC corresponds to 0.15M NaCl +0.015M sodium citrate solution), 50% formamide, 7% Sodium Dodecyl Sulfate (SDS), 10 XDenhardt's, 5% dextran sulfate, and 1% salmon sperm DNA; (2) the initial hybridization is for 20 hours, the hybridization temperature depends on the probe length (for i.e. > 100 nucleic acid probe is 42 ℃), followed by 20 ℃ in 2 XSSC + 2% SDS washing two times, 20 minutes each, at 20 ℃ in 0.1 XSSC + 0.1% SDS washing one time 20 minutes. The last wash for probes > 100 nucleic acids in length was performed in 0.1 XSSC + 0.1% SDS at 60 ℃ for 30 minutes. The highly stringent hybridization conditions described above for polynucleotides of defined size can be adapted by those skilled in the art to longer or shorter oligonucleotides according to the protocol described by Sambrook et al (molecular: a Laboratory manual, Cold Spring Harbor Laboratory; 3rd edition, 2001).

The invention also relates to vectors comprising the nucleic acids according to the invention.

The object of the present invention is in particular to clone and/or express vectors comprising such nucleotide sequences.

The vectors of the present invention preferably comprise elements which allow the expression and/or secretion of the nucleotide sequence in a given host cell. Thus, the vector must contain a promoter, translation initiation and termination signals, and appropriate transcriptional regulatory regions. It must be able to persist in a stable manner in the host cell and optionally have specific signals for the specific secretion of the translated protein. These several elements can be selected and optimized by the skilled person depending on the host cell used. For this purpose, the nucleotide sequence may be inserted into a vector that replicates itself in the chosen host or into an integrating vector of the chosen host.

Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods (e.g., liposome, electroporation, heat shock, or chemical methods).

The vector is, for example, a plasmid or a virus-derived vector. These vectors are used to transform host cells to clone or express the nucleotide sequences of the present invention.

The invention also encompasses a host cell transformed with or comprising a vector of the invention.

The host cell is selected from prokaryotic or eukaryotic systems such as bacterial cells, for example, and also yeast cells or animal cells, in particular mammalian cells. Insect or plant cells may also be used.

The invention also relates to animals, not including humans, having transformed cells according to the invention.

Another aspect of the invention relates to a method for producing an antibody according to the invention or a functional fragment thereof, characterized in that said method comprises the following steps:

a) culturing the host cell of the invention in a culture medium under suitable culture conditions; and

b) recovering said antibody or a functional fragment thereof from the culture medium or from said cultured cells.

The transformed cells of the invention are used in a method for producing the recombinant polypeptide of the invention. The method for producing the recombinant form of the polypeptide of the present invention is characterized in that the method uses a vector and/or a cell transformed with the vector of the present invention, and the method is also included in the present invention. Preferably, the cells transformed with the vector of the present invention are cultured under conditions allowing the above-described polypeptide to be expressed and the recombinant peptide to be recovered.

As already mentioned, the host cell may be selected from prokaryotic or eukaryotic systems. In particular, it is possible to identify nucleotide sequences of the invention that promote secretion in such prokaryotic or eukaryotic systems. The vectors of the invention with such sequences can thus advantageously be used for the production of recombinant proteins to be secreted. Indeed, the purification of these recombinant proteins of interest may be facilitated by the fact that they are present in the supernatant of the cell culture broth rather than in the host cells.

The polypeptides of the invention may also be prepared by chemical synthesis. A process of this type is also an object of the present invention. Methods of chemical synthesis are known to the person skilled in the art, such as Solid phase techniques (see, in particular, Steward et al, 1984, Solid phase peptides synthesis, Pierce chem. company, Rockford, 111, 2nd ed.) or partial Solid phase synthesis techniques (by fragment condensation or conventional synthesis in solution). The invention also encompasses polypeptides obtained by chemical synthesis and capable of containing the corresponding unnatural amino acid.

The invention also comprises antibodies or derived compounds or functional fragments thereof, obtainable by the method according to the invention.

According to another aspect, the invention relates to the above-mentioned antibody, characterized in that it is additionally capable of specifically binding to a receptor of the human tyrosine kinase family and/or of specifically inhibiting the tyrosine kinase activity of such a receptor.

According to a new embodiment, the invention relates to an antibody or a derived compound or functional fragment thereof, consisting of an antibody having a bispecific nature in that it comprises a second motif capable of interacting with any receptor involved in tumor development (e.g. VEGFR, VEGF, EGFR, IGF-1R, HER2neu, HGF, cMET, FGF, transmembrane four superfamily (tetraspanins), integrins, CXCR4 or CXCR 2).

According to a first embodiment, one such antibody consists of a bispecific antibody and comprises a second motif which specifically inhibits the binding of EGF to the human Epidermal Growth Factor Receptor (EGFR) and/or which specifically inhibits the tyrosine kinase activity of said EGFR. According to another even more preferred aspect of the invention, the second anti-EGFR motif is derived from the monoclonal antibody cetuximab (C225 or erbitux), matuzumab, huR3, HuMax-EGFR or panitumab.

According to a second embodiment, the antibody of the invention consists of a bispecific antibody and comprises a second motif specifically inhibiting the HER2/neu receptor-modulated activity and/or specifically inhibiting the tyrosine kinase activity of the HER2/neu receptor. More particularly, the second anti-HER 2/neu motif is from the mouse monoclonal antibody 4D5 or 2C4 or from the humanized antibody trastuzumab or pertuzumab.

According to a third embodiment, the antibody of the invention consists of a bispecific antibody and comprises a second motif specifically inhibiting the binding of Hepatocyte Growth Factor (HGF) to the cMET receptor and/or specifically inhibiting the tyrosine kinase activity of the cMET receptor.

According to a fourth embodiment, the antibody of the invention consists of a bispecific antibody and comprises a second motif that specifically inhibits the IGF-1R receptor-mediated activity and/or that specifically inhibits the tyrosine kinase activity of said IGF-IR receptor. More particularly, the second anti-IGF-1R motif is from the mouse monoclonal antibody 7C10, from the corresponding humanized antibody H7C10(Goetsch et al, International patent application WO 03/059951), from the hEM164 antibody (Maloney et al, Cancer Res., 2003, 63 (16): 5073-5083), from the anti-IGF-1R antibody developed by Abgenix (see U.S. patent application 2005/281812) or from Mab39, 1H7(Li et al, Cancer Immunol. Immunotherer., 2000, 49 (4-5): 243-252) or 4G11(Jackson-Booth et al, Horm. Metab. Res., 2003, 35 (11-12): 850-856).

Finally, according to a final embodiment, the antibody according to the invention consists of a bispecific antibody and comprises a second motif capable of interacting with any type of receptor involved in tumor development, such as, by way of non-limiting example, VEGFR, VEGF, FGF (fibroblast growth factor) or any member of the CXCR (chemokine receptor) family, such as CXCR2 or CXCR 4.

Also mentioned as suitable are anti-CD 20 antibodies, such as rituximab, ibritumomab or tositumomab; anti-CD 33 antibodies such as gemtuzumab or lintuzumab; anti-CD 22 antibodies, such as epratuzumab; anti-CD 52 antibodies, such as alemtuzumab; anti-EpCAM antibodies such as edrecolomab, Ch17-1A, or IGN-101; anti-CTP 21 or 16 antibodies, such as Xactin; anti-DNA-Ag antibodies, e.g.¹³¹I-Cotara TNT-1; anti-MUC 1 antibodies, such as pemtumumab or R1150; anti-MUC 18 antibodies, such as ABX-MA 1; anti-GD 3 antibodies, such as mitumomab; anti-ECA antibodies, such as CeaVac or labtuzumab; anti-CA 125 antibodies, such as OvaRex;anti-HLA-DR antibodies such as apolizumab; anti-CTLA 4 antibodies, such as MDX-010; anti-PSMA antibodies, e.g., MDX-070,¹¹¹In&⁹⁰Y-J591、¹⁷⁷Lu J591, J591-DM 1; anti-lewis y antibodies, such as IGN 311; anti-angiogenic antibodies, such AS1405 and 90YmuBC 1; an anti-Trail-R1 antibody, such as Trail R1mAb or Trail R2 mAb.

Bispecific or bifunctional antibodies constitute second generation monoclonal antibodies in which two different variable regions are combined in the same molecule (Hollinger and Bohlen, 1999, Cancer and metastasis, rev. 18: 411-419). The usefulness in this respect has been demonstrated due to the use of several molecules capable of increasing new effector functions or capable of targeting the surface of tumor cells in the diagnostic and therapeutic fields. Such antibodies can be obtained by chemical methods (Glennie MJ et al, 1987, J.Immunol.139, 2367-2375; Repp R. et al, 1995, J.Hemat., 377-382) or by somatic methods (Staerz U.D. and Bevan M.J., 1986, PNAS 83, 1453-1457; Suresh M.R. et al, 1986, Method Enzymol, 121: 210-228), but can also preferably be obtained by genetic engineering techniques which make heterodimerization possible and thus facilitate the purification of the antibody of interest (Merchand et al, 1998, Nature Biotech., 16: 677-681).

These bispecific antibodies can be constructed with the structure of intact IgG, bispecific Fab '2, Fab' PEG, diabodies or bispecific scFv, but also as tetravalent bispecific antibodies, where two binding sites are present for each target antigen (Park et al, 2000, MoL. Immunol, 37 (18): 1123-30) or fragments of the above items.

In addition to the specific economic advantages, the production and use of bispecific antibodies is more economical than the production of two specific antibodies, and the use of such bispecific antibodies also has the advantage of reducing therapeutic toxicity. Indeed, it is possible to reduce the total amount of circulating antibody and the consequent potential toxicity using bispecific antibodies.

In a preferred embodiment of the invention, the bispecific antibody is a bivalent or tetravalent antibody.

Finally, the invention relates to the use of the above-mentioned antibody or a derivative compound or functional fragment thereof as a medicament.

The invention also relates to pharmaceutical compositions comprising as active ingredient a compound consisting of the antibody of the invention or one of its derivative compounds or functional fragments. Preferably, the antibody is supplemented with an excipient and/or a pharmaceutically acceptable carrier.

According to another preferred embodiment, the present invention also relates to the above pharmaceutical composition comprising at least one second anti-neoplastic compound selected from compounds capable of specifically inhibiting the tyrosine kinase activity of a receptor (such as IGF-IR, EGFR, HER2/neu, cMET, VEGFR or VEGF or any other anti-neoplastic compound known to the person skilled in the art). In a second preferred aspect of the invention, said second compound may be selected from the group consisting of anti-EGFR, anti-IGF-IR, anti-HER 2/neu, anti-cMET, VEGFR, VEGF, etc. antibodies, isolated antibodies or functional fragments and derivative compounds thereof, capable of inhibiting the proliferation and/or anti-apoptotic and/or angiogenic and/or inductive activity of said receptor-initiated metastatic spread.

According to another preferred embodiment of the invention, the composition further comprises at least one inhibitor of tyrosine kinase activity of receptors such as IGF-IR, EGFR, HER2/neu, cMET and VEGFR, for use as a combined product in a simultaneous, separate or extended (extended) manner.

In another preferred embodiment, the inhibitor of tyrosine kinase activity of these receptors is selected from the group consisting of the derivatized natural agents dianilinophthalimide, pyrazole-or pyrrolo-pyridopyrimidine or quinazoline. Such inhibitors, which are well known to the person skilled in the art, are described in the literature (Ciardiello F., Drugs 2000, supply.1, 25-32).

Another complementary embodiment of the invention consists of the above composition, further comprising a cytotoxic/cytostatic agent as a combined product for simultaneous, separate or prolonged use.

By "simultaneous use" is meant the use of two compounds contained in a composition in a single dosage form.

"separately used" refers to the simultaneous use of two compounds in a composition contained in different dosage forms.

By "prolonged use" is meant the sequential use of two compounds in a composition contained in different dosage forms.

In general, the compositions of the present invention significantly improve the effectiveness of cancer therapy. In other words, the therapeutic effect of the antibodies of the invention is enhanced in an unexpected manner by the use of cytotoxic agents. Another major subsequent advantage produced by the compositions of the present invention may be related to the use of lower effective doses of the active ingredient, thus making it possible to avoid or reduce the risk of side effects, in particular the effects of cytotoxic agents. Moreover, the composition may achieve the desired therapeutic effect more quickly.

"therapeutic anti-cancer agent" or "cytotoxic agent" refers to a substance that, when administered to a patient, treats or prevents the development of cancer in the patient. Non-limiting examples of such agents include "alkylating" agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogenic agents, antiandrogenic agents, and immunomodulators.

For example, in VIDAL, reference is made to such agents under the "cytotoxic" topic on the page of oncologically and hematology related compounds; the cytotoxic compounds cited by reference in this document are cited herein as preferred cytotoxic agents.

