AU616195B2 - Creación de Inventos y Patentes con Inteligencia Artificial

AU616195B2 – Lectines fixing beta-d-galactoside
– Google Patents

AU616195B2 – Lectines fixing beta-d-galactoside
– Google Patents
Lectines fixing beta-d-galactoside

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Publication number
AU616195B2

AU616195B2
AU21214/88A
AU2121488A
AU616195B2
AU 616195 B2
AU616195 B2
AU 616195B2
AU 21214/88 A
AU21214/88 A
AU 21214/88A
AU 2121488 A
AU2121488 A
AU 2121488A
AU 616195 B2
AU616195 B2
AU 616195B2
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Australia
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ala
val
asn
ile
asp
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1987-07-21
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AU2121488A
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Inventor
Arthur Donny Strosberg
Vivian Teichberg
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Ideon Corp

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Ideon Corp
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1987-07-21
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1988-07-18
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1991-10-24

1988-07-18
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Ideon Corp

1989-02-13
Publication of AU2121488A
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patent/AU2121488A/en

1991-10-24
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1991-10-24
Publication of AU616195B2
publication
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patent/AU616195B2/en

2008-07-18
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legal-status
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Classifications

G—PHYSICS

G01—MEASURING; TESTING

G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 – G01N31/00

G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient

A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates

A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent

A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment

A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent

A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates

A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin

A61K47/6817—Toxins

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient

A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site

A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used

C07K14/4726—Lectins

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K38/00—Medicinal preparations containing peptides

Abstract

The present invention relates to amino acid sequences reproducing at least in part the sequence of animal and human lectines. The sequence of amino acids comprises a skeleton which is comprised of at least the following amino acids and in the following positions: . Application particularly to the diagnostic of tumoral affections.