"alkylating agent" refers to a substance that can be covalently bound to any molecule (preferably a nucleic acid, such as DNA) within a cell or that is capable of alkylating such molecules. Examples of such alkylating agents include nitrogen mustards such as mechlorethamine (mechlorethamine), chlorambucil, melphalan, mechlorethamine hydrochloride, pipobroman (pipobroman), prednimustine (prednimustine), disodium phosphate or estramustine phosphate (estramustine); oxazaphosphorines, such as cyclophosphamide, hexamethylmelamine, trofosfamide, sulpho-phosphoramide or ifosfamide; aziridines or aziridines, such as thiotepa, triethylenediamine or altetramine; nitroso ureas such as nitrosourea mustard, streptozocine, fotemustine or chlorocyclohexenetustine; alkyl sulfonates such as busulfan, treosulfan or improsulfan; triazenes, such as dacarbazine (dacarbazine); or platinum complexes such as cisplatin, oxliplatin (oxaliplatin) or carboplatin.

By "antimetabolite" is meant an agent that blocks growth and/or cellular metabolism by interfering with certain activities, typically DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluorouracil, floxuridine, 5-fluorodeoxyuracil, capecitabine (capecitabine), cytarabine, fludarabine (fludarabine), cytosine arabinoside, 6-mercaptopurine (6-MP), 6-mercaptoguanine (6-TG), chlorodeoxyadenosine, 5-azacytidine, gemcitabine (gemcitabine), cladribine (cladribine), deoxycofomycin (deoxyfumycin), and pentostatin (pentostatin).

"antitumor antibiotic" refers to a compound that prevents or inhibits DNA, RNA, and/or protein synthesis. Examples of such antitumor antibiotics include doxorubicin (doxorubicin), daunorubicin (daunorubicin), idarubicin (idarubicin), valrubicin (valrubicin), mitoxantrone (mitoxantrone), actinomycin (dactinomycin), mithramycin (mithramycin), plicamycin (plicamycin), mitomycin C, bleomycin (bleomycin), and procarbazine (procarbazine).

"mitotic inhibitors" prevent the normal progression of the cell cycle and mitosis. In general, microtubule inhibitors or "taxanes" (such as paclitaxel and docetaxel) are capable of inhibiting mitosis. Vinca alkaloids such as vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), and vinorelbine (vinorelbine) also inhibit mitosis.

"chromatin suppressors" or "topoisomerase inhibitors" are substances that inhibit the normal function of chromatin-forming proteins (e.g., topoisomerase I and II). Examples of such inhibitors include camptothecin (camptothecine) and its derivatives for topoisomerase I, such as irinotecan (irinotecan) or topotecan (topotecan); for topoisomerase II there are etoposide, etoposide phosphate and teniposide.

An "anti-angiogenic agent" is any drug, compound, substance or agent that inhibits the growth of blood vessels. Examples of anti-angiogenic agents include, but are not limited to, ranizoxan (razoxin), marimastat (marimastat), batimastat (batimastat), prinomastat (prinomastat), tanostat (tanomastat), ilomastat (ilomastat), CGS-27023A, halofuginone (halofuginone), COL-3, neovastat (neovastat), BMS-275291, thalidomide, CDC 501, DMXAA, L-651582, squalamine (squalamine), endostatin, SU5416, SU6668, interferon- α, EMD121974, interleukin-12, IM862, angiostatin (angiostatin), and vivaxin.

By "antiestrogen" or "estrogen antagonist" is meant any substance that reduces, antagonizes, or inhibits the activity of estrogen. Examples of such agents are tamoxifen (tamoxifene), toremifene (toremifene), raloxifene (raloxifene), droloxifene (droloxifene), iodoxyfene, anastrozole (anastrozole), letrozole (letrozole) and exemestane (exemestane).

By "antiandrogen" or "androgen antagonist" is meant any substance that reduces, antagonizes, or inhibits the activity of androgen. Examples of antiandrogens include flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride and cimetidine.

Immunomodulators are substances that stimulate the immune system. Examples of immunomodulators include interferons, interleukins (such as aldesleukin, OCT-43, denileukin diftotox or interleukin-2), tumor necrosis factors (such as tasonermine) or other types of immunomodulators, such as lentinan, azopyran (sizofian), roquinacre (roquinimex), pidotimod (pidotimod), pegamase (pegademase), thymopentine, poly I: c or levamisole is used in combination with 5-fluorouracil.

For further details, one skilled in the art can refer to the handbook entitled Therapeutic Chemistry, vol.6, antitumoral and perspectives in the treatment of cancer, TEC and DOC edition, 2003[ in French ] "published by the French Association of Therapeutic Chemistry Teachers.

In a particularly preferred embodiment, the composition of the invention as a combined product is characterized in that the cytotoxic agent is chemically bound to the antibody for simultaneous use.

In a particularly preferred embodiment, said composition is characterized in that said cytotoxic/cytostatic agent is chosen from spindle inhibitors or stabilizers, preferably vinorelbine and/or vinflunine and/or vincristine.

To facilitate the binding between the cytotoxic agent and the antibody of the invention, a spacer molecule, such as a polyethylene glycol, or an amino acid, may be introduced between the two compounds bound; alternatively, in another embodiment, active derivatives of the cytotoxic agent into which a function capable of reacting with the antibody has been introduced may be used. These bonding techniques are known to those skilled in the art and details thereof will not be discussed in detail herein.

Other EGFR inhibitors include, without limitation, monoclonal antibody C225 and anti-EGFR 22mab (Imclone Systems incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200(Merck KgaA) or compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166(Novartis), PKI-166/CGP-75166(Novartis), PTK787(Novartis), CP701(Cephalon), flunomide (Pharmacia/Sugen), CI-1033(Warner Lambert Davis), CI-CI 3/103183, Warner Lambert Davis, CL-387, 785(Wyeth-Ayerst), BBR-1611 (Manehringer BH/GMbh), Brirner Lambert-Davis), Bcrystal-EGF (Szergerx-3), and Bgehrink-Blok-33 (Szergerk-Marc), and Bgehrzerich-EGF (Szerich-II), and the like fusion protein B-103 (Szerich-R-103, Szerich protein-Szerich protein A2, and the like, DAB-389(Seragen/Lilgand), ZM-252808(Imperial Cancer), RG-50864(INSERM), LFM-A12(Parker Hughes Center), WHI-P97(Parker Hughes Center), GW-282974(Glaxo), KT-8391(Kyowa Hakko), or "EGFR vaccine" (York Medical/Central of immunology Molecular).

Another aspect of the invention relates to a composition characterized in that at least one of said antibodies or derived compounds or functional fragments thereof is conjugated to a cytotoxin and/or a radioisotope.

Preferably, said toxin or said radioisotope is capable of preventing the growth or proliferation of a tumor cell, in particular of completely inactivating said tumor cell.

Also preferred, the toxin is an intestinal bacterial toxin, in particular Pseudomonas exotoxin a.

Preferred radioisotopes for use in combination with therapeutic antibodies are gamma-emitting isotopes, preferably iodine¹³¹Yttrium, yttrium⁹⁰Gold, gold¹⁹⁹Palladium, palladium¹⁰⁰Copper, copper⁶⁷Bismuth, bismuth²¹⁷And antimony²¹¹. Isotopes that emit alpha and beta radiation may also be used therapeutically.

"toxin or radioisotope used in combination with at least one antibody or functional fragment thereof according to the invention" means any method that makes it possible to bind said toxin or said radioisotope to at least one antibody, in particular to covalently bind (with or without the introduction of a binding molecule) two compounds.

Examples of reagents that form chemical (covalent), electrostatic, or non-covalent bonds between all or part of the binding members include, inter alia, benzoquinone, carbodiimide, and more particularly EDC (1-ethyl-3- [ 3-dimethyl-aminopropyl ] -chlorocarbodiimide), bismaleimide (dimaleimide), dithiobis-nitrobenzyl (DTNB) acid, N-succinimidyl S-acetylthioacetate (SATA), bridging reagents having one or more groups, bridging reagents having one or more pendant phenyl (phenylside) groups, reagents that react with Ultraviolet (UV), most preferably N- [ -4 (azidosalicylamido) butyl ] -3 '- (2' -dithiopyridyl) -propionamide (APDP), N-succinimidyl-3 (2-dithiopyridyl) propionate (SPDP), and 6 -hydrazino-nicotinamide (HYNIC).

In particular for radioisotopes, another form of conjugation may consist of a bifunctional ion chelating agent.

Examples of such chelating agents include EDTA (ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaacetic acid) derived chelating agents that have been developed to bind metals, particularly radioactive metals, to immunoglobulins. Thus, DTPA and its derivatives can be substituted with various groups on the carbon chain to increase the stability and rigidity of the ligand-metal complex (Krejcarek et al, 1977; Brechbiel et al, 1991; Gansow, 1991; U.S. Pat. No.4,831,175).

For example, DTPA (diethylenetriaminepentaacetic acid) and its derivatives, in their free form or in their complexed with metal ions, have been widely used for a long time in the fields of medicine and biology, showing the remarkable feature of forming stable chelators with metal ions, which can be coupled with proteins, such as antibodies, for therapeutic or diagnostic purposes, in order to develop radio-immune cross-linking agents for cancer therapy (Meases et al, 1984; Gansow et al, 1990).

Also preferably, said at least one antibody according to the invention forming said cross-linking agent is selected from functional fragments thereof, in particular fragments missing its Fc component, such as scFv fragments.

The invention also comprises the use of the composition for the manufacture of a medicament intended for the prevention or treatment of cancer.

The invention also relates to the use of an antibody or a derivative compound or a functional fragment thereof (preferably a humanized antibody) and/or a composition according to the invention for the manufacture of a medicament for inhibiting the growth of tumor cells. In general, the present invention relates to the use of an antibody or a derivative compound or a functional fragment thereof (preferably a humanized antibody) and/or a composition for the manufacture of a medicament for the prevention or treatment of cancer.

Preferably, the cancer that can be prevented and/or treated comprises prostate cancer, osteosarcoma, lung cancer, breast cancer, endometrial cancer, colon cancer, multiple myeloma, ovarian cancer, pancreatic cancer or any other cancer.

In a preferred manner, the cancer is selected from estrogen-related breast cancer, non-small cell lung cancer, colon cancer and/or pancreatic cancer.

Another aspect of the invention relates to the use of said antibodies in a method for the diagnosis, preferably in vitro, of a disease associated with the expression level of JAM-A. Preferably, in the diagnostic method, the JAM-a protein-related disease is cancer.

The antibody or derivative compound or functional fragment thereof according to the invention can therefore be used in a method for the in vitro detection and/or quantification of the JAM-a protein in a biological sample, in particular for the diagnosis of diseases associated with an abnormal expression of this protein (such as cancer), wherein said method comprises the following steps:

a) contacting a biological sample with an antibody or derivative compound or functional fragment thereof according to the present invention;

b) confirming the possible formation of antigen-antibody complexes.

Therefore, the invention also comprises a kit or a kit for carrying out said method (said method being a method for detecting gene expression of Legionella pneumophila (Legionella pneumophila Paris) or a related organism, or a method for detecting and/or identifying a bacterium of Legionella pneumophila or a related microorganism) comprising the following components:

a) a polyclonal antibody or a monoclonal antibody according to the present invention;

b) optionally, an agent that constitutes a mediator beneficial for the development of an immune response;

c) alternatively, an agent that displays an antigen-antibody complex generated by an immune reaction.

Preferably, the antibody or functional fragment thereof may be immobilized on a support, in particular on a protein chip. One such protein chip is an object of the present invention.

Preferably, the protein chip can be used in a kit or kit of parts required for detecting and/or quantifying the JAM-a protein in a biological sample.

It must be noted that the term "biological sample" as used herein relates to a sample taken from a living organism (in particular blood, tissue, organ or other sample taken from a mammal, in particular from a human) or any sample (such as a cell sample, if desired a transformed cell sample) which may contain one such JAM-A protein.

The antibody or functional fragment thereof may be in the form of an immuno-cross-linker or in the form of a labeled antibody to obtain a detectable and/or quantifiable signal.

The labeled antibody or a function or fragment thereof according to the present invention includes, for example, an antibody cross-linking agent (immune cross-linking agent) may be bound to, for example, enzymes such as peroxidase, alkaline phosphatase, α -D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and glucose-6 phosphate dehydrogenase, or may be bound to molecules such as biotin, digoxin or 5-bromodeoxyuridine. Fluorescent labels may also be conjugated to the antibodies or functional fragments thereof of the present invention, including, inter alia, fluorescein and its derivatives, fluorochrome, rhodamine (rhodamine) and its derivatives, Green Fluorescent Protein (GFP), dansyl, umbelliferone, and the like. In such a cross-linking agent, the antibody or functional fragment thereof according to the present invention is also prepared by a method known to those skilled in the art. They can be directly bound to an enzyme or fluorescent label; bound through a spacer or linker group, such as polyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DPTA); or in the presence of a binding agent, such as those mentioned above for the therapeutic cross-linking agent. The cross-linking agent with the fluorescent marker can be prepared by reacting with isothiocyanate.

Other cross-linking agents also include chemiluminescent labels, such as luminol and diepoxide (dioxetane), bioluminescent labels, such as luciferase and luciferin, or radioactive labels, such as iodine¹²³Iodine, iodine¹²⁵Iodine, iodine¹²⁶Iodine, iodine¹³³Bromine, bromine⁷⁷Technetium, technetium^99mIndium, indium¹¹¹Indium, indium^113mGallium, gallium⁶⁷Gallium, gallium⁶⁸Ruthenium (II) and (III)⁹⁵Ruthenium (II) and (III)⁹⁷Ruthenium (II) and (III)¹⁰³Ruthenium (II) and (III)¹⁰⁵Mercury, mercury¹⁰⁷Mercury, mercury²⁰³Rhenium^99mRhenium¹⁰¹Rhenium¹⁰⁵Scandium (III)⁴⁷Tellurium^121mTellurium^122mTellurium^125mThulium, thulium¹⁶⁵Thulium, thulium¹⁶⁷Thulium, thulium¹⁶⁸Fluorine¹⁸Yttrium, yttrium¹⁹⁹And iodine¹³¹. The existing methods of binding radioisotopes to antibodies, either directly or via chelating agents such as the above-mentioned EDTA or DTPA, known to those skilled in the art, can be used as diagnostic radioisotopes. It should therefore be mentioned that the technique of chloramine (chloramine) -T is followed by [ iodine ]¹²⁵]Methods of sodium labeling (Hunter W.M. and Greenwood F.C. (1962) Nature 194: 495); technetium as described in Crockford et al^99mLabeling (U.S. Pat. No.4,424,200) or conjugation via DTPA as described by Hnatowich (U.S. Pat. No.4,479,930).

The invention also relates to the use of the antibody according to the invention for producing a medicament for specifically targeting a biologically active compound to cells expressing or overexpressing the JAM-A protein.

In the sense of the present specification, a "biologically active compound" is any compound capable of modulating (in particular inhibiting) cellular activity, in particular growth activity, proliferation activity, transcription and gene translation activity.

The invention also relates to an in vivo diagnostic reagent consisting of the antibody or a functional fragment thereof, preferably a labeled antibody or a functional fragment thereof, particularly a radiolabeled antibody or a functional fragment thereof, and the use thereof in medical imaging, particularly in the detection of cancers associated with the expression or overexpression of JAM-A protein by cells.

The invention also relates to a composition as a combination product or to an anti-JAM-a/toxin cross-linker or radioisotope as described herein for use as a medicament.

Preferably, supplementary excipients and/or pharmaceutical carriers are added to the composition as a combined product or to the cross-linking agent.

In the present specification, "pharmaceutical carrier" refers to a compound or combination of compounds which is included in a pharmaceutical composition, does not cause secondary reactions, and, for example, facilitates the use of the active compound, prolongs its lifespan and/or effectiveness in the body, enhances its solubility in solution, or improves its storage. Such pharmaceutical carriers are well known to those skilled in the art and may be adapted according to the nature of the active compound selected and the route of use.

Preferably, such compounds are administered by the systemic route, in particular by the intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or oral route. More preferably, the composition consisting of the antibody according to the invention is used in several doses equally spaced in time.

The route of use, the dosage schedule and the optimal galenic form of the composition are determined according to the conditions generally considered in the formulation of a treatment suitable for the patient, for example, the age or weight of the patient, the severity of his general state, his tolerance to the treatment and the side effects experienced.

The invention therefore relates to the use of an antibody or one of its functional fragments for the preparation of a medicament for specifically targeting a compound with biological activity to cells expressing or overexpressing JAM-a.

Other features and advantages of the invention are further described in the embodiments of the description and in the accompanying drawings (hereinafter the figure description).

Drawings

FIG. 1 shows the murine 6F4 antibody heavy and light chain sequences, respectively. Underlined and bolded parts are CDRs (numbering according to Kabat).

FIGS. 2A and 2B represent the alignment of the V-region (FIG. 2A) and J-region (FIG. 2B) of murine 6F4 antibody, respectively, to the selected murine cell line, i.e., IGKV19-93 x 01 for the V-region (SEQ ID No.39) and IGKJ 1x 01 for the J-region (SEQ ID No. 40).

FIGS. 3A and 3B represent the alignment of the V-region (FIG. 3A) and J-region (FIG. 3B) of the murine 6F4 antibody, respectively, with the selected human cell line, i.e., IGKV1-33 x 01 for the V-region (SEQ ID No.41) and IGKJ 1x 01 for the J-region (SEQ ID No. 42).

Figure 4 represents the protein sequence of the 6F4 antibody light chain with reference to the KABAT and IMGT coding systems, respectively.

Fig. 5A, 5B and 5C represent the alignment of the V-region (fig. 5A), D-region (fig. 5B) and J-region (fig. 5C) of murine 6F4 antibody, respectively, with selected murine cell lines, i.e. IGHV1S 135A 01 for the V-region (SEQ ID No.43), IgHD-ST4 a 01 for the D-region (SEQ ID No.44) and IgHJ 2a 01 for the J-region (SEQ ID No. 45).

FIGS. 6A, 6B and 6C represent the alignment of the V-region (FIG. 6A), D-region (FIG. 6B) and J-region (FIG. 6C) of murine 6F4 antibody, respectively, with the selected human cell line, i.e., IGHV 1-F01 for the V-region (SEQ ID No.46), IGHD 1-1.01 for the D-region (SEQ ID No.47) and IGHJ 4.01 for the J-region (SEQ ID No. 48).

Figure 7 represents the protein sequence of the heavy chain of antibody 6F4 with reference to the KABAT and IMGT coding systems, respectively.

FIGS. 8A and 8B represent 6F 4-Sepharose immunopurification of the 6F4 antigen from HT-29 cell membranes. Analysis of the collected fractions by SDS-PAGE electrophoresis (FIG. 8A) and western hybridization (FIG. 8B) is also shown.

FIGS. 9A and 9B show the analysis of the immunopurified protein by SDS-PAGE electrophoresis (FIG. 9A) and western hybridization (FIG. 9B). Two purifications (#1 and #2) were performed and analyzed under reducing or non-reducing conditions.

FIG. 10 shows MALDI-TOF mass spectrometry analysis of the extracted peptide mixture after trypsin hydrolysis.

FIGS. 11A and 11B confirmation of the proteins identified by western hybridization (under non-reducing conditions): revealing with 6F4 antibody (FIG. 11A) and anti-human JAM-A polyclonal antibody (FIG. 11B).

FIG. 12 shows the specificity of the 6F4 antibody for human JAM-A protein. The amounts used for each protein were 250ng, 25ng and 10 ng.

FIG. 13 represents the sensorgrams obtained after injection (double arrow) of 100nm of 6F4 antibody in HBS-EP buffer onto murine JAM1Fc protein (flow cell #1, bottom curve) and murine JAM1Fc protein (flow cell #2, top curve) for 2 minutes, at 25 ℃ for 5 minutes of dissociation time and at a flow rate of 30. mu.l/min (CM 4: m-JAM1-Fc 501.6RU (Fc1) and 511.5RU (Fc 2)).

FIG. 14 represents a sensorgram obtained using dual reference, (Fc2-Fc1)6F4(Fc2-Fc1) HBS-EP. Curves were fitted using a langmuir a + B binding model. The kinetic parameters calculated were as follows: ka ═ 10 (1.38 ± 0.001) ×⁵M^-1s^-1；kd＝(0.25±1.58)*10^-6s^-1(ii) a Rmax (global fit) 371 RU; kappa²＝0.853。

FIG. 15 shows the anti-tumor activity of the 6F4 antibody in a Swiss nude mouse MCF-7 cell xenograft model. The 6F4 antibody was detected twice a week by the IP route, in unpurified form (peritoneal cavity fluid), at a theoretical dose of 250. mu.g/mouse. The 9G4 antibody was of the same isotype (IgG1) and was not related to the activity measured.

FIG. 16 shows JAM-A protein expression recognized by Mab 6F4 on the surface of various tumor cell lines.

Figure 17 represents a humanized 6F4VL domain sequence wherein: bands are those corresponding to amino acids that are actually altered compared to the human counterpart, band 1 corresponds to the amino acid whose ability to be humanized was analyzed, the human residues are noted with a label thereunder, and band 2 corresponds to the murine amino acid remaining in the humanized 6F4VL domain.

Figure 18 represents a humanized 6F4 VH domain sequence in which: bands are those corresponding to amino acids that are actually altered compared to the human counterpart, band 1 corresponds to the amino acids whose ability to be humanized was analyzed, the human residues are noted with a label thereunder, and band 2 corresponds to murine amino acids that remain in the humanized 6F4 VH domain.

FIG. 19 shows JAM-A downregulation in vitro induced by 6F4 MAb.

Fig. 20 shows inhibition of tumor cell proliferation in vivo induced by 6F4 MAb.

FIG. 21 represents JAM-A in vivo downregulation induced by 6F4 Mab.

FIG. 22 represents in vivo models, 6F4 and its F (ab')₂Graph comparing the effect of fragments on MCF-7.

FIG. 23 shows a comparison of JAM-A expression in thyroid normal tissues and tumor tissues.

FIG. 24 shows a comparison of JAM-A expression in lung normal tissues and tumor tissues.

FIG. 25 shows a comparison of JAM-A expression in normal breast tissue and in tumor tissue.

FIG. 26 represents a graph showing the in vivo activity of 6F4 against nude mouse A431 epidermoid carcinoma xenografts.

Fig. 27 shows a: the effect of the 6F4 antibody on PHA-induced non-specific lymphocyte proliferation; b: effect of the 6F4 antibody on antigen presentation process. The first experiment had 2 independent donors.

Fig. 28 shows a: the effect of the 6F4 antibody on PHA-induced non-specific lymphocyte proliferation; b: effect of the 6F4 antibody on antigen presentation process. The second experiment had 2 independent donors.

Fig. 29 shows platelet aggregation of 10 normal human donors. The results are expressed as mean +/-sd.

FIG. 30 represents the serum toxin release from 10 normal donors. The results are expressed as mean +/-sd.

Figure 31 represents the alignment between the 6F4 VH domain and the IGHV1-03 × 01 germline gene (SEQ ID No. 49).

Examples

Example 1: production of 6F4 antibody

For the production of murine monoclonal antibodies (Mab), 5x10 from ATCC was used⁶MCF-7 cells were immunized against BALB/C mice. Final booster injection 10⁷After MCF-7 cells, cells from the lymph nodes of mice were fused with Sp2/O-Ag14 myeloma cells using the classical technique described by Kohler and Milstein. The supernatants of the fused hybridomas were then screened for functional activity, i.e., their activity in inhibiting MCF-7 cell proliferation in vitro.

For this screening, MCF-7 cells were cultured in 96-well plates at a density of 5 × 10 in 100 μ l per well of hybridoma culture medium without fetal bovine serum³And (4) cells. Plates were incubated in 5% CO₂Was incubated at 37 ℃ for 24 hours in air. After 24 hours, 50. mu.l of hybridoma supernatant to be screened are added to each well. The last row of the plate was left as control:

three wells were supplemented with 50 μ l hybridoma supernatant not related to the activity of interest (which was cultured in the same medium as used for the fusion cells). These cells will be used to correct for the effect of inactive supernatants on tritiated deoxythymidine;

add 50 μ l hybridoma medium to three wells.

After approximately 52 hours of incubation, 0.25. mu. Ci per well was added³H deoxythymidine, and incubated at 37 ℃ for a further 20 hours.³Introduction of H-deoxythymidine into DNA is indicative of cell proliferation which can be quantified by measuring liquid scintillation counting. Background noise and threshold values were determined for each plate functioning as a control well (containing culture medium only and irrelevant hybridoma supernatant).

By this method, 43 hybridomas secreting antibodies that inhibit the growth of MCF-7 cells were selected after the first screen. 11 of these 43 hybridomas were poorly or non-viable and discarded. In proliferation assays performed after expansion and cloning of hybridomas, only hybridomas whose supernatants have an inhibitory activity of 20% or more on MCF-7 cell proliferation were selected. At the end of the cloning/selection process, only one clone was confirmed to have the desired properties, clone 6F 4.

Example 2: humanization procedure by CDR-grafting of the light chain variable region (6F4VL) of the 6F4 antibody

a) Comparison between 6F4VL nucleotide sequence and all known murine cell line sequences

As a preliminary step in humanization of CDR-grafts, the 6F4VL nucleotide sequence was first compared to all murine cell line sequences present in the IMGT database (website http:// IMGT. cines. fr).

The V and J regions of murine cell lines having 98.56% sequence identity of the V region and 100% sequence identity of the J region were identified as IGKV19-93 x 01(SEQ ID No.39, EMBL nomenclature: AJ235935) and IGKJl 01(SEQ ID No.40, EMBL nomenclature: V00777), respectively.

Taking these percentages of identity into account, it was decided to use the 6F4VL sequence directly.