Description

AU-Al -21214/88 ISATION MONDIALE DE LA PROPRIETE INTELLECTUELLE 6 1 0 4 90R5 -Bureau intlernational i DEMANDE INTERNATIONALE PUBLIEE EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCT) (51) Clssification Internationale des brevets 4 (11) Numiro de publication Internationale: WO 89/ 00581 C07K 13/00,7/06, 7/08 C07K 7/10, 3/20, A61K 39/395 Al G01N 33/577 C07K 15/0~6 (43) Date de publication internationale: C07K 15/08 26 janvier 1989 (26.01.89) (21) Numiro de ]a demande Internationale: PCT/FPR88/00370 (22) Date de d~p6t international: 18 juiliet 1988 (18.07.88) (31) Numfiro de la demande prioritaire: 87/1 0288 (32) Date de priorit6: (33) Pays de priorit6: 21 juillet 1987 (2 1.07.87)
FR
(71) Dr~posant (pipur toils les Etoats d~sign~s sauf US): IDEON CORPORAT iON [US/US]; 301 Penobscott Drive, Redwood City, CA 94063 (US).
(72) Invente’trs; et Inventeurs/D~posants (US seulement) STROSBERG, Arthur, Donny [BE/FR]; 66, rue de Javel, F-75015 Paris (FR1). TEICHB RG, Vivian [IL/IL]; Institut Weizmann, 76 100 Rehovot (IL), (74) Mstndataires: ORES, Irene etc.; Cabinet Ores, 6, avenue de Messine, F-75008 Paris (FR).
(81) Etats disignis: AT (bre~vet e ;rop~en), AU, BE (breve t europ~en), CH (brevet vurop~en), DE (brevet europ~en), DK, FI, FR (brevet europ~en), GB (brevet europ~en), IT (b’revet europ~en), JP, KR, LU (brevet europ~en), NL (brevet europ~en), NO, SE (brevet europ~en), US.
PuhI,1e Avec rapport de recAerche internationale.
A. 0. J. P. 6 APR 1989 [ASTRAI
A
1 3 FEN89 8 9 PATENT OFFICri (54) Title: LECTINES FIXING IIBETA D-GALACTOSlDE (54) Titre: LECTINES FIXANT LE BETA-D-GALACTOSIDE 110 20 30 SER PMOAsm GLY VAL, VAL Asp GLuAa ri io UR pik Lys ALA C,.Y GLit All. Cit lte VAL Lys GL.A VAL. -N10 SEA M ASP 50, I.1 Asi Pit ALA Itt 5 Asm(YP)(01M L .(AAl)(tA GLV CLa Pt PROa P.t Wall G11( GLU All- Pit It'( (Lit LE tIl Pi Asi SIR tw K.GIL _sPI (57) Abstract t11t0.rjS.CQI,0 to.A il1 f1 I A L L R IE11 E t L L y I -II The presen; invention relates to amino acid sequences reproducing at iea~t in part the sequenee of animal and human lectires. Th~i sequence of amino acids comprises a skeleton which is comprised of at least the following amino acids and in t~te follo~’4ng positions: 2 L4 14 19 23 32 is 37 40 43 46 paragino glyolia. Leudo l.uetno biottin.
46a 46 50 51 asparagine Pratt ne &rqli iin pli~nylatanine acid» $4 61 63 64 70 &tpariqi Valine Asparagila. wine1 tryptophaa.
71 7) 765 76 ft 64 9 3 93 104 to 9tyqwit khilosiint phiyllni. 1ucino ,prollna 104 17 112 13 4cld. asParqt. glyohri. phi6ltnyllanine proline *Application particjarly to the diagnostic of tumoral affections.
AMINO ACID SEQUENCES REPRODUCING AT LEAST IN PART THE SEQUENCE OF ANIMAL AND HUMAN LECTINS, PROCESSES FOR OBTAINING THEM AND THEIR DIAGNOSTIC AND THERAPEUTIC
APPLICATIONS
The present invention relates to the isolation of a lectin of human origin, to the determination of the amLino acid sequence of lectins from diverse sources, including lectins of human origin, and to their synthesis, to the application of the said sequences in therapy as an immunostimulant and immunosuppressant and as an interferon-like agent, for the treatment of certain viral diseases and certain forms of cancer, to the production of anti-lectin polyclonal and monoclonal antibodies and to their application to immunodiagnosis, to the identification and isolation of the genes for lectins which recognize the abovementioned amino acid sequences, and to the use of the expression of these genes for the production of lectins, of amino acid sequences and of oligonucleotides appropriate to the latter.
Lectins are proteins which have been found in a wide variety of vertebrates, ranging from the electric eel (whence their name) to birds and mammals.
Lectins are proteins which bind sugars and combine with mono- and with oligosaccharides via bonds which are other than covalent. All lectins are oligomeric proteins containing several saccharide-binding sites per molecule, that is to say they are multivalent. This multivalent structure endows lectins with the capacity to agglutinate cells carrying the appropriate saccharide fractions on their outer membrane.
During the past twenty years, several hundred lectins, mainly extracted from plants, have been purified and characterized. Lectins are used as reagents for the purification and characterization of glycoconjugates or as mitogens for specific classes of lymphocytes.
At the present time, some lectins are used in blood banks for blood Typing, mainly on account of the lack Of availability of iNatural anti-O(H) antibodies and on account of the fact that some of them distinguish A, i
AZ
4 dAI o1 2 and A z blood subgroups. Occasionally, lectins are used for the separation of mixed erythrocyte populationsf for example in the rare cases of mosaics of blood groups resulting from chimera formation, from somatic mutations or from bone marrow transplantations. Moreover, since some lectins [for example phytohaemagglutinin (PHA) and concanavalin Al stimulate B lymphocytes (for example Phytolacca mitogen), lectins are also used for identifying the main lymphocyte subpopulations. In contrast to artigens, which stimulate only a small proportion of lymphocytes (usually from 0.02 to lectins stimulate a high proportion (up to 70-80%) of B cells, irrespective of the antigenic specificity of the receptor lymphocytes. Lectins are hence polyclonal activators or polyclonal ligands. They are used in the study of the mechanism by which an antigen acting at the surface of the lymiphoid cell specifically promotes or specifically inhibits clonal expansion and immunoglobulin synthesis.
Various lymphokines such as interleukin-2 (11-2) and interferon should be mentioned is other products synthesized by l.tin-stimulated lymphocytes. Mitogenic stimulation, especially by PHA, is also used as a diagnostic tool for the detection of congenital and acquired immunological deficiencies, for the detection of sensitizations due to infectious agents or linked to certain autoimmune diseases, and for the control of the effects of various immunosuppressant and immunotherapeutic treatments. Another especially useful application of the stimulation of lymphocytes by these lectins is represented by cytogenetic studies of human and animal chromosomes, Lectins are also widely used by immunologists for studying the properties and constitution of lymphocyte membranes. By using appropriate lectin derivatives, it is possible to examine not only the distribution of lectin receptors on the lymphocyte surface, but also the mobility of the receptors in the membrane, and to demonstrate, for example, that the redistribution and capping 4 of the receptors on the lymphocyte surfaces result from 3 binding of the lectins.
Lectin receptors, that is to say the membrane constituents which react with the lectins, may be detected on electrophoretograms or isolated in purified form by the same methods as are used for isolatinsurface antigens with the aid of the corresponding antibodies. The detection is preferably carried out by staining the lectin in the electropIoretogrens of membrane preparations. The receptors may be isolated from solubilized membranes, by precipitation either with the lectin alone, or using the latter combined with an antilectin antibody; the best results are obtained by isolation of the said receptors by affinity chromatography on immobilized lectins.
The existence in many vertebrate tissues of an endogenous lectin activity specifically binding p-Dgalactoside was demonstrated in 1974 and attributed to the family of proteins known as «electrolctins» (on account of the fact that the first animal lectin described had been isolated from the electric organ of the electric eel Electrophorus electricus). Lectins have since been isolated from chick embryo muscle, kidney, heart and intestin, from calf heart, lung and spleen, from rat lung, from monkey muscle and from human lung and muscle. Remarkably, the different lectins identified to date have properties very similar to those described for the electrolectin of the electric eel. They all agglutinate trypsin-treated rabbit erythrocytes, they have all been shown to have chiefly a cytoplasmic intercellular (sic) localization, they all have a molecular weight of between 15,000 and 30,000 daltons and are composed of two to four subunits, and they all possess the same affinity for the following saccharides, in decreasing order: p-D-galactosylthiogalactopyranoside (TDG) lactose D-galactose.