FIG. 2A represents an alignment of V regions and FIG. 2B represents an alignment of J regions.

b) Comparison of the 6F4VL nucleotide sequence with all known human cell line sequences

To identify the best human candidate for CDR-grafting, the germline of human origin with the greatest possible identity to 6F4VL was sought. For this purpose, the mouse 6F4VL nucleotide sequence was compared to all human sequences present in the IMGT database.

The V and J regions of the human cell line were identified as having a V region sequence identity of 81.36%, i.e., IGKV1-33 x 01(SEQ ID No.41, EMBL nomenclature: M64856), and a J region sequence identity of 86.84%, i.e., IGKJ 1x 01(SEQ ID No.42, EMBL nomenclature: J00242).

Thus, cell line IGKV1-33 x 01 was selected for sector V and cell line IGKJ 1x 01 was selected for sector J as the human acceptor sequence for the mouse 6F4VL CDR.

FIG. 3A shows the alignment of V region and FIG. 3B shows the alignment of J region.

c) Humanized versions of 6F4VL

The humanization process consisted of the following steps: IGKV1-33 x 01 was serially linked to IGKJ 1x 01 cell line sequences, and then the mouse 6F4VL CDRs were attached to the scaffold region of the isoline antibody.

Molecular modeling of the murine 6F4 Fv region during this stage of the humanization process is particularly useful for selecting from the murine residues to be retained, as they play an important role in maintaining the three-dimensional structure of the molecule (CDRs, canonical structure of VH/VL interface, etc.) or in binding antigen. In the scaffold region, every difference between mouse (6F4VL) and human (IGKV1-33 x 01/IGKJ 1x 01) nucleic acids was examined very carefully.

For greater clarity below, FIG. 4 represents the 6F4VL sequence with reference to the KABAT and IMGT classifications.

3 murine residues were identified that had to be retained.

According to IMGT, residue 33(Ile) is involved in CDR1 anchoring, which is part of CDR1 according to Kabat.

According to IMGT, residue 49(His) is involved in CDR2 anchoring, in constituting the VH/VL interface and belonging to the Vernier zone.

Residue 53(Thr) participates in CDR2 anchoring according to IMGT, which is part of CDR2 according to Kabat.

Three changes in the scaffold regions of IGKV1-33 x 01 and IGKJ 1x 01 will be initially studied. These alterations involve residues 24, 69 and 71(IMGT nomenclature). It will be appreciated that, of course, these three changes are studied independently of one another and may also form various combinations. The goal was to have available all possible mutants to test these and select those that retained the best binding properties. Each mutant was therefore subjected to an ELISA/Biacore binding assay.

Residue 24(Lys/Gln) is close to CDR1 and is therefore important for maintaining the configuration that enables correct presentation of CDR 1. More particularly, the residue may interact with residues 69-70 in the Vernier zone. Lys is the only less important in the human VL, but it is part of the CDR1 according to Kabat.

Although residue 69(Arg/Thr) is within the Vernier zone and thus directly involved in the canonical structure of CDR1, this residue is always Thr in human VL.

Although residue 71(Tyr/Phe) is directly involved in the canonical structure of CDR1, this residue is always Phe in the human VL.

Second, modification of residue 56(Ala) to Thr is contemplated. The residues, although outside the CDRs, according to IMGT also belong to CDR2 according to Kabat.

Third and finally, residues 34 and 55(IMGT nomenclature) can be subject to two additional changes. These two residues, outside the IMGT defined CDR, but included in Kabat defined CDR.

According to Kabat, residue 34(Ala/Asn) belongs to the CDR1 and is partly involved in forming the VH/VL interface. This mutation remains relevant despite the strong representation of Ala in humans.

Residue 55(Gln/Glu) is part of the CDR2 according to Kabat, and is also involved in forming the VH/VL interface. This mutation remains relevant despite the strong representation of Gln in humans.

As mentioned above, these three mutations can be tested independently or in various combinations.

Example 3: humanization procedure by CDR-grafting of the heavy chain variable region (6F4 VH) of the 6F4 antibody

a) Comparison between 6F4VL nucleotide sequence and all known murine cell line sequences

As a preliminary step in humanization of CDR-grafts, the 6F4 VH nucleotide sequence was first compared to all murine cell line sequences present in the IMGT database (website http:// IMGT. cines. fr).

The sequence identity of the murine V-, D-and J-domains was 99.30% for the V-domain (IGHV1S 135X 01; SEQ ID No. 43; EMBL nomenclature: AF304556), 80% for the D-domain (IgHD-ST 4X 01; SEQ ID No. 44; EMBL nomenclature: M23243) and 100% for the J-domain (IgHJ 2X 01; SEQ ID No. 45; EMBL nomenclature: V00770).

FIG. 5A represents the alignment of V region, FIG. 5B represents the alignment of D region, and FIG. 5C represents the alignment of J region.

Taking these identity percentages into account, it was decided to use the 6F4 VH sequence directly, as was the case with 6F4 VL.

FIG. 2A represents an alignment of V regions and FIG. 2B represents an alignment of J regions.

b) Comparison of the 6F4 VH nucleotide sequence with all known human cell line sequences

To identify the best human candidate for CDR-grafting, the germline of human origin with the greatest possible identity to each of the three V, D and J regions of the 6F4 VH was sought. For this purpose, the mouse 6F4 VH nucleotide sequence was compared to all human cell line sequences present in the IMGT database.

Species of human origin were identified which had a sequence identity of 75.34% for the V-region (IGHV1-f 01; SEQ ID No. 46; EMBL nomenclature: Z12305), 71.42% for the D-region (IGHD 1-1 01; SEQ ID No. 47; EMBL nomenclature: X97051) and 87.51% for the J-region (IGHJ 4A 01; SEQ ID No.48, EMBL nomenclature: J00256).

For each of regions V, D and J, the above lines were selected and the regions rearranged.

FIG. 6A represents the V region alignment, FIG. 6B represents the D region alignment, and FIG. 6C represents the J region alignment.

c) Humanized version of 6F4 VH

The humanization process consisted of the following steps: IGHV 1-F01, IGHD 1-1-01 were linked to IGHJ 4-01 cell line sequences, and mouse 6F4 VH CDRs were attached to the scaffold regions of these homogeneous antibodies.

Molecular modeling of the mouse 6F4 Fv region during this stage of the humanization process is particularly useful for the selection of mouse residues to be retained, as they play an important role in maintaining the three-dimensional structure of the molecule (canonical structures for CDRs, VH/VL interfaces, etc.) or in binding antigen. In the scaffold region, every difference between mouse (6F4 VH) and human (IGHV 1-F01, IGHD1-1 01 and IGHJ4 01) nucleic acids was examined very carefully.

For greater clarity below, FIG. 7 represents the 6F4 VH sequence with reference to the KABAT and IMGT classifications.

As with the light chain, it was identified that the 4 murine residues must remain unchanged.

Residue 2(Ile) is part of the Vernier zone and is involved in forming the CDR3 structure.

According to IMGT, residue 35(Tyr) is involved in CDR1 anchoring, which is part of CDR1 according to Kabat, and is also involved in forming the VH/VL interface and interacting with CDR 3.

According to IMGT, residue 50(Tyr) is involved in the CDR2 anchoring, which is part of CDR2 according to Kabat, or part of Vernier zone and is involved in forming the VH/VL interface.

Residue 59(Arg) is involved in CDR2 anchoring according to IMGT, is part of CDR2 according to Kabat and is involved in forming the VH/VL interface.

The first humanized version will be able to include three mutations at residues 61, 62 and 65, respectively (IMGT classification).

These three residues are located in the CDR2 and participate in forming the VH/VL interface according to Kabat.

Residue 61(Asn/Ala) is not directly involved in antigen recognition. Mutations at this position can therefore be taken into account.

Residue 62(Gln/Glu) and residue 65 (Lys/Gln).

Second, two additional changes will be evaluated. These two changes involve residues 48 and 74(IMGT nomenclature).

Residue 48(Ile/Met) belongs to the scaffold region and is involved in the formation of the VH/VL interface.

Residue 74(Lys/Thr) is part of the Vernier zone and may participate in the formation of the CDR2 structure.

Third and finally, a third series of mutations, i.e., changes at residues 9(Pro/Ala) and 41(His/Pro) can be considered. Thus, in a similar manner to the mutations planned on 6F4VL, the goal was to come as close as possible to the human germline without altering the anchoring function of the CDRs.

For summary purposes only, the cell lines used and their amino acid and nucleotide sequence numbers are listed below in tables 4 and 5, respectively.

TABLE 4

Germline (EMBL number)	SEQ ID No.
		IGKV19-93*01(AJ235935)	39
IGKJ1*01(V00777)	40
		IGKV1-33*01(M64856)	41
IGKJ 1*01(J00242)	42
		IGHV1S135*01(AF304556)	43
IGHDST4*01(M23243)	44
		IGHJ2*01(V00770)	45
IGHV1-f*01(Z12305)	46
		IGHD1-1*01(X97051)	47
IGHJ4*01(J00256)	48
		IGHV1-03*01(X62109)	49

TABLE 5

Germline (EMBL number)	SEQ ID No.
		IGKV19-93*01(AJ235935)	50
IGKJ1*01(V00777)	51
		IGKV1-33*01(M64856)	52
IGKJ 1*01(00242)	53
		IGHV1S135*01(AF304556)	54
IGHD-ST4*01(M23243)	55
		IGHJ2*01(V00770)	56
IGHV1-f*01(Z12305)	57
		IGHD1-1*01(X97051)	58
IGHJ4*01(J00256)	59
		IGHV1-03*01(X62109)	60

Example 4: purification and characterization of 6F4 antibody antigen targets for immunoaffinity purification

The antigen target of the 6F4 antibody was purified from HT-29 cells, which are rich in membrane fraction. After dissolving in 50mM Tris/HCl buffer (pH7.4, containing 150mM NaCl, Triton X-100 and IGEPAL), the membrane proteins were gently mixed with 6F4 antibody immobilized on agarose beads at 4 ℃ and incubated overnight. The 6F4-Ag complexes formed on the microbeads were then washed with various detergent-containing solutions to remove non-specifically bound proteins. The 6F4 antigen target was eluted from the 6F 4-agarose support using 0.1M Gly/HCl buffer, pH 2.7. The collected fractions were subjected to SDS-PAGE (10% gel, non-reducing conditions), and after transferring the gel onto nitrocellulose membrane, western hybridization analysis (using a concentration of 0.5. mu.g/ml of the first antibody 6F4, chemiluminescence detection) was performed to select fractions enriched with the antigen of interest (FIGS. 8A and 8B). The western hybridization analysis demonstrated that the non-selective fraction and wash did not contain the protein of interest, which eluted specifically at acidic pH.

The enriched fractions from the two purifications were then subjected to SDS-PAGE (10% gel) and subjected to western hybridization analysis under the conditions described previously. The apparent molecular weight (apparent molecular weight) of the antigen recognized by the 6F4 antibody in the western blot after analysis under reducing conditions was 35kDa (FIGS. 9A and 9B). Differences in apparent molecular weights may be noted when electrophoresis is carried out under non-reducing conditions, under which conditions the apparent molecular weights are indeed slightly lower than those observed under reducing conditions.

Identification of antigen targets

After SDS-PAGE (10% gel) electrophoresis, the proteins were stained with colloidal blue (colloid) using a method compatible with mass spectrometry (FIG. 10). The band of interest corresponding to the protein detected by western hybridization was excised with a scalpel, and then decolorized by incubation in 25mM ammonium bicarbonate solution. After reduction (DTT)/alkylation (iodoacetamide), the protein was hydrolyzed "on gel" (overnight hydrolysis at 37 ℃) with trypsin (Promega), a proteolytic enzyme that hydrolyzes the protein at the lysine and arginine residues, thus releasing peptides with lysine or arginine residues at the C-terminal position, and the resulting protein was extracted with an acetonitrile/water mixture (70/30, v/v) in the presence of formic acid. These proteins were then placed on MALDI targets in a mixture with a matrix (α -cyano-hydroxycinnamic acid, Bruker daltons) in the presence of ATFA and then subjected to MALDI-TOF mass spectrometry (Auto flex, Bruker daltons). The mass spectrum obtained is shown in FIG. 10. The peptide list deduced from this analysis was used to identify proteins by looking up the database with a Mascot lookup engine (Matrix Sciences).

NCBInr database search results, limited to proteins of human origin, indicate that three proteins have significant scores (score > 64):

1. crystal structure of human type I ligation adhesion molecules

Score 116

This protein corresponds to the extracellular domain of the F11R/JAM-A protein used in structural studies.

F11 receptor (human)

Score 116

This protein corresponds to the precursor of the protein F11/R isoform a.

F11 receptor subtype b (human)

Score of 65

This is a precursor of protein F11R, which has two 20 amino acid deletions relative to subtype a.

Thus, the proteins identified by this method are referred to as F11R or F11 receptors. In fact, this is the official designation adopted by the protein when it was first described as a receptor for the so-called F11 antibody (Naik et al, 1995, biochem. J., 310, 155-162). This protein is now widely known under its name JAM-A or "adaptor adhesion molecule A", also known as JAM1, PAM-1, CD321 or antigen 106.

Of the peptides released by trypsin hydrolysis and analyzed by mass spectrometry, the experimental molecular weights of 9 peptides correspond (within 0.1 Da) to those obtained by theoretical hydrolysis of the human form of JAM-A/subtype a. These 9 peptides contained 37% of the primary sequence of the protein. Furthermore, the theoretical molecular weight of the JAM-A precursor (. about.32.9 kDa) was consistent with the experimentally determined apparent molecular weight of SDS-PAGE.

Identification of targets for western hybridization identification

Then, JAM-A identified by the proteolytic method was determined by western hybridization performed on a 10% SDS-PAGE gel under non-reducing conditions, with 0.5. mu.g/ml of 6F4 antibody, detected by chemiluminescence.

As shown in fig. 11A, the 6F4 antibody recognized native JAM-a protein in HT-29 membrane extracts and in immune purification process-enriched fractions (apparent MW ═ 35kDa), as well as dimeric recombinant protein JAM-a/Fc (R & DSystems ref.1103-JM, apparent MW — 120 kDa). This recognition was equivalent to dilution of commercial anti-human JAM-a goat polyclonal antibody (R & D Systems, ref. af1103) to 1/1000 (fig. 11B).

Example 5: specificity of the 6F4 antibody to human JAM-A

The specificity of the 6F4 antibody was determined by performing western hybridization under the conditions described above.

FIG. 12 shows that the 6F4 antibody is specific for human form JAM-A, since it recognizes recombinant proteins hJAM-A/Fc (R & D Systems ref.1103-JM), but does not recognize human form JAM-B and JAM-C (recombinant proteins hJAM-B/Fc and hJAM-C/Fc, R & D Systems ref.1074-VJ and 1189-J3) nor murine form JAM-A (recombinant proteins mJAM-A/Fc, R & D Systems ref.1077-JM).

Example 6: determination of the affinity of the 6F4 antibody by BIAcore (surface plasmon resonance technique) Principle of

Affinity constant K for soluble protein JAM-1-Fc (extracellular domain fused to Fc fragment of this antibody and produced in recombinant form in NSO cells) Using BIAcore, 6F4 antibody_D(M) can be according to formula K_D＝k_d/k_a(Rich and Myszka, J.mol.Recog., 2005, 18, 431) by binding kinetics (k)_a) (1/m.s) and dissociation kinetics (k)_d) (1/s) was calculated.

Materials and methods

The apparatus used was: BIAcore X and BIAevaluation 3.1X software (Uppsala, SW)

Reagent:

murine monoclonal 6F4 antibody: 1.3mg/ml

Human JAM-1-Fc (ref.1103-JM R & D system): 50 μ g of non-carrier

Mouse JAM-1-Fc (ref.1077-JM R & D Systems): 50 μ g of non-carrier

-running buffer: HBS-EP (BIAcore)

-binding kit: "amine" (BIAcore)

-binding buffer: acetate salt pH5.0(BIAcore)

-a capture antibody: goat IgG Fc anti-human (GAH goat anti-human) (Bioscience)

-regeneration buffer: glycine, HCl pH1.5, action 30 seconds (BIAcore)

Discussion and conclusions

FIG. 13 shows that murine 6F4 antibody binds to the extracellular portion of human JAM-1 protein, but does not bind to the extracellular portion of murine JAM-1 protein

The data of FIG. 14 make it possible to calculate the K of the 6F4 antibody for the human JAM-1 protein under these experimental conditions_DIs 22 pM.

The slow dissociation kinetics show the involvement of the affinity phenomenon of the antibody for the antigen (bivalent analytical model).

Example 7: activity of the 6F4 antibody in the MCF-7 xenograft model

The 6F4 antibody (unpurified and injected by IP route at a dose of 250. mu.g/mouse) demonstrated that the antibody significantly inhibited the growth of MCF-7 cells in vivo by a percentage of 56% compared to PBS-injected mice (FIG. 15). The irrelevant 9G4 antibody used as the IgG1 control isotype was, as expected, without anti-tumor activity.

Example 8: study of the distribution of antigens recognized by 6F4 on a series of tumor cells

To determine the potential effect of the 6F4 antibody, the 4 types of tumors were studied by flow cytometry for their membrane expression characteristics. The selected cell lines were MCF-7 (estrogen-related breast cancer), A549 (non-small cell lung cancer), HT29 and Colo205 (colon cancer) and BxPC3 (pancreatic cancer). For labeled cells, a series of doses (10. mu.g/ml, 5. mu.g/ml, 1. mu.g/ml, 0.5. mu.g/ml, 0.25. mu.g/ml and 0.125. mu.g/ml) were tested.

The results in fig. 16 show that the 6F4 antibody recognizes an antigen that is significantly expressed on the surface of all the cells tested. The obtained label was saturated, which confirmed its specificity. Saturation of sites was obtained from the antibody at a concentration of 1. mu.g/ml, demonstrating that the affinity of the 6F4 antibody for the JAM-A antigen is high.

Example 9: CDR-grafted humanization by the variable region of the 6F4 antibody light chain (6F4VL)

Summary of the immunogenetic analysis

Detailed data of the closest human V Gene identification

The closest V region (calculated from the first nucleotide of the V region to the second-CYS codon plus the 15nt CDR 3-IMGT)

	Score of	Identity of each other
			M64856 IGKV1-33*01	922	81.36％(227/279nt)

M64855 IGKV1D-33*01	922	81.36％(227/279nt)
			X63398 IGKV1-27*01	868	79.21％(221/279nt)
Y14865 IGKV1-NL1*01	841	78.14％(218/279nt)
			X72817 IGKV1D-43*01	841	78.14％(218/279nt)

Detailed data of the identification of the closest human J genes

The closest J-region:

	score of	Identity of each other
			J00242 IGKJ1*01	140	86.49％(32/37nt)
AF 103571 IGKJ4*02	122	81.08％(30/37nt)
			J00242 IGKJ4*01	113	78.38％(29/37nt)
Z70260 IGKJ2*02	104	75.68％(28/37nt)
			Z46620 IGKJ2*04	95	72.97％(27/37nt)

Identification of the Key residues

Several criteria are included in the definition and ranking of key residues in the outer CDRs. These criteria include, at a minimum, known involvement in the formation of the VH/VL interface, antigen binding or CDR structures, amino acid class changes between murine and human residues, positioning of residues in the 3D structure of the variable domain, etc.

The 6F4VL domain was found to differ in 21 amino acids from the closest IGKV1-33 x 01 human germline V gene, all of which were outside the CDR residues. From among these 21 residues, analysis of the above-referenced parameters resulted in the identification of the 9 most likely to function. These murine residues are K24, I39, a40, H55, T66, Q68, a69, R85 and Y87. Of these 9 residues, 3 of them are assumed to be even more important so as to retain their murine origin in the humanized form. These residues are I39 and H55 and T66, located at the CDR1 and CDR2 anchors, respectively. Finally, the 6 amino acids are analyzed individually and/or in combination to determine whether they are humanized or retain their murine origin.

Since the J-region is not involved in antigen binding and V-region structure formation, it was decided to use the primary human IGKJ1 × 01 germline gene.

In the design sequence of the humanized 6F4VL domain depicted in fig. 17:

amino acids that correspond to changes in their actual human counterparts;

1 corresponds to the amino acid for which the humanisation ability was analysed, the human residues being indicated below with a label;

2 corresponds to the amino acid remaining murine in the humanized 6F4 VH domain.

Example 10: first version of CDR-grafted humanised variable region of the 6F4 antibody heavy chain (6F4 VH)

Summary of the Immunogenetic analysis

The D-gene belongs strictly to the CDR3 region of the VH domain. The humanization process is based on a "CDR-grafting" approach. The closest human D gene analysis is useless in this strategy.

Detailed data of the closest human V Gene identification

The closest V-region (calculated from the first nucleotide of the V-region to the second-CYS codon)

	Score of	Identity of each other
			Z12305 IGHV1-f*01	796	75.35％(217/288nt)
X62106 IGHV1-2*02	787	75.00％(216/288nt)
			X92208 IGHV1-2*03	782	74.65％(215/288nt)
Z12310 IGHV1-2*04	778	74.65％(215/288nt)
			M99642 IGHV1-24*01	760	73.96％(213/288nt)

- -detailed data of the closest human J Gene identification

The closest J-region:

	score of	Identity of each other
			J00256 IGHJ4*01	181	87.23％(41/47nt)
X86355 IGHJ4*02	172	85.11％(40/47nt)
			M25625 IGHJ4*03	172	85.11％(40/47nt)
J00256 IGHJ1*01	138	74.51％(38/51nt)
			J00256 IGHJ5*01	133	74.00％(37/50nt)

Identification of the Key residues

Several criteria are included in the definition and ranking of key residues in the outer CDRs. These criteria include, at a minimum, known involvement in the formation of the VH/VL interface, antigen binding or CDR structures, amino acid class changes between murine and human residues, positioning of residues in the 3D structure of the variable domain, etc.

It was found that there were 31 amino acids differences between the 6F4 VH domain and the closest IGHV 1-F01 human germline V gene, all of which are residues outside the CDRs. Of these 31 residues, analysis of the above referenced parameters resulted in the identification of the 9 most likely to function. These murine residues are I2, Y40, I53, Y55, R66, N68, Q69, K72 and K82. Of these 9 residues, 2 are envisaged to be even more important so as to retain their murine origin in the humanized form. These residues are residues Y55 and R66, located on the CDR2 anchor. Finally, these 7 amino acids were combined and/or combined to determine whether they could be humanized or whether they retained their murine origin.

Since the J-region is not involved in antigen binding and V-region structure formation, it was decided to use the native human IGHJ4 × 01 germline gene.

In the designed sequence of the humanized 6F4 VH domain depicted in fig. 18:

amino acids that correspond to changes in their actual human counterparts;

1 corresponds to the amino acid for which the humanisation ability was analysed, the human residues being indicated below with a label;

2 corresponds to the amino acid remaining murine in the humanized 6F4 VH domain.

Example 11: second edition of CDR-grafted humanization by the variable region of the 6F4 antibody heavy chain (6F4 VH) Book (I)

Another method for identifying CDR-grafted candidate genes for the human V-gene is to find human homology at the amino acid level using the IMGT/DomainGapAlign tool.

The results of the IMGT/DomainGapAlign immunogenetic analysis are summarized below:

alleles	Species (II)	Structural domains	Smith-Waterman score	Percent identity	Overlap
						IGHV1-3*01	Human being	1	451	64.3	98

Identification of key residues in IGHV 1-03X 01 germline (SEQ ID No.49, EMBL nomenclature: X62109).

Figure 31 shows an alignment between the 6F4 VH domain and the IGHV1-3 x 01 protein sequence.

The selection and ranking of these residues is based on different criteria based on: each single position is based on the relative importance of its structural relatedness, its known structure-function relationship, the relatedness of amino acid class switching (if it occurs), and also takes advantage of the results obtained during the first humanization process.

In the first purpose, all the different "outside of the CDR" amino acids have been changed relative to their human counterparts, except for residues Y55 and R66, which strongly suggest that these two residues be included in the binding, as are the CDR 2-anchor assigned residues. When all other subsequently described analyses were performed, the humanisation ability of these two residues at the end of the procedure was investigated. Indeed, disclosure of the full activity of the parent antibody, the VH domain of 6F4 Hz2 double humanization must be modified as follows; the "de-humanization" process will consist of (if necessary) back-mutations of these amino acids on their murine counterparts:

the first set of residues, E1Q, K43R and K75R, showed standard strong binding and corresponded to the first position to evaluate "de-humanization" (if benefit differentiation was sought).

Then, residues from group 2, namely K48Q, S49R, F88Y and H90R, which are chemically related mutations but structurally somewhat unimaginable key residues, were tested in a second round of experiments.

The six residues of the third group are presumably more involved in all and/or core-directed residues and are therefore assumed to be less involved in binding and are therefore studied whenever necessary in a third round of improvement.

It is envisaged that the residues of group 4 are less structurally and/or amino acid class switch related and for which "de-humanisation" will be studied later.

Finally, the following 6 residues, I2V, Y40H, I53M, N68S, K72Q and K82T, correspond to amino acids that are humanized without altering (at least in the initial association) the binding activity of the VH domain that was first humanized. "Dehumanization" of these residues will be performed in the last round of improvement.

The D-gene belongs strictly to the CDR3 region of the VH domain. The humanization process is based on a "CDR-grafting" approach. Analysis of the closest human D-gene is not useful in this strategy.

Since the J region is not involved in the antigen binding and structure formation of the V-region, it was decided to use the primary human IGHJ4 × 01 germline gene.