All lectins identified to date have been shown to require the prestl-t of reducing agents for maintenaince of their agglutinating activity.
FurtheKrSe, it has been shown that the lectins I L 4 of various tissues of the same animal and the lectins of homologous tissues of different mammals exhibit antigenic cross-reactivity.
The biological functions of vertebrate lectins have not yet been definitely established. It is however, noteworthy, that their concentration in the tissues is regulated in accordance with development, and that they are associated with neoplastic processes. In adult animals, vertebrate lectins are mainly present in organs involved in the immune defences (that is to say the thymus, placenta and skin). Lectins exhibiting specificity with respect to lactose exhibit immunomodulatory properties in experimentally induced autoimmune diseases (such as myasthenia gravis and type I diabetes).
The data at present available indicate that lectins exhibit a structural ressemblance to interferons, which are lymphokines which have already been tested in human therapy for certain viral diseases and certain forms of cancer. Furthermore, as stated above, a functional study of the eel electrolectin has shown immunosuppressant properties in an experimental form of myasthenia gravis induced by the injection of acetylcholine receptor. These two types of observations suggest that lectins might be used clinically as lymphokines and/or as immunosuppressant proteins, and in immunodiagnosis using the reaction of antigenic tumour proteins with antilectin antibodies.
With the object of studying the regulation of the expression and biological functions of vertebrate lectins, GITT and BARONDES (PROC. iATL. ACAD. SCI. USA, Vol.
83, p. 7603-7607, October 1986) set out to determine the sequences of lectins and more especially that of a human dimeric lectic binding p-D-galactoside, the subunit of which has a molecular weight of 14,000 daltons, this lectin originating from human lung. These authors isolated two cDNA clones by the imrunoscreening of a human hepatoma cDNA library, with the aid of an antiserum which GA binds specifically to a lectin binding p-D-galactoside, A of M.W. 14,000 daltons. r-Aey found that the amino acid I/ I -I 5 sequences of the inserts of these two clones, which they thereby deduced, exhibit substantial homology with one another, with the sequence of chick skin lectin which binds p-D-galactoside, and with 8 peptides derived from purified human lung lectin of M.W. 14,000 daltons.
However, differences between the sequences of the two hepatoma clones and between each of these clones and the human lung peptides suggested to them that at least three variants of the gene which codes for this lectin might exist and might be expressed in human tissue, it being noted, however, that they did not establish the nature of the proteins encoded by the human hepatoma clones I and 2.
In view of the importance of the physiological role of lectins in the ontogenic events and the stages of differentiation, it became apparent that there was a need to establish the homology between the various vertebrate lectins by making every possible effort to determine their sequences, and to broaden the available sources of homologous lectins, in particular for the purpose of their therapeutic use and for diagnosis.
These were the conditions under which the Inventors determined the structural homolocg of different lectins which bind p-D-galactoside, by establishing the amino acid sequences of these lectins, isolated from the electric eel and from human placenta; they compared the sequences which they established with previously published sequences deduced from cDNAs coding for lectins binding p-D-galactoside, isolated from chick embryo skin and from human hepatoma, respectively, and with the partial amino acid sequence of the lectin of human lung; they thus revealed the substantial homologies between these lectins of diverse origin.
The subject of the present invention is an amino acid sequence constituting a peptide, characterized in that it corresponds to part of the sequence of lectins binding B-D-galactoside, and in that the said peptide comprises a fragment which contains a tryptophan as well S as two glutamic acid residues, which fragment forms at S Cw -6a- Ile-Ile-Leu-Pro-Asp-Gly-X 2 -X 2b-X 2 c-X -Phe-Pro-Asn- Arg-Leu (c) in which X 2 is chosen from the group comprising Ser and Leu, X 2 b is Glu, X 2 c is chosen from the group comprising Ile and Phe, X 2 d is chosen in the group comprising Lys and His, with the proviso that when X 2 b -X2c- X 2 d represents Glu-Ile-His, X 2 a is Ser and when X 2 b -X -X2 represents 2a 2pb 2rc 2d Glu-Phe-Lys, X 2 a may be Leu.
According to another advantageous provision of the invention, the said sequence is located downstream of the sequence at a distance of about 30 amino acid residues.
According to yet another provision of the invention, the peptide corresponding to part of the sequence of said lectins additionally comprises, immediately upstream of the said sequence the following amino acid sequence Asp-Gly-Gly-Al (d) According to another provision of the invention, the peptide according to the invention additionally includes, immediately downstream of the said sequence the following amino acid sequence telb Diak b/1.68 MG 7 Ala-Val-Phe-Pro-Phe-Gln-Pro-Gly-Ser-Val-Ala-Glu-Val (e) According to yet another provision of the invention, the said peptide according to the invention comprises an amino acid skeleton which is composed of at least the following amino acids, in the following positions: 2 14 16 19 23 32 serine lysine glycine leucine glycine phenylalanine 37 43 45 46 asparagine glycine lucine leucine histidine 48 49 50 51 56 asparagine proline arginine phenylalanine aspartic acid 61 63 64 70 71 valine asparagine serine tryptophan glycine 73 75 76 81 84 glutamine arginine glutamine phenylalanine glycin3 92 93 104 105 106 threonine phenylalanine leucine proline aspartic acid 107 112 113 114 glycine phenylalanine proline asparagine.
According to a variant of this provision, the said peptide is characterized in that its skeleton additionally comprises the following amino acids in the following positions: 3 5 28 53 54 asparagine valine aspartic acid alanine histidine 67 68 72 74 glycine glycine glycine threonine glutamine proline 91 102 103 109 isoleucine isoleucine isoleucine glutamic acid 132 133 isoleucine lysine.
According to another variant of this provision, the said peptide is characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 8 10 26 27 29 threonine asparagine alanine proline alanine lysine
F.»
«Ir ii-4 8 31 32 36 38 39 serine valine leucine lysine aspartic acid serine 47 53 54 55 58 phenylalanine alanine histidine glycine asparagine 60 62 72 74 78 isoleucine cysteine threonine glutamine valine 82 91 110 115 proline glutamine isoleucine phenylalanine arginine 116 118 128 129 leucine leucine glycine aspartic acid According to yet another variant of this provision, the said peptide is characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 4 6 18 20 21 22 glycine Valine glutamine threonine valine lysine 52 53 54 55 72 aspartic acid alanine histidine glycine threonine 74 80 91 96 glutamine proa,-ne isoleucine asparagine.
According to another variant of this provision, the -aid peptide is characterized in that its skeleton additionally comprises the following amino acids, in the following positions; 28 67 68 74 aspartic acid glycne glycine glutamine 79 phenylalanine.