Experimental data of the obtained re-humanized 6F4 antibody

In the following experiments, re-humanization involved only the heavy chain, the light chain always corresponding to the 6F4VL domain of QTY/AET humanization, as exemplified in example 9, this finally selected humanized VL domain exhibited anti-JAM-a binding activity, similar to the recombinant chimeric 6F4 antibody. Similarly, a re-humanization version improvement assay was performed with reference to the anti-JAM-a binding activity of the recombinant chimeric 6F4 antibody as defined by an ELISA assay (data not shown).

Example 12: 6F4 MAb in vitro downregulation of JAM-A expression

MCF-7, HT29 and A549 cell lines were selected to determine the effect of 6F4 Mab on JAMA expression. Briefly, cells were plated at 75cm²The flask was incubated at 37 ℃ in a medium containing 5% CO2 in air and 10% Fetal Calf Serum (FCS) for 24 hours. The cells were then washed 3 times with PBS and incubated for one more day in serum-free medium. After the second incubation, the serum-free medium was removed and replaced with fresh serum-free medium alone or with fresh serum-free medium containing 6F4 or an IgG1 isotype control (described as 9G 4). After 5 or 16 hours of incubation, the cells were disrupted by placing them on ice in cold lysis buffer (10mM Tris HCl buffer, pH7.5, 15% NaCl 1M (Sigma Chemical Co.), 10% detergent mixture (10mM Tris-HCl, 10% Igepal lysis buffer) (Sigma Chemical Co.), 5% sodium deoxycholate (Sigma Chemical Co.), 1 protease inhibitor cocktail complete TM tablet (Roche) and 1% phosphatase inhibitor cocktail Set II (Calbiochem), pH 7.5). The lysate was clarified by centrifugation at 4 ℃. Proteins were quantified by BCA protein assay, and 25. mu.g of protein was loaded in each lane of a Biorad 4-12% Bis-Tris gel. Samples were heated at 100 ℃ for 5 minutes and either held at-20 ℃ or loaded directly onto 4-12% SDS-PAGE gels and transferred to nitrocellulose membranes. Hybridization first blocked all antibodies with 5% BSA. With specific anti-JAMA primary antibody was incubated at room temperature for 2 hours. The filter was washed in TBST and incubated with the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed in TBST before protein development with ecl (amersham).

As shown in fig. 19, significant down-regulation of JAM-a was observed for 3 cell lines treated with 6F4 MAb. MCF-7 appears to be the most sensitive cell line, with complete and stable downregulation occurring as early as 5 hours after 6F4 incubation. JAM-A was noted to be partially, but persistently, downregulated in HT29 cells. The kinetics of down-regulation of a549 cells differed in that no significant effect was observed for a549 cells early in incubation with 6F4 Mab, whereas a complete inhibitory effect appeared after 16 hours of incubation with 6F4 Mab. As expected, no significant difference was observed between untreated cells and cells incubated with 9G4 isotype control.

Example 13: effect of a Single injection of 6F4 on tumor proliferation in vivo

To determine the mechanism of action of 6F4 Mab in vivo, MCF-7 cells were injected into 7-week-old female mice fed estrogen pills. When the tumor reaches 80 to 100mm³At volume of (a), 3 groups of mice with comparable tumors were generated. Prior to injection of any substance, tumors were removed from one mouse in these groups to examine the basal proliferation of tumor cells in untreated tumors. The other 2 groups of mice were injected with 1mg of 6F4 or the same dose of the IgG1 isotype control (described as 9G 4).

At 6 hours post-injection, tumors were excised, fixed in formalin, paraffin embedded, cut into 5 μm sections, and stained with anti-Ki 67 antibody to determine the level of proliferation of the treated versus control tumors.

As shown in figure 20, no difference was observed between the tumors excised before injection (noted as T0, i.e., 0 th) and the tumors injected with isotype control 9G 4. On the other hand, significant inhibition of tumor cell proliferation was observed after a single injection of 6F 4.

Example 14: effect of a Single injection of 6F4 on JAM-A expression in vivo

The in vivo protocol for this study was identical to the in vivo proliferation assay described, except that excised tumors were quickly frozen in liquid nitrogen for western hybridization analysis. The western hybridization experiments were performed as described in example 13 above.

Figure 21 demonstrates that no difference was observed between untreated mice (noted T0, i.e. 0 th) and mice injected once with the 9G4 isotype control. Note that the antibody was significantly down-regulated when 6F4 Mab treated mice, suggesting that a potential mechanism for the in vivo anti-tumor effect of this antibody may be to down-regulate the receptor. This result is consistent with the results observed in vitro and the results described below in example 13.

Example 15: 6F4 and its F (ab') ₂ Comparison of the antitumor Activity of the fragments

Since MCF-7 cells highly expressed JAM-A and despite the fact that 6F4 is IgG1 (an isotype known to be rarely involved in effector function in mice), 6F4 and its F (ab')₂In vivo comparisons between fragments to determine whether there may be involvement of effector functions in vivo activity.

For this purpose, five million MCF7 cells were transplanted into 7-week-old female mice fed estrogen pills. 5 days after cell engraftment, with 300. mu.g of 6F4 or with 200. mu.g of 6F4F (ab')₂Mice were treated three times per week. The first injection was injected with 600. mu.g of antibody and 400. mu.g of 6F4F (ab')₂. Tumor volumes were measured twice weekly for 4 weeks.

FIG. 22 shows 6F4 and 6F4F (ab')₂Tumor growth in treated mice was significantly different from that in control mice, from D3 to D27(6F 4: p.ltoreq.0.03; 6F4F (ab')₂: p is less than or equal to 0.015). At 6F4 and 6F4F (ab')₂No difference was observed between the groups of mice, indicating that effector function was not involved in the formation of 6F4 activity.

Example 16: evaluation of expression of JAM-A in human tissues

Comparison of JAM-A expression in tumor tissues with normal patient tissues was performed to select tumor types that overexpress JAMA. The study selected pairs of normal and tumor tissues from the same patient. In these patients, normal tissue picks up tissue near the tumor. JAM-A expression was determined by Immunohistochemistry (IHC) using tissue arrays (Supershirs). Briefly, slides were deparaffinized and antigen reduced using Dakocytomation solution S1699 at 98 ℃ for 20 minutes. Inactivation of endogenous peroxidase (0.3% H)₂O₂Solution 5 min) and blocking non-specific sites (Ultra-V-Block; labvision, ref. TA-125-UB), the primary antibody (anti-hJAM-A, AF1103 (R)&Dsytem) or goat IgG isotype control (Zymed)) was incubated at room temperature for 1 hour. After washing with TBS-tween, the binding of anti-hJAM-A was shown using LSAB + kit (dakocytomation). The complex of primary Ab and LSAB + was developed by the color reaction HRP-DAB. The slides were then counterstained with hematoxylin.

Samples of thyroid cancer, lung cancer and breast cancer were analyzed. For the thyroid samples (FIG. 23), no expression was observed in normal thyroid tissue, whereas JAM-A appeared to be strongly expressed on tumor sections (membrane staining) of the same patient. In normal tissues of the lung, alveolar epithelial cells (pneumocytes) express JAM-A. However, strong membrane expression was observed in all tumor samples (fig. 24). For breast cancer, weak expression of JAM-A was observed in normal breast tissue, localized to the lobular ducts. On cancer sections, 3 cancer samples (invasive ductal carcinoma, atypical medullary carcinoma and invasive papillary carcinoma) shown in FIG. 25 demonstrated that JAM-A was overexpressed in breast cancer tissues.

These data suggest that thyroid, breast and lung cancer may be good targets for JAMA treatment.

Example 17: in vivo Activity of 6F4 in A431 epidermoid carcinoma xenografted nude mice

A-431 cells were routinely cultured in DMEM (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Sigma). Two days prior to transplantation, cells were passaged to be in exponential growth phase. 1 million A-431 cells were transplanted into 7-week-old athymic nude mice. 5 days after transplantation (D5), mice were randomly grouped into the following treatments (i.p.): the control group received two injections of PBS a week, the 6F4 treated group, first injected i.p. with a 2mg loading dose of antibody, and then injected twice a week with a 1mg dose of antibody. Tumors were measured twice a week and tumor volume was calculated using the following formula: π/6x length x width x height. Statistical analysis was performed for each time point using the Mann-Whitney test and SigmaStat software. FIG. 26 shows that 6F4 Mab was able to significantly inhibit the growth of A-431 cell line in vivo (day 38 to day 56, p < 0.009).

Example 18: evaluation of antigen presenting Activity of 6F4 against Antigen Presenting Cells (APC)

JAM protein is expressed in various tissues throughout the body of humans, and is also expressed on the surface of platelets, leukocytes and erythrocytes (Naik 1995; Malergue 1998; Korneki 1990; Williams 1999; Gupta 2000). JAM-a appears to be expressed on platelets, neutrophils, monocytes, lymphocytes and erythrocytes (reviewed in Mandell 2005).

To determine whether 6F4 treatment could disrupt antigen presentation in patients, an assessment of potential intervention with Antigen Presenting Cells (APCs) including macrophages and dendritic cells was performed. During presentation, the APC internalizes the antigen (internalise) and degrades the antigen to produce a peptide that binds to CMH class II molecules within the cytosol. This complex is then expressed on the APC membrane and presented to specific T lymphocytes that respond to the stimulus by proliferating.

In the following of the present study, the potential effect of 6F4 on tetanus toxoid presentation to human PBMC was evaluated. PBMCs were isolated from blood by Ficoll gradient centrifugation for the above purpose. The cells were washed with PBS, counted and suspended in RPMI 1640 medium supplemented with 10% heat-inactivated Fetal Calf Serum (FCS), glutamine and antibiotics at a concentration of 0.25X 10⁶Cells/ml. Adding to wells in a 96-well plateAntigen or antibody to be tested to a final concentration of 10. mu.g/ml, and then 100. mu.l PBMC were inoculated per well. 9G4 Mab was used as IgG1 isotype control, phytohemagglutinin PHA (final concentration 2.5. mu.g/ml), a lymphocyte polyclonal activator, was introduced, as a positive control.

Specific antigen activator Tetanus Toxoid (TT) was selected and added to PBMCs at 100. mu.g/ml. The plates were then incubated at 37 ℃ for 96 hours in air containing 5% CO 2. Then, the mixed solution is mixed with 0.25. mu. Ci³H deoxythymidine was added to the wells and incubated for 24 hours. After incubation, cells were harvested, the filters were dried and the amount of radioactivity was counted using a liquid scintillation counter.

For figures 27A and 28A, two independent experimental values are shown, PHA (polyclonal activator), used as a positive control for PBMC preparations, is a potent inducer of lymphocyte proliferation, with indices ranging from 30 to 70, depending on the donor and the experiment. Under these conditions, the lymphocyte proliferation index did not change, and 6F4 did not exhibit any significant agonist or antagonist activity regardless of the antibody incubated. Figures 27B and 28B, showing two independent experimental values, show that significant differences can occur between donors proliferating to TT-activated lymphocytes. In these experiments, the index ranged from 2 to 5, depending on the donor and the experiment. However, no intervention in antigen presentation was observed in the presence of 6F 4.

In summary, despite the significant expression of JAM-a on APCs and lymphocytes, the use of antibodies directed against this target does not disrupt neither the specific proliferation of lymphocytes nor the antigen presentation process.

Example 19: evaluation of platelet aggregation and post-6F 4 incubation activation

To investigate whether 6F4 bound to human platelets can have any biological activity, two parameters were determined: platelet aggregation and serotonin release.

For this purpose, human platelets from 10 normal donors were incubated with several antibodies to be tested (5. mu.g/ml).

PM6/248 (anti. alpha. IIb. beta.3) has been reported to induce platelet aggregation. 9G4 was used as a negative isotype control.

Thrombin (thrombin) and ADP induce aggregation as expected in assays of human platelets. PM6/248 also induces platelet aggregation.

After incubation with 6F4, no platelet aggregation was detected. This effect was comparable to that of incubation with 9G4 (used as a positive control, fig. 29).

In a similar manner, 6F4 failed to induce serotonin release (fig. 30), whereas thrombin induced 5-HT release as expected.

Taken together, these results suggest that no biological function was stimulated on human platelets following 6F4 activation, despite human platelets expressing JAM-a.

Sequence listing

<110> Pierel-method Boley pharmaceutical Co

L. Geqi

N. Kelvia

J, F, black jew

C Beth

<120> novel antiproliferative antibody

<130>D24958

<140>PCT/EP2007/062760

<141>2007-11-23

<150>FR 06/10329

<151>2006-11-24

<160>64

<170>PatentIn version 3.3

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<211>11

<212>PRT

<213> species of little rat (mus musculus)

<400>8

Lys Ala Ser Gln Asp Ile Asn Asn Tyr Ile Ala

1 5 10

<210>9

<211>6

<212>PRT

<213> species of little rat (mus musculus)

<400>9

Thr Asp Tyr Ser Met Tyr

1 5

<210>10

<211>7

<212>PRT

<213> species of little rat (mus musculus)

<400>10

Tyr Thr Ser Thr Leu Gln Ala

1 5

<210>11

<211>17

<212>PRT

<213> species of little rat (mus musculus)

<400>11

Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210>12

<211>9

<212>PRT

<213> species of little rat (mus musculus)

<400>12

Ala Arg Gln Thr Asp Tyr Phe Asp Tyr

1 5

<210>13

<211>106

<212>PRT

<213> species of little rat (mus musculus)

<400>13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Gly Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>14

<211>116

<212>PRT

<213> species of little rat (mus musculus)

<400>14

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Ser Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210>15

<211>215

<212>PRT

<213> species of little rat (mus musculus)

<400>15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Gly Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys Asn His

210 215

<210>16

<211>440

<212>PRT

<213> species of little rat (mus musculus)

<400>16

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Ser Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala

115 120 125

Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu

130 135 140

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly

145 150 155 160

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp

165 170 175

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro

180 185 190

Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys

195 200 205

Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile

210 215 220

Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val

245 250 255

Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val

260 265 270

Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala

305 310 315 320

Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro

325 330 335

Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala

340 345 350

Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu

355 360 365

Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr

370 375 380

Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr

385 390 395 400

Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe

405 410 415

Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys

420 425 430

Ser Leu Ser His Ser Pro Gly Lys

435 440

<210>17

<211>106

<212>PRT

<213> species of little rat (mus musculus)

<400>17

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>18

<211>116

<212>PRT

<213> species of little rat (mus musculus)

<400>18

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>19

<211>116

<212>PRT

<213> species of little rat (mus musculus)

<400>19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Arg Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Thr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210>20

<211>18

<212>DNA

<213> species of little rat (mus musculus)

<400>20

caagacatta acaattat 18

<210>21

<211>12

<212>DNA

<213> species of little rat (mus musculus)

<400>21

actgactaca gc 12

<210>22

<211>9

<212>DNA

<213> species of little rat (mus musculus)

<400>22

tacacatct 9

<210>23

<211>24

<212>DNA

<213> species of little rat (mus musculus)

<400>23

attgatcctt acaatggtgg tact 24

<210>24

<211>24

<212>DNA

<213> species of little rat (mus musculus)

<400>24

ctacagtatg ataatctgtg gacg 24

<210>25

<211>21

<212>DNA

<213> species of little rat (mus musculus)

<400>25

cagacggact actttgacta c 21

<210>26

<211>24

<212>DNA

<213> species of little rat (mus musculus)

<400>26

ggttactcat tcactgacta cagc 24

<210>27

<211>27

<212>DNA

<213> species of little rat (mus musculus)

<400>27

gcaagacaga cggactactt tgactac 27

<210>28

<211>33

<212>DNA

<213> species of little rat (mus musculus)

<400>28

aaggcaagcc aagacattaa caattatata gct 33

<210>29

<211>21

<212>DNA

<213> species of little rat (mus musculus)

<400>29

tacacatcta cattacaagc a 21

<210>30

<211>18

<212>DNA

<213> species of little rat (mus musculus)

<400>30

actgactaca gcatgtac 18

<210>31

<211>51

<212>DNA

<213> species of little rat (mus musculus)

<400>31

tatattgatc cttacaatgg tggtactagg tacaaccaga agttcaaggg c 51

<210>32

<211>318

<212>DNA

<213> species of little rat (mus musculus)

<400>32

gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 60

atcacttgca aggcaagcca agacattaac aattatatag cttggtacca acacaagcct 120

ggaaaaggtc ctaggctgct catacattac acatctacat tacaagcagg catcccatca 180

aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 240

gaagatattg gaacttatta ttgtctacag tatgataatc tgtggacgtt cggtggaggc 300

accaagctgg aaatcaaa 318

<210>33

<211>348

<212>DNA

<213> species of little rat (mus musculus)

<400>33

gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta ctcattcact gactacagca tgtactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagacagacg 300

gactac tttg actactgggg ccaaggcacc actctcacag tctcctca 348

<210>34

<211>639

<212>DNA

<213> species of little rat (mus musculus)

<400>34

gacatccaga tgacacagtc tccatcctca ctgtctgcat ctctgggagg caaagtcacc 60

atcacttgca aggcaagcca agacattaac aattatatag cttggtacca acacaagcct 120

ggaaaaggtc ctaggctgct catacattac acatctacat tacaagcagg catcccatca 180

aggttcagtg gaagtgggtc tgggagagat tattccttca gcatcagcaa cctggagcct 240

gaagatattg gaacttatta ttgtctacag tatgataatc tgtggacgtt cggtggaggc 300

accaagctgg aaatcaaacg ggctgatgct gcaccaactg tatccatctt cccaccatcc 360

agtgagcagt taacatctgg aggtgcctca gtcgtgtgct tcttgaacaa cttctacccc 420

aaagacatca atgtcaagtg gaagattgat ggcagtgaac gacaaaatgg cgtcctgaac 480

agttggactg atcaggacag caaagacagc acctacagca tgagcagcac cctcacgttg 540

accaaggacg agtatgaacg acataacagc tatacctgtg aggccactca caagacatca 600

acttcaccca ttgtcaagag cttcaacagg aatgagtgt 639

<210>35

<211>1320

<212>DNA

<213> species of little rat (mus musculus)

<400>35

gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta ctcattcact gactacagca tgtactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagacagacg 300

gactactttg actactgggg ccaaggcacc actctcacag tctcctcagc caaaacaaca 360

gccccatcgg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 420

ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 480

tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 540

agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 600

gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgtggttgt 660

aagccttgca tatgtacagt cccagaagta tcatctgtct tcatcttccc cccaaagccc 720

aaggatgtgc tcaccattac tctgactcct aaggtcacgt gtgttgtggt agacatcagc 780

aaggatgatc ccgaggtcca gttcagctgg tttgtagatg atgtggaggt gcacacagct 840

cagacgcaac cccgggagga gcagttcaac agcactttcc gctcagtcag tgaacttccc 900

atcatgcacc aggactggct caatggcaag gagttcaaat gcagggtcaa cagtgcagct 960

ttccctgccc ccatcgagaa aaccatctcc aaaaccaaag gcagaccgaa ggctccacag 1020

gtgtacacca ttccacctcc caaggagcag atggccaagg ataaagtcag tctgacctgc 1080

atgataacag acttcttccc tgaagacatt actgtggagt ggcagtggaa tgggcagcca 1140

gcggagaact acaagaacac tcagcccatc atggacacag atggctctta cttcgtctac 1200

agcaagctca atgtgcagaa gagcaactgg gaggcaggaa atactttcac ctgctctgtg 1260

ttacatgagg gcctgcacaa ccaccatact gagaagagcc tctcccactc tcctggtaaa 1320

<210>36

<211>318

<212>DNA

<213> species of little rat (mus musculus)

<400>36

gacatacaga tgactcagag cccatcatca ttgagcgcgt ctgtcggcga tcgggttacc 60

attacctgcc aggcaagtca agatatcaac aactatattg cttggtatca acagaagccc 120

ggtaaagccc caaagctgct gatacactac acctccaccc tggagaccgg cgtgccttct 180

agattttctg gaagcgggtc cggaaccgat tatacgttca caatctccag ccttcagccc 240

gaagacatcg ccacatacta ctgtctgcaa tacgacaatc tgtggacatt tggccagggg 300

actaaggtgg agatcaaa 318

<210>37

<211>345

<212>DNA

<213> species of little rat (mus musculus)

<400>37

gaagtgcagc tggttcagag cggcgccgag gtaaaaccag gggcgacggt gaagataagc 60

tgcaaggtga gtgggtactc attcaccgac tattcaatgc actgggtcca acaggcccct 120

ggtaaaggac tggagtggat gggatacatc gatccctaca atggaggcac taggtacgcc 180

gagaagttcc aggggagagt cactattacc gcagatactt ctaccgatac tgcctacatg 240

gaactcagca gtctgcggtc cgaggacaca gcagtctact attgtgctcg ccaaacagac 300

tattttgact attggggcca gggaaccttg gtgacagtgt cctct 345

<210>38

<211>348

<212>DNA

<213> species of little rat (mus musculus)

<400>38

caggtgcaat tggtacagtc aggcgcggag gtgaagaagc ctggggctag tgttaaagtc 60

tcctgtaaag cctccggata ttccttcact gactactcta tgcattgggt tcgccaggca 120

ccagggcagc ggctggaatg gatggggtac attgatccct acaacggagg cacgcgatat 180

agtcagaagt tccagggtcg ggtgacaatc acagccgata cgtccaccag caccgcctac 240

atggagttga gcagtctcag gtcagaagac acagccgtgt actattgcgc aagacagacc 300

gattatttcg actactgggg ccaaggcact ctcgtgaccg tctctagc 348

<210>39

<211>115

<212>PRT

<213> species of little rat (mus musculus)

<400>39

Met Arg Pro Ser Ile Gln Phe Leu Gly Leu Leu Leu Phe Trp Leu His

1 5 10 15

Gly Ala Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro

50 55 60

Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser

85 90 95

Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp

100 105 110

Asn Leu Leu

115

<210>40

<211>12

<212>PRT

<213> species of little rat (mus musculus)

<400>40

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210>41

<211>117

<212>PRT

<213> human (homo sapiens)

<400>41

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Gln Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser

35 40 45

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Tyr Asp Asn Leu Pro

115

<210>42

<211>12

<212>PRT

<213> human (homo sapiens)

<400>42

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210>43

<211>98

<212>PRT

<213> species of little rat (mus musculus)

<400>43

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

Asn Met Tyr Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Phe

65 70 75 80

Met His Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>44

<211>2

<212>PRT

<213> species of little rat (mus musculus)

<400>44

Gln Thr

1

<210>45

<211>16

<212>PRT

<213> species of little rat (mus musculus)

<400>45

Asp Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

1 5 10 15

<210>46

<211>98

<212>PRT

<213> human (homo sapiens)

<400>46

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr

<210>47

<211>121

<212>PRT

<213> human (homo sapiens)

<400>47

Met Ser Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp

1 5 10 15

Gly Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val

20 25 30

Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser

35 40 45

Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser

50 55 60

Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr

65 70 75 80

Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp

85 90 95

Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu

100 105 110

Asp Thr Ala Val Tyr Tyr Cys Ala Arg

115 120

<210>48

<211>15

<212>PRT

<213> human (homo sapiens)

<400>48

Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10 15

<210>49

<211>97

<212>PRT

<213> human (homo sapiens)

<400>49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala

<210>50

<211>643

<212>DNA

<213> species of little rat (mus musculus)

<400>50

cagatgaagc tgatttgcat gtgctgagat catattctac tgccccagag atttaataat 60

ctgatcatac acactccaac agtcattctt ggtcaggaga cgttgtagaa atgagaccgt 120

ctattcagtt cctggggctc ttgttgttct ggcttcatgg taaggagttt aacattgaat 180

atgctaaaaa gagtatgtga tcaggaattt ctggtccttc agaaaaatct tctttgaata 240

taattaattt catagggatt tgtgttcttt ttaattatag gtgctcagtg tgacatccag 300

atgacacagt ctccatcctc actgtctgca tctctgggag gcaaagtcac catcacttgc 360

aaggcaagcc aagacattaa caagtatata gcttggtacc aacacaagcc tggaaaaggt 420

cctaggctgc tcatacatta cacatctaca ttacagccag gcatcccatc aaggttcagt 480

ggaagtgggt ctgggagaga ttattccttc agcatcagca acctggagcc tgaagatatt 540

gcaacttatt attgtctaca gtatgataat cttctaccca cagtgataca aatcataaca 600

aaaaccaccc agggaagcag aagtgagagg ctaggttgcc cac 643

<210>51

<211>39

<212>DNA

<213> species of little rat (mus musculus)

<400>51

tggacgttcg gtggaggcac caagctggaa atcaaacgt 39

<210>52

<211>667

<212>DNA

<213> human (homo sapiens)

<400>52

ctgcagctgt gcccagcctg ccctatcccc tgctgatttg catgttcgca gagcacagcc 60

ccctgccctg aagacttatt aataggctgg tcgcaccctg tgcaggagtc agtcccaacc 120

aggacacagc atggacatga gggtccctgc tcagctcctg gggctcctgc agctctggct 180

ctcaggtaag gaaggataac actaggaatt ttctcagcca gtgtgctcag tacagcctgg 240

ctcttgatgg aagccttcct ataatatgac taatagtatg aatatttgtg tttatgtttc 300

taatcgcagg tgccagatgt gacatccaga tgacccagtc tccatcctcc ctgtctgcat 360

ctgtaggaga cagagtcacc atcacttgcc aggcgagtca ggacattagc aactatttaa 420

attggtatca gcagaaacca gggaaagccc ctaagctcct gatctacgat gcatccaatt 480

tggaaacagg ggtcccatca aggttcagtg gaagtggatc tgggacagat tttactttca 540

ccatcagcag cctgcagcct gaagatattg caacatatta ctgtcaacag tatgataatc 600

tccctcccac agtgtaacaa gtcataacat aaatcaccca ggggagcaga tgcgtgaggc 660

tcagctg 667

<210>53

<211>37

<212>DNA

<213> human (homo sapiens)