According to another variant of this provision, the said peptide is characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 26 27 29 30 31 33 alankne proline alanine lysine ser.ne va.ine 3 38 39 40 74 78 lexcine lysine aspartic acid sarine gluitamine valine 79 109 3110 115 phenylalanine glUtanmine phenylalanine arginine -I 9 116 118 123 leucine leucine tyrosine.
According to yet another provision of the invention, the amino acid sequenca constituting a peptide is characterized in that it comprises at least the following 129 amino acids, which are present according to the same sequence in the lectin of the electric eel: 2 (SER MET) ASN GLY PHE LYS ALA GLY VAL PRO SER ILE ASN VAL GLY ASN HIS ILE ASN PRO GLN GLN ALA VAL GLY ASN (TRP) GL PHE PRO PHE LYS GL$ ILE THR PHE ILE LEU PRO ASP VAL VAL ASP GLU ARG MET SER GLN ASN LEU THR VAL ASP SER THR ASN PHE SER ALA GLU ASP LEU ARG PHE ASP ALA HIS VAL VAL ASN SER PHE (THR) GLU GLN (ARG) GLN GLY GLU ASP PHE ASN SER GLU GL’ PHE GLY SER GLU ILE HIS LYS GLY
ALA
ALA
GLY
GLN
GLU
LYS
ARG
110
PHE
ILE
LEU
ASP
GLY
GLY GLY
ILE
100
ILE
PRO
120 ASN ASN ARG TYR MET HIS PHE GLU GLY GLU ALA ARG ILE TYR SER ILE GLU ILE LYS…
According to yet another advantageous provision of the invention, the amino acid sequence is characterized in that it comprises at least the following amino acids, which appear according to the same sequence in the lectin of human plasma: 1 4 6 8 10 11 SER ASN TYR VAL SER THR ASN 18 20 21 23 S( ILE GLY GLU VAL
S,
t I r i
I;
26
ALA
28 PRO ASP GLY LYS ASP 48 50 ASN PRO ARG 61 THR ILE VAL 71 72 TRP GLY THR 81 82 83 PHE GLN PRO 92 93 94 THR PHE ASP 103 104 105 ILE LEU PRO 114 115 116 ASN ARG LEU 128 ALA ASP GLY
ALA
SER
51
PHE
30 31 LYS SER 41 42 ASN ASN 53 34 35 36 PHE VAL LEU ASN LEU 43 45 46 LEU CYS LEU HIS PHE 54 55 56
ASN
LYS ASN 74 GLU GILN 84 85 GLY SER 95 96 GLN ALA 107 1 ASP GLY LI 118 1 ASN LEU 130 ASP PHE ALA HIS GLY LA ASN b6, 68 SER ASP GLY GLY ALA 78 ARG GLU ALA VAL PHE PRO 86 87 88 89 90 9: VAL ALA GLU VAL I 97 100 101 102 ASN LEU LEU VAL LLE 109 110 111 112 113 I GLU PHE LYS PHE PRO 120 121 123 125 U ALA ILE ASN LEU MET ALA 31 132 133 134 135 YS ILE LYS
LE
08 19
GL
1
L
-4 The amino acid sequences according to the present invention correspond to part of the sequence of lectins which bind p-D-galactoside, exhibit greater affinity for p-D-galactosylthiogalactopyranoside than for lactose and greater for the latter than for galactose, and require reducing agents in order to maintain their agglutinating activity; these sequences comprise essential determinants of the said lectins and exhibit cross-reactivity with respect to antibodies directed towards lectins of div.se origin, both human and animal.
According to the present invention, the amino acids of the sequence which reproduces that of the lectin of the electric eel participate in the composition of several peptides which have been analyzed by degradation in a gas phase microsequencer, and of which,. the aminc acids of which they are composed have been identified by high pressure liquid chromatography (HPLC), the following peptides having thereby been identified: E Asn Se Glu Glu Phe Arg
I
«4 11 Ala Phe Tyr Glu le Ile Phe Val lie Phe I1l H7 s (11) Gly Phe Glu Arg.
Gly Asn Met Gly Tyr Tyr Asp Val Gin Arg Ile Phe Val Ala Asp Gin Asn Leu Thr Val Ser Glu His Gly Ser Ser Ala Val Ile Leu Pro Pro Ile Leu Phe Phe Ile Ile His Asn Thr Pro Asn Ser Asn Ala Glu Phe Arg Glu Gly Glu Ala Arg Pro Glu Ile Gl Ile Lys Gly Asp Gin Gin Ala Ser Phe Gin Gin Asn Phe Asn Ser Glu Glu Asp Gly Ser Glu Asn Arg Ile Asp Ser Thr Val Gly Asn Ser Leu His lie Asn Ile Asn Ala Pro According to the invention, the amino acid sequences according to the present invention, which correspond to part of the sequence of lectins as defined above, have a p-pleated sheet structure, the said pleated sheets being at least 10 in nuber, in positions corresponding to the positions of amino acids common to lectins of diverse origin.
The amino acid sequences according to the present invention are additionally distinguished by their hydrophobic character, established with the aid of the K1rE AND DOOLITTLE hydropathy scale.
The amino acid sequences according to the present invention are also distinguished by the fact that the tryptophan which they contain is in a position between Sposition 69 and position 76 of the sequence, as well as two glutamic acid residues, and in that the region 69-76 torms at least in part the p-D-galactoside-binding site.
The said amino acid sequences are distinguished, furthermore, by the fact that they contain at least two uysteine or half-cystine residues which are in positions 44 and 62, and whose chemical state should play an important part in the functional integrity of the L protein.
r Ile Tyr Ser Ile Glu Ile i Ile Tyr Ser Ile Glu Ile Lys /3
-I
12 The amino acid sequences according to the present invention are additionally distinguished by the fact that they contain a terminal peptide blocked on the nitrogen, which is composed of an N-acetylated Ser-Met sequence.
The subject of the present invention is also a process for the purification of lectins binding 6-Dgalactoside, starting from tissues of vertebrate animals and in particular the electric eel and of human organs, such as, in particular, the placenta, by homogenization and fractionation of the animal tissues, followed by isolation by affinity chromatography with the aid of a matrix of lactosyl-Sepharose, which process is characterized in that the elution buffer used to isolate the lectins consists of a phosphate-buffered saline solution of pH 7.2 to which lactose and 2-mercaptoethanol have been added.
The subject o the invention is, in addition, a process for the determination of the sequences or sequencing of amino acids corresponding to part of the sequence of lectilis binding p-D-galactoside which are defined above, which is characterized by the use of an automatic degradation method by coupling phenyl isothiocyanate with the terminal nitrogen of the peptide to be sequenced, cleavage of the said amino-terminal residue by cyclization in an acid medium and conversion of the thiazolinom (sic) derivative formed to a phenylthiohydantoin derivative, which degradation method is carried out in the presence of polybrene.
The subject of the present invention is also a process for obtaining, by synthesis, amino acid sequences A q corresponding to part of the sequence of lectins binding 3-D-galactoside, characterized in that the said synthesis is carried out using a method derived from the method of BERGMAN and ZERVAS and using protective groups to protect the reactive functions of the amino acids to be couple!, and appropriate coupling methods for coupling the protected amino acids with one another in order to form 1RA 4 the sequences sought.
S .The subject of the present invention is, U 13 additionally, anti-human placental lectin polyclonal Santibodies, characterized in that they essentially consist of sera obtained from blood of animals immunized by injection of lectin as defined above, purified by the process for the purification of lectins a.s defined above.
The subject of the present invention is also anti-human placental lectin monoclonal antibodies, characterized in that they consist of clones isolated from hybridomas obtained by fusion of appropriate tumour cells, in particular SP 2-0 or NS-1, with spleen cells of mice immunized against the lec:in of human placenta as defined above, appropriatel. purified, in particular by means of the purification process as defined above.
The subject of the present invention is also an agent for the diagnosis of tumour conditions, characterized in that it comprises polyclonal antibodies and/or monoclonal antibodieo as defined above.
The subject of the present invention is, in addition, a therapeutic agent characterized in that it comprises a lectin comprising the amLno acid sequence (a) and, immediately downstream of the said sequence the amino acid sequence optionally in combination with at least one pharmraceutically acceptable excipient.
The subject of the present invention is, additionally, a therapeutic agent characterized in that it comprises polyclonel antibodies and/or monoclonal antibodies as defined above, optionally in combination with toxins and/or enzymes directed in the same way as the ectin.
Apart from the foregoing provisions, the invention also comprises other provisions which will become apparent from the description which follows, which comprises examples of obtaining the amino acid sequences defined in the foregoing and methods for monitoring the p-D-galactoside-binding lectin activity of these sequences, reference being made to the attached drawings, wherein: Qf Figure 1 shows the amino acid sequence of the S electric eel lectin, bringing out the alignment of the I L^ 1 I I-
F
14 peptides and of the fragments of which it is composed; Figure 2 shows a typical HPLC profile of a digestion of the electrolectin with trypsin, n which each of the fractions obtained by digestion has been analyzed in a gas phase sequencer and in which the corresponding sequence is indicated above the peak; Figure 3 shows a comparison of the sequences obotained, respectively, for the lectins binding A-D-galactoside isolated from electric eel, from human placenta (Hum.p.) and from human lung (Hum. with those obtained from the cDNAs of chick embryo skin and of Li-7 human hepatoma («Hum.hep.1», «Hum.hep.2»); Figure 4 shows the p-pleated sheet structures and the hydropathy profiles of the lectins binding p-Dgalactoside isolated from chick and from electric eel.
The top two curves have been calculated with the aid of the method of CHOU FASMAN [(BIOCHEMISTRY, 13, (1974), 211-222]; the bottom two curves have been calculated using the KYTE DOOLITTLE hydropathy scale MOL.
BIOL. 157 (1982) 105-132]; Figure 5 shows the migration of the human lectin in polyacrylamide gel, obtained by electric focusing; Figure 7 shows the molecular species identified by hybridization, by SOUTHERN’s method; and Figure 8 shows an immunoblot of migration of purified lectin.
It should be clearly understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation.
SEXAMPLE I, Purification of lectin binding p-D-galactoside from electric eel Electric eels, Electrophorus electricus, (obtained Salive from WORLDWIDE PARAMOUNT AQUARIUM/ARDSLEY, were decapitated and their main electric organ was cut up into small cubes and frozen at -2C’C.
Homogenization of the tissues and fractionation *i were carried out as described by LEVI TEICHBERG, J.
BIOL. CHEM., 256 (1981), 5735-5740. The lectins were 15 isolated by affinity chromatography on a matrix of lactosyl-Sepharose as described in the latter publication. The elution buffer consisted of a saline solution of pH 7.2, buffered with 0.01 M phosphate and to which 100 mM lactose and 14 mM 2-mercaptoethanol had been added.
The purity of the eluted lectin was checked by electrophoresis on sodium dodecyl sulphate-polyacrylam .de, and its activity after dialysis against phosphatebuffered saline solution was monitored on trypsinized rabbit erythrocytes in a quantitative haemagglutination test performed on microtitration plates as also described in the publication cited above.
EXAMPLE II Purification of lectin binding p-D-galactoside from human placenta Fresh human placentas originating from nocturnal deliveries were kept on ice until they were collected and treated, starting the following morning.
The lectin was recovered using the procedure described in Example I.
The purity and homogeneity of the human placental lectin were checked by polyacrylamide electrophoresis in the presence of a pH gradient (so-called «isoelectric focusing» technique) according to the following procedure: The pHi of the human lectin (ELH) is determined by isoelectric focusing (IEF), proceeding as follows: Technique Polyacrylamide gel stock solution 24.25 g acrylamide.
S0.75 g bisacrylamide.
2.50 g Amberlite MB-6 (Pharmacia).
250.00 ml distilled water.
Preparation of the gel (plates 115 x 230 mm) 15 ml of the stock solution are filtered.
1.90 ml Pharmalyte (pH gradient 3.10 (sic)).
4.00 ml glycerol.
The mixture is adjusted to 30 ml with H 2 0.
The mixture is outgassed.
ii i 16 300 Pi N,N,N’,N’-tetramethylethylenediamine (TEMED) are added.
The gel is poured.
The gel is left to polymerize for one hour.
Migration: The gel is placed on the cooling stage of the apparatus.
The electrodes are arranged on either side, a strip of LKB paper impregnated with 1 M H 3
PO
4 solution for the anode and 1 M NaOH for the cathode.
Prefocusing.
minutes at 40 mW constant power.
Sample spotting (at 1 cm from the anode).
On LKB paper, 1 x 0.5 cm.
ELH: 20 yl spotted of a solution containing 600 pg/ml, i.e. 12 pg.
Reference proteins: 20 pl Pharmacia standard solution.
Migration: 1 h 30 min, 40 6W constant power.
Visualization: Fixation 45 minutes 10% trichloroacetic acid followed by 39 minutes in a 30% ethanol/5% acetic acid solution.
Staining 1 hour with Coomassie blue.
Decolorization in 40% ethanol 5% acetic acid.
Drying.
The pHi f (migration distance) (cm) is then drawn for the standard proteins.
The pHi of the human le tin determined in this manner is 5.25, as shown in attached Figure EXAMPLE III Separation and purification of the peptides 1. Enzyme digestions,, a) Digestion with trypsin was carried out in 1% NH 4 HC03 by adding 10 pg of trypsin treated with tosylphenylalanyl chloromethyl ketone, dissolved in 0.G’i iM HC1, to I mg of lectin isolated from electric eel ^SIAQN or from human placenta, as described in Examples I and II above. After enzyme digestion for 6 hours at 37*C, a S further 10 Mg of trypsin were added, and the digestion 17 was stopped 6 hours later by lowering the pH to 4. The digestion product was separated by HPLC on an RP 300 column using the following gradient: solvent A: H 2 0/0.1% TFA solvent B: 80% CH 3 CN/20% H20/0.1% TFA (TFA Trifluoroacetic acid).
The two solvents were outgassed by passing a stream of helium through them. The separation was brought to completion using a gradient from 0% of solvent B to 40% of solvent B in the space of 8 hours, at a flow rate of 0.5 ml/minute. The variable wavelength detector was adjusted to 206 nm with a sensitivity of 0.2 nm. The temperature of the operation was 25 0
C.
b) Digestion with Staphylococcus aureus protease (described in HOUMARD DRAPEAU, PROC. NATL.
ACAD. SCI., 69, 3506-3509) was carried out under conditions suitable for producing cleavage at Glu-X peptide bonds: 10 pg of the enzyme (supplied by MILES LABORATORIES) were added to 1 mg of lectin in 50 mM
NH
4
HCO
3 at pH 7.8 and digestion was continued for 18 hours at 37°C. The resulting peptides were separated by HPLC as described above.
2. Cyanogen bromide cleavage.
This cleavage was carried out in 70% formic acid at 25 0 C for 24 hours in the dark, using 10 mg of cyanogen bromide per 1 mg of lecti°,.
The resulting fragments were separated by gel filtration on Sephadex G-50 Superfine in the presence of M guanidine hydrochloride. The fragments were released on Sephadex G-25 in 1% NH 4
HCO
3 and lyophilized.
The peptides obtained by digestion with trypsin and with Staphylocccus aureus protease were separated by reversed-phase chromatography on a WATERS instrument and monitored at 206 nm using a 440 spectrophotometer. Most j 35 of the separations were carried out on an RP 300 column (supplied by BROWNLEE) in 1% trifluoroacetic acid (TFA) buffers, using the gradients described above.
NA4~ EXAMPLE IV Sequencing of the amino acids present in I I the lectin peptides.
I Th ii 18 1. Nequence analysis The different peptides were analyzed by automated EDMAN degradation in a gas phase microsequencer (marketed by APPLIED BIOSYSTEMS). Cheaiically treated polybrene (sold under the trade name «CHEMUBRENE» by CHEMUNEX, PARIS) were added in order to prevent extraction of the peptides and to improve the yields. The phenylthiohydantoin amino acids were identified by HPLC on a WATERS instrument equipped with an RP 18 column 5 p in diameter (supplied by BROWNLEE), using a sodium acetate/acetonitrile gradient.