<400>53

tggacgttcg gccaagggac caaggtggaa atcaaac 37

<210>54

<211>294

<212>DNA

<213> species of little rat (mus musculus)

<400>54

gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta ctcattcact gactacaaca tgtactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactagctac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagccttc 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aaga 294

<210>55

<211>163

<212>DNA

<213> species of little rat (mus musculus)

<400>55

aagcttgccc aggaaccact agtgctcaca cagctctgcc cacaggggaa acctaaccat 60

gcctgccccc tactcagcag gaaggctctg aagctctgag aggattttga acaagttact 120

gtcacagtga gacagctcgg gctaccatgt aagaaaagct caa 163

<210>56

<211>45

<212>DNA

<213> species of little rat (mus musculus)

<400>56

tactttgact actggggcca aggcaccact ctcacagtct cctca 45

<210>57

<211>294

<212>DNA

<213> human (homo sapiens)

<400>57

gaggtccagc tggtacagtc tggggctgag gtgaagaagc ctggggctac agtgaaaatc 60

tcctgcaagg tttctggata caccttcacc gactactaca tgcactgggt gcaacaggcc 120

cctggaaaag ggcttgagtg gatgggactt gttgatcctg aagatggtga aacaatatac 180

gcagagaagt tccagggcag agtcaccata accgcggaca cgtctacaga cacagcctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aaca 294

<210>58

<211>17

<212>DNA

<213> human (homo sapiens)

<400>58

ggtacaactg gaacgac 17

<210>59

<211>46

<212>DNA

<213> human (homo sapiens)

<400>59

tactttgact actggggcca aggaaccctg gtcaccgtct cctcag 46

<210>60

<211>291

<212>DNA

<213> human (homo sapiens)

<400>60

ggggcctcag tgaaggtttc ctgcaaggct tctggataca ccttccaggt ccagcttgtg 60

cagtctgggg ctgaggtgaa gaagcctact agctatgcta tgcattgggt gcgccaggcc 120

cccggacaaa ggcttgagtg gatgggatgg atcaacgctg gcaatggtaa cacaaaatat 180

tcacagaagt tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac 240

atggagctga gcagcctgag atctgaagac acggctgtgt attactgtgc g 291

<210>61

<211>299

<212>PRT

<213> human (homo sapiens)

<400>61

Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe Ile

1 5 10 15

Leu Ala Ile Leu Leu Cys Ser Leu Ala Leu Gly Ser Val Thr Val His

20 25 30

Ser Ser Glu Pro Glu Val Arg Ile Pro Glu Asn Asn Pro Val Lys Leu

35 40 45

Ser Cys Ala Tyr Ser Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe

50 55 60

Asp Gln Gly Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr

65 70 75 80

Ala Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe

85 90 95

Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser

100 105 110

Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu Ile Val

115 120 125

Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile Pro Ser Ser Ala Thr

130 135 140

Ile Gly Asn Arg Ala Val Leu Thr Cys Ser Glu Gln Asp Gly Ser Pro

145 150 155 160

Pro Ser Glu Tyr Thr Trp Phe Lys Asp Gly Ile Val Met Pro Thr Asn

165 170 175

Pro Lys Ser Thr Arg Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro

180 185 190

Thr Thr Gly Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly

195 200 205

Glu Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser

210 215 220

Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val

225 230 235 240

Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu Val Phe Gly

245 250 255

Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp Arg Thr Lys Lys Gly

260 265 270

Thr Ser Ser Lys Lys Val Ile Tyr Ser Gln Pro Ser Ala Arg Ser Glu

275 280 285

Gly Glu Phe Lys Gln Thr Ser Ser Phe Leu Val

290 295

<210>62

<211>897

<212>DNA

<213> human (homo sapiens)

<400>62

atggggacaa aggcgcaagt cgagaggaaa ctgttgtgcc tcttcatatt ggcgatcctg 60

ttgtgctccc tggcattggg cagtgttaca gtgcactctt ctgaacctga agtcagaatt 120

cctgagaata atcctgtgaa gttgtcctgt gcctactcgg gcttttcttc tccccgtgtg 180

gagtggaagt ttgaccaagg agacaccacc agactcgttt gctataataa caagatcaca 240

gcttcctatg aggaccgggt gaccttcttg ccaactggta tcaccttcaa gtccgtgaca 300

cgggaagaca ctgggacata cacttgtatg gtctctgagg aaggcggcaa cagctatggg 360

gaggtcaagg tcaagctcat cgtgcttgtg cctccatcca agcctacagt taacatcccc 420

tcctctgcca ccattgggaa ccgggcagtg ctgacatgct cagaacaaga tggttcccca 480

ccttctgaat acacctggtt caaagatggg atagtgatgc ctacgaatcc caaaagcacc 540

cgtgccttca gcaactcttc ctatgtcctg aatcccacaa caggagagct ggtctttgat 600

cccctgtcag cctctgatac tggagaatac agctgtgagg cacggaatgg gtatgggaca 660

cccatgactt caaatgctgt gcgcatggaa gctgtggagc ggaatgtggg ggtcatcgtg 720

gcagccgtcc ttgtaaccct gattctcctg ggaatcttgg tttttggcat ctggtttgcc 780

tatagccgag gccactttga cagaacaaag aaagggactt cgagtaagaa ggtgatttac 840

agccagccta gtgcccgaag tgaaggagaa ttcaaacaga cctcgtcatt cctggtg 897

<210>63

<211>259

<212>PRT

<213> human (homo sapiens)

<400>63

Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu Leu Cys Leu Phe Ile

1 5 10 15

Leu Ala Ile Leu Pro Glu Asn Asn Pro Val Lys Leu Ser Cys Ala Tyr

20 25 30

Ser Gly Phe Ser Ser Pro Arg Ala Ala Ser Tyr Glu Asp Arg Val Thr

35 40 45

Phe Leu Pro Thr Gly Ile Thr Phe Lys Ser Val Thr Arg Glu Asp Thr

50 55 60

Gly Thr Tyr Thr Cys Met Val Ser Glu Glu Gly Gly Asn Ser Tyr Gly

65 70 75 80

Glu Val Lys Val Lys Leu Ile Val Leu Val Pro Pro Ser Lys Pro Thr

85 90 95

Val Asn Ile Pro Ser Ser Ala Thr Ile Gly Asn Arg Ala Val Leu Thr

100 105 110

Cys Ser Glu Gln Asp Gly Ser Pro Pro Ser Glu Tyr Thr Trp Phe Lys

115 120 125

Asp Gly Ile Val Met Pro Thr Asn Pro Lys Ser Thr Arg Ala Phe Ser

130 135 140

Asn Ser Ser Tyr Val Leu Asn Pro Thr Thr Gly Glu Leu Val Phe Asp

145 150 155 160

Pro Leu Ser Ala Ser Asp Thr Gly Glu Tyr Ser Cys Glu Ala Arg Asn

165 170 175

Gly Tyr Gly Thr Pro Met Thr Ser Asn Ala Val Arg Met Glu Ala Val

180 185 190

Glu Arg Asn Val Gly Val Ile Val Ala Ala Val Leu Val Thr Leu Ile

195 200 205

Leu Leu Gly Ile Leu Val Phe Gly Ile Trp Phe Ala Tyr Ser Arg Gly

210 215 220

His Phe Asp Arg Thr Lys Lys Gly Thr Ser Ser Lys Lys Val Ile Tyr

225 230 235 240

Ser Gln Pro Ser Ala Arg Ser Glu Gly Glu Phe Lys Gln Thr Ser Ser

245 250 255

Phe Leu Val

<210>64

<211>777

<212>DNA

<213> human (homo sapiens)

<400>64

atggggacaa aggcgcaagt cgagaggaaa ctgttgtgcc tcttcatatt ggcgatcctt 60

cctgagaata atcctgtgaa gttgtcctgt gcctactcgg gcttttcttc tccccgtgca 120

gcttcctatg aggaccgggt gaccttcttg ccaactggta tcaccttcaa gtccgtgaca 180

cgggaagaca ctgggacata cacttgtatg gtctctgagg aaggcggcaa cagctatggg 240

gaggtcaagg tcaagctcat cgtgcttgtg cctccatcca agcctacagt taacatcccc 300

tcctctgcca ccattgggaa ccgggcagtg ctgacatgct cagaacaaga tggttcccca 360

ccttctgaat acacctggtt caaagatggg atagtgatgc ctacgaatcc caaaagcacc 420

cgtgccttca gcaactcttc ctatgtcctg aatcccacaa caggagagct ggtctttgat 480

cccctgtcag cctctgatac tggagaatac agctgtgagg cacggaatgg gtatgggaca 540

cccatgactt caaatgctgt gcgcatggaa gctgtggagc ggaatgtggg ggtcatcgtg 600

gcagccgtcc ttgtaaccct gattctcctg ggaatcttgg tttttggcat ctggtttgcc 660

tatagccgag gccactttga cagaacaaag aaagggactt cgagtaagaa ggtgatttac 720

agccagccta gtgcccgaag tgaaggagaa ttcaaacaga cctcgtcatt cctggtg 777

Claims (18)

1. An antibody, or its F (ab') capable of binding to JAM-A₂A fragment, characterized in that it contains:

a light chain comprising the following three CDRs defined according to IMGT:

CDR-L1 of sequence SEQ ID NO. 1;

CDR-L2 of sequence SEQ ID NO. 3; and

CDR-L3 of sequence SEQ ID NO. 5;

and

a heavy chain comprising the following three CDRs defined according to IMGT:

CDR-H1 of sequence SEQ ID NO. 7;

CDR-H2 of sequence SEQ ID NO. 4; and

CDR-H3 of sequence SEQ ID NO. 12.

2. The antibody of claim 1, or an F (ab') capable of binding to JAM-A₂A fragment, characterized in that it comprises a light chain variable region sequence consisting of the amino acid sequence SEQ ID No.13 and it comprises a heavy chain variable region sequence consisting of the amino acid sequence SEQ ID No. 14.

3. The antibody of claim 1, or an F (ab') capable of binding to JAM-A₂A fragment, characterized in that it comprises a light chain sequence consisting of the amino acid sequence SEQ ID No.15 and in that it comprises a heavy chain sequence consisting of the amino acid sequence SEQ ID No. 16.

4. The antibody of claim 1, or an F (ab') capable of binding to JAM-A₂A fragment, characterized in that it consists of a humanized antibody comprising a light chain variable region sequence consisting of the amino acid sequence SEQ ID No.17 and comprising a heavy chain variable region sequence consisting of the amino acid sequence SEQ ID No.18 or 19.

5. A murine hybridoma, accession number I-3646, of CNCM submitted to the paris pasteur institute on 6.7.2006.

6. An antibody secreted by the hybridoma of claim 5.

7. The antibody of claim 1, or an F (ab') capable of binding to JAM-A₂Fragment, characterized in that its Kd for the JAM-A protein is between 1pM and 1 nM.

8. The antibody of claim 1, or an F (ab') capable of binding to JAM-A₂Fragment, characterized in that it has a Kd for the JAM-A protein of between 10pM and 40 pM.

9. An isolated nucleic acid, characterized in that it encodes the antibody according to any one of claims 1 to 4 or 6 to 8 or its F (ab') capable of binding to JAM-A₂A nucleic acid of a fragment.

10. A vector consisting of the nucleic acid of claim 9.

11. A host cell comprising the vector of claim 10.

12. Preparation of an antibody according to any one of claims 1 to 4 or 6 to 8 or F (ab') thereof capable of binding to JAM-A₂Method of fragmentation, characterized in that it comprises the following steps:

a) culturing the host cell of claim 11 in a culture medium and under suitable culture conditions; and

b) recovering said antibody or one of F (ab') capable of binding to JAM-A from the culture medium or from said cultured cells₂And (3) fragment.

13. An antibody according to any one of claims 1 to 4 or 6 to 8, or an F (ab') thereof capable of binding to JAM-A₂The fragment compound as the active ingredient.

14. A composition according to claim 13, characterized in that it further comprises as a combined product an anti-tumor antibody, other than an antibody directed against JAM-a protein, for simultaneous, separate or prolonged use.

15. A composition according to claim 13 or 14, characterized in that it further comprises as a combined product a cytotoxic or cytostatic agent, for simultaneous, separate or prolonged use.

16. A composition according to claim 15, characterised in that said cytotoxic or cytostatic agent is chemically bound to at least one of the components of said composition used simultaneously.

17. A composition according to any one of claims 13, 14 or 16, characterized in that at least one of said antibodies or its F (ab') capable of binding to JAM-a₂The fragments are conjugated to a cytotoxin and/or a radioisotope.

18. The antibody of any one of claims 1 to 4 or 6 to 8 or an F (ab')₂Use of a fragment, and/or a composition according to any one of claims 13 to 17, in the manufacture of a medicament for the prevention or treatment of thyroid, breast and lung cancer.