a) Analysis of the lectin fragments cleaved with cyanogen bromide.
The amino acid composition of the eel lectin indicates the presence of three methionine r,i-‘dues. To take advantage of the presence of these residues, the protein was hydrolyzed using cyanogen bromide in formic acid. Only a single long fragment and a small fragment were isolated, the fragments being purified and partially sequenced. The third peptide with a (blocked) terminal nitrogen was not recovered.
The results of these cleavages are summarized in attached Figure 1, which shows the aligment of the peptides and of the fragments of lectin isolated from electric eel; in this Figure, C 1
C
2 and C 3 denote the fragments obtained by cyanogen bromide cleavage. By replacing 70% formic acid by a 50:50 HFBA/forMiIc acid solution (HFBA heptafluorobutyric acid), cleavage was observed at a further site which corresponds, in all probability, to a tryptophan -X peptide peptide bond (X: unidentified amino acid). This new peptide was partially sequenced to obtain the sequence Gly-(Thr)-Glu-Gln.
b) Analysis of the peptides resulting from digestion with trypsin.
The majority of the sequence of the electric eel lectin and all the results relating to the human protein wore obtained by analysis of the peptides obtained by digestion with trypsin, separated on an RP 300 column as described in section 1.a) of the present Example.
T
-19 Attached Figure 2 shows a typical separation obtained on an RP 300 column; the sequences of the corresponding peptides are indicated along the peaks, the meanings being as follows: A Ala Alanine B Asx Asp or Asn C Cys Cysteine or half-cystine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histicline I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine 0 PCA Cyclized Gln P Pro Proline Q Gin Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan X ()unknown Y Tyr Tyrosine Z Glz Glu or Gin The peaks have been numberedl in accordance with the position of the peptides in th’e whole sequence shown in Figure 1, in which the said peptides are designated TI,
T
2 1 T 3
T
4 1 T51 To, T 7 1 T 8 T9, T 10 1 and T 1 (T =digestion with trypsin), A further two peptides (CT 8) were obtamned, probably by the action of chymotrypsin present in the trypsin.
The sequences indicated at each of the peaks of Figure 2 are as follows:
CT
8 N. S.E. E. F.R=Asn, Ser Giu. Giu. Arg.
T3:A.G.Q.N.L.T.V. iAla.Gly.Gln.Asn.Leu.Thr.Val, CT: F.N. S.E.F. F. R. Phe.Asn. Ser. Glu. Glu. Phe.Arg.
T
10 Y. X.H. F.E. G. E.A. R.=Tyr. Met. His. Phe. Glu. Gly Glu.Ala.Arg.
TO E. G. G.F. P *=Glu.Gly.Gly.Phe.Pro
T
11 S. I.E. I.=Ile. Tyr. Ser. Ile. Glu. Ile T:F D A. H. G. D Q Q V. V. N. S F Q G. G. N=Phe.
Asp. Ala. His. Gly. Asp. Gln -Gln.Ala. Val. Val.
Val.Asn.Ser.Phe.Gln.Gly.Gly.Asn.
T
8 I.Q.1. T. F.N. S. E. E.F. R.=Ile. Gln. Ile. Thr. Phe., As n.Ser. Glu. Glu. Phe. Arg.
T: I. I. L.P. D. G. S-E.I. H.F. P.N. N.R. =Ile. Ile. Leu.
Pro. Asp. Gly. Ser. Glu. Ile. His. Phe. Pro. Asn.
Asn.Arg.
TO G. V. P.S.I. N. F.A. I.N.V. G.N.S. A. E.D. L.
A.L. H. I P. R.Gly. Val. Pro. Ser. Ile. Asp. Ser.
Thr. Asn. Phe .Ala. Ile. As n.Val. Gly, Asn. Ser.
Ala. Glu. As p.Leu, Al aLeu. His. Ile. As n.Pro. Arg.
a’ Analysis of the peptides resulting from digestion with Stphylococcus aureus protease.
Further lectin sequences and overlapping sequences were obtained by analysis of the peptides resulting from the digestion of electric eel lectin with the protease extracted from Staphylococcus aureus which, under the experimental. coiiditions employed, cleaves the proteins at Glu-X peptide bonds. The paptides were separated by HPLC before their sequence was analyzed using the automated sequencer. The peptides cleaved with Staphylococcus aureus protease are designated SPI, SP 2
SP
3 anid SP 4 in Figure 1.
Analyses of the sequen~ces of the peptides obtained both by chemical cleavage and by proteolytic cleavages enabled the whole of the amino acid sequence of the lectin binding p-D-galactoside isolated from the electric eel, and more than 60% of that of thet human placental lectin, to be established. The alignment of the peptides as shown in attached Figure 3 for the lectin of chick, of electric eel, of human placenta of.
21 human lung (Hum.l.) and of human hepatoma (Hum. Hep. 1 and 2) is based on the substantial homologies exhibited by the other lectins with the 14 kD protein which binds P-D-galactoside isolated from chick embryo.
2. Secondary structures of the lectins The characteristic parameters of the secondary structu:es of the lectins were calculated on chick and eel lectins with the aid of software supplied by the University of Wisconsin group for the application of computrers in genetic engineering and a «Microwax II» computer. The results of these calculations are shown in attached Figure 4, which shows the propensity of the lectins to form p-pleated sheet structures and which also shows their hydropathy profiles. The upper two curves which show the p-pleated sheet structures as a function of the number of amino acid residues were calculated with the aid of the method of CHOU FASMAN 11IO- CHEMISTRY, 133, 211-222], and ‘he lower two curves which show the hydropathy as a function of the number of amino acid residues were obtained with the aid of the KYTE VOOLI’TTL, hyropathy scale MOL. BIOL., 157 105-132].
I( Figure 4, the portions of each of the curves ‘which are above the threshold line whose value is 1.00 indicate the amino acid residues probably involved in the p-pleated zheet structures. It follows ftt the chick and eel lectinsi both appear to be composed of at least p-pleated sheets which all consist of amino acid residues in equivalent positions in both proteins.
The same applies to the hydropathy profiles, the similarity of which is vory great in both proteins.
The amino acid sequences which it has been possible to establish, as well the propensity of the i lectins to form p-pleated sheets and their hydropathy profile9, as shown in Fig. 4, demonstrate the homology of the structure of the lectins of vertebrate animals.
The chick and eel lectins contain 51 identical residues out of the 130 positions which have been compared, which corresponds to a 39% homology. The identical residues are distributed over the en’ire length of the proteins, i r 22 except over the final third of the chains, which exhibits much less similarity than the remainder of the polypeptides.
Apart from the 51 identical residues, 27 positions are occupied by homologous residues, encoded by codons which differ by only a single nucleotide. The terminal peptide blocked on the nitrogen is composed of an N-acetylated Ser-Met sequence. Analysis of the peptides resulting from CNBr cleavage in an HFBA/formic acid mixture has enabled position 70 to be assigned to tryptophan. This position 70 assigned to the tryptophan residue is corroborated by the fact that all the sequences of lectins shown in Fig. 3 have the same region 76.
Furthermore, the alignment of the peptides as shown in Figures 1 and 3 is corroborated by the similarity of the regions forming -pleated sheets and of the hydropathy profiles, especially of the chick and eel sequences.
Comparison between the lectins of chick and of human placenta brings out 59 identical residues out of 111 positions compared. In fact, several peptide sequences are identical in both proteins: the sequences 29-33, 35-40, 45-51, 53-56, 70-76, 78-82, 89-91, 100-111 and 125-127.
Comparison between the lectin of eel and the lectin of human placenta brings out 47 identical residues out of 1ll positions compared, Comparison between the partial sequences of the four lectins of human origin shown in Figure 3 brings out significant homologies: 50 residues out of 54 comparable positions (93% homology) are identical in the lectins of human lung rnd placenta, whereas only 41% homology isi found between the lectin of human placenta and the product of the human heipatoma 1 cDNA clone (42 residues out of the 103 residues compared). These rasults show that at least four different genes coding for the lectins Ji. M^ which bind p-D-galactoside may be present in the hutan 4 genoame, and that they have a common origin, the ,n r i P- I- a 23 consequence of which is that the same essential structural determinants, such as, in particular, the B-Dgalactoside-binding site which appears to be located, at least in part, at positions 70-76, 4. to be found in the peptide sequences of the lectina of different animal origin. This peptide sequence contains a tryptophan residue and two glutamic acid residues in positions 73 and 76, respectively.
EXAMPLE V Preparation, by synthesis, of an amino acid sequence corresponding to at least part of a lectin binding p-D-galactoside The peptides sequenced as described in Example IV above were synthesized using the MERRIFIELD solid phase method [see J. AM. CHEM. SOC. (1963), 85, 2194].
The complete synthesis of the electric eel lectin and of the lectin of human placenta will permit a better approach to the properties and constitution of lymphocyte membranes, and the synthesis of the latter lectin may make available an entirely synthetic source for the preparation of an immunostimulant and immunosuppressant therapeutic agent of great value.
EXAMPLE VI Preparation of polyclonal antibodies to the human placental lectin.
Preparation of polyclonal antibodies to the human placental lectin.
Rabbits were injected at time zero with 50 Mg of plac ntal lectin, purified by affinity chromatography, in Freund’s complete adjuvant by multipoint intradermal injection.
The second immunization took place 15 days later with ag of lectin in Freund’s incomplete adjuvant injected subcutaneously.
The third immunization took place 15 days later with ug of lectin in Freund’s incomplete adjuvant injected subcutaneously.
Bleeding was performed regularly every 3 weeks.
From these sera, the immunoglobulin fractions were S purified by DEAE and then tested to determine their antibody activity by the ELISA method and by p< 7 I I -24 immunoblotting. Testing for polyclonal response by the ELISA method: The lectin, diluted in bulffer containing NaCl/,amercaptoethanol to a roiicentration of 5 ug/mi, is bound to a Nunc 96-well ixnxnanplate on the basis of 50 pl per well for 1 h at -3 washes in PBS/l% skimmed milk/0.l% Tween (PX.T). -Saturation 1 h at p1 per well of the different antisera and at diffeorent dilutions (in PMT) are distributed on the plate and incubated for 1 h at 30 0 C. 3 washes in. PMT. 50 p 1 per, well of p~sroxcidase-coupled goat anti-rabbit serum diluted to 1/1,000, GP R-pox. 3 washes in PBS. Visualizatioti with 100 pl of ABTS substrate at a concentration of 1% in acetate buffer pH 4.7 containing 0.2% of 30 vol. UI2O2. The absorpt..on is measured at a wavelength of 405 m. in a Titertek Multiskan analyzer. The results obtained are shown in attached Fig. 6. Test for polyclonal response, demonstrated by iinmunoblotting: Electrophoresils is carried out by SDS-PAGE, which consists of migro-t'ion of, the purified lectin in, thes presence of p-inercaptoethanol on the basis of 8 pg per well on acrylamide gel containing 0,1% of SDS. 250 volts, 4 h. The lectin is then transferred onto nitroc' llulose, at volts for 16 h. Saturation with PMT is carried out for 5 h at 4VC. The nitrocelluilose is cut into strips 1 cm wide. Incubation with antisera is carried out. Washing Is performed wilth PM~ T GAR-pox 1/1,000 (2 mi/strip). washes in PBS/0,1% Triton. Visualization is carried out with chloronaphthol. The immunoblot is shown in attached Fig. 7 (siC), which (N demonstrates that the rabtantisera (purified IgG Sfractions) reatt with the lectint the visualization 1 __II 25 demonstrates a band of molecular weight 14 kD. In Fig. 7 (sic), the incubation schemes are as 'allows: Strip No. 1: physiological fluid, 0.15 M NaC1, 0.01 M sodium phosphate, pH 7.4 (PBS). Strip No. 2: normal mouse serum, 1/50. Strip No. 3: mouse anti-ELH prefusion seruin, 1/50. Strip No. 4: mouse anti-ELH serum, 1/50. EXAMPLE VII -Preparation of anti-human placental lectin monoclonal antibodies. 1. Imiunization Balb/c female mice were immunized by injecting them intravenously once per week for three consecutive weeks with 100 pg of purified lectin. After a three-month rest period, the mice received an intravenous booster with the same quantity of lectin three days before removal of the spleen cells for fusion. 2. Fusion NS 1 mouse myeloma cells are used for the fusion. They were selected for their sensitivity to aminopterin and cultured on RPMI medium containing 10% of foetal calf serum, 2 r.1 glutamine, 1 mM sodium pyruvate, 100 IU/ml of penicillin and 100 Ag/ml of streptomycin. The spleen cells were dispersed by injection of serum-free RPMI medium into the spleen of the hyperimmunized mice and washed thzee times in RPMI medium before fusion. The myeloma cells and ,:he spleen cells were then fused in a ratio of one myeloma cell per 10 spleen cells, in the presence of 41% of polyethylene glycol of weight 1,500 (Merck), according to the method of KOHLER and MILSTEIN (Nature, 256, 495-497, 1975), and then washed in RPMI medium and resuspended in 100 ml of complete RPMI medium. The cells were then redistributed in Nunclon microplates (24 wells per plate) on the basis of 1 ml/ well containing A.2 x 105 cells. After 24 hours, 1 ml/well of medium consisting of complete RPMI medium containing 0.1 mM hypoxanthine, 0.4 M aminopterin and qRBA 16 pM thymidine was added. Aliquot portions of selected tk medium were replaced on days 3, 6 and 10 after fusion. -26 After 15 days, the surviving hybridomas were cultured on complete RPMI medium supplemented with hypoxanthine and thymidine and, 15 days after fusion, the presence of anti-human placental lectin antibodies was tested for in the culture supernatants. The clones were then cultured progressively on normal complete RPMI medium. The positive cultures were cloned by the limiting dilution method according to OI V.T. and HERZENBERG, 1980 Immunoglobulin-producing hybrid cell lines, in "Selected Methods in Cellular Immunoloyv" (Eds. Mishell, 2i R.B. and Shrigi, p. 351, Fillman, San Francisco). The cells were redistributed in the wells of a Nunclon microplate on the basis of 0.5 cell on average per well, with 3 x 105 thymocytes by way of nutritive cells. After 10 days, the wells containing single clones were selected and, 5 days late::, the supernatants were tested to determine the presence of anti-yeast antibodies. Three clones derived from a positive primary culture were selected and injected into Balb/c mice to obtain large amounts of antibodies. To this end, elderly Balb/c female mice were stimulated with an intraperitoneal injection of 0.3 ml of tetramethylpentadecane. After 4 days, 20 million hybrid cells were injected into the mice. After 15 days, the ascites fluids were harvested and their anti-yeast activity was tested. EXAMPLE VIII Cloning o oligonucleotide probes. Cloning was carried out by synthesizing two oligonucleotide probes based on the peptide sequences of the human placental lectin, between residues 48 and 56 and 70 and 82, respectively. 4 major molecular species were identified in a genome library by SOUTHERN's hybridization method, and their cloning; was carried out. More specifically, hybridization of A 4 31 cell DNA, digested with EcoRI, and phage A DNA, digested with HindIII, was carried out with the 32 P-labelled P38 probe. The specific activity of the P38 probe is V A 3 x i0 5 cpm/pmole. 27 27 Figure 7 shows the SOUTHERN's hybridization carried out under the above conditions. 1 Phage A DNA 10 pg per well 2 A A 4 5 DNA 10 Ag per well. EXAMPLE IX Applications of anti-human placental lectin polyclonal and monoclonal antibodies. 1. The polyclonal antibodies to the human placental lectin, prepared as described in Example VI above, have been used for revealing the presence and size of the placental lectin in a mixture of proteins subjected to polyacrylamide gel electrophoresis followed by electrophoretic transfer onto a cellulose nitrate membrane and absorption of the antibodies (so-called ''immunoblotting" technique), by the immunoblotting technique, which is carried out as described above in Example VI. 2. The polyclonal antibodies to the human placental lectin, prepared as described in Example VI above, have been used for detecting the presence of lectin at the surface of certain lymphoid cells, both normal and tumour cells. The results obtained to date suggest that the level of lectin expressed at the surface of tumour cells varies in accordance with the capacity of these cells to spread and form metastases. These polyclonal antibodies accordingly constitute efficaceous detection agents for the presence of metastases. 3. The antilectin polyclonal or monoclonal antibodies additionally constitute useful diagnostic agents for determination of the metastatic capacity of tumours, using one of the known techniques such as Ammunofluorescence and ELISA, in particular. 4. The antilectin polyclonal and monoclonal antibodies additionally constitute valuable therapeutic 1 products, in that they may be used as carriers for targeting toxins or enzymes to tumour cells expressing the lectin at their surface. Claims (7) 1. Amino acid sequence constituting a peptide, characterized in that it corresponds to part of the sequence of lectins binding p-D-galactoside, and in that the said peptide comprises a fragment which contains a tryptophan as well as two glutamic acid residues, which fragment forms at least in part the p-D-galactoside- binding site and comprises the amino acid sequence (a) Trp-Gly-Thr-Glu-Gln-Arg-Glu (a) or an amino acid sequence in which not more than one of the amino acif,..s Thr or Gin has been replaced by another amino acid, with the proviso that the said peptide does not contain the sequences Gly-Ser-Asn, Met-Glu-Glu, Ala- Gly-Ala or the amino acid Thr immediately upstream of the said fragment. 2. Peptide according to Claim 1, characterized in that it additionally comprises the sequence: X1-Xi-Asn-X,-Gly (b) in which X i is a hydrophobic amino acid chosen from the group comprising Val, Ala, Met, Ile and Leu upstream of the sequence 3. Peptide *cording to Claim 2, characterized in that the said sequence is Irated upstream of the sequence at a distance of about 35 amino acid resi- dues. 4. Peptide according to Claim 1, characterized in that it additionally includes, downstream of the said sequence the following amino acid sequence: .lLou Pre goAcap GlyX X rro -AX. X. Lo (jp)' Peptide according to Claim 4, characteriz d in that the said sequence is located downst of the sequence at a distance of about 30 no acid resi- dues. 6. Peptide according to aim 1 or Claim 2, charac- terized in chat it a d-ionally comprises, immediately upstream of the d sequence the following amino acid seque Asp-Gly-Gly-Ala (d) 'f nnt fn artm~f-n^ fcf~it~aii 1 phAraeter zed f-4-i- 1 -28a- Ile-I ie-Leu-Pro-Asp-Giy-X 2 a b -x 2 c -x 2 d Phe-Pro-Asn- Arg-Leu (c) in which X 2 a is chosen from the group comprising Ser and Leu, X 2b is Giu, X 2 cis chosen from the group comprising Ile and Phe, X 2d is chosen in the group comprising Lys and His, with the proviso that when X 2 b -X 2 c X 2 d represents Glu-Ile-His, X 2 a is Ser and when x 2 b -X 2 c- X2d represents Glu-Phe-Lys, X 2 a may be Leu. Peptide accord-ing to claim 4, characterized in that the said sequence is located downstream of sequence (a) at a distance of about 30 amino acid residues. 6. Peptide according to ciaim 1 or claim 2, characterized in that it additionally comprises, immediately upstream of the said sequence the following amino acid sequence Asp-Gly-Gly-Ala (d) S S S S. S 0S SS S S S. S S S *S Peptide according to claim 1, characterized in S 55.555 S *SSS S S 55 S 55.5 B. S S S. S 55.5.. 4, Malb Disk~ 9/1.68 MG I u r-~p 29 that it additionally includes, immediately downstream of the said sequence the following amino acid sequence Ala-Val-Phe-Pro-Phe-Gln-Pro-Gly-Ser-Val-Ala-Glu-Val (e) 8. Peptide according to Claim 1, characterized in that it comprises an amino acid skeleton which is com- posed of at least the following amino acids, in the following positions: 2 14 16 19 23 32 serine lysine glycine leucine glycine phenylalanine 37 43 45 46 asparagine glycine leucine leucine histidine 48 49 50 51 56 asparagine proline arginine phenylalanine aspartic acid 61 63 64 70 71 valine asparagine serine tryptophan glycine 73 75 76 81 84 glutamine arginine glutamine phenylalanine glycine 92 93 104 105 106 threonine phenylalanine leucine proline aspartic acid 107 112 113 114 glycine phenylalanine proline asparagine. 9. Peptide according to Claim 8, characterized in that its skeleton additionally comprises the following amino acids in the following positions: 3 5 28 53 54 asparagine valine aspartic acid alanine histidine 67 68 72 74 glycine glycine glycine threonine glutamine proline 91 102 103 109 isoleucine isoleucine isoleucine gl:tamic acid 132 133 isoleucine lysine. Peptide according to Claim 8, characterized in that its skeleton additionally comprises the following amino acids, in the following positions: /6 4 t I 30 8 10 26 27 29 threonine asparagine alanine proline alanine lysine 31 32 36 38 39 serine valine leucine lysine aspartic acid serine 47 53 54 55 58 phenylalanine alanine histidine glycine asparagine 62 72 74 78 isoleucine cysteine threonine glutamine valine 82 91 110 115 proline glutamine isoleucine phenylalanine arginine 116 118 128 129 leucine leucine glycine aspartic acid 11. Peptide according to Claim 8, characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 4 6 18 20 21 22 glycine valine glutamine threonine valine lysine 52 53 54 55 72 aspartic acid alanine histidine glycine threonine 74 80 91 96 glutamine proline isoleucine asparagine. 12. Peptide according to Claim 8, characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 28 67 68 74 aspartic acid glycine glycine glutamine 79 phenylalanine. 13. Peptide according to Claim 8, characterized in that its skeleton additionally comprises the following amino acids, in the following positions: 26 27 29 30 31 33 alanine proline alanine lysine serine valine 36 38 39 40 74 78 leucine lysine aspartic acid serine glutamine valine 79 109 110 115 phenylalanine glutamine phenylalanine arginine S116 118 123 leucine leucine tyrosine. 31 14. Amino acid sequence according to Claim 1, charac- terized in that it comprises at least the following 129 amino acids, which are present according to the same sequence in the lectin of the electric eel: 2 (SER MET) ASN GLY VAL VAL ASP GLU ARG MET SER PHE LYS ALA GLY GLN ASN LEU THR VAL LYS GLY VAL PRO SER ILE ASP SER THR ASN PHE ALA ILE ASN VAL GLY ASN SER ALA GLU ASP LEU ALA LEU HIS ILE ASN PRO ARG PHE ASP ALA HIS GLY ASP GLN GLN ALA VAL VAL VAL ASN SER PHE GLN GLY GLY ASN (TRP) GLY (THR) GLU GLN (ARG) GLU GLY GLY PHE PRO PHE LYS GLN GLY GLU ASP PHE LYS ILE 100 GLN ILE THR PHE ASN SER GLU GLU PHE ARG ILE 110 ILE LEU PRO ASP GLY SER GLU ILE HIS PHE PRO 120 ASN ASN ARG TYR MET HIS PHE GLU GLY GLU ALA ARG ILE TYR SER ILE GLU ILE LYS... Amino acid sequence according to Claim 1, charac- terized in that it comprises at least the following amino acids, which appear according to the same sequence in the lectin of human placenta: ()0 32 1 SEFL 4 6 ASN TYR VAL SER 8 THR 10 11 AS9N 21 23 )ILE GLY GLU VAL 26 ALA GLr 48 ASN THR TRP 81 PE 92 THR 103 ILE 114 ASN ALA 16. 28 PRO ASP LYS ASP 50 PRO ARG 61 ILE VAL 71 72 GLY THR 82 83 GLN PRO 93 94 PHE ASP 104 105 LET! PRO 115 116 ARG LET) 128 ASP GLY Amino ALA SER 51 PHE 30 31 LYS SER 41 42 ASN ASN 53 ASN ALA PE 43 LEU 54 HIS 34 35 VAL LEU ASN 45 CYS LET) HIS 55 56 36 LET) 46 PE LYS ASN 74 GLU GLN 84 85 GLY SER 95 96 GLN ALA SER( 107 ASP GLY 118 ASN LET) ASP 10~ LET 11~ GLI 13( PHI ARG 86 VAL 97 ASN 3 109 J GLU )120 J ALA 131 SLYS GLU 87 ALA LEU 11 PH GLY ASP ALA ASN 67 68 ASP GLY GLY ALA 78 ALA VAL PilE PRO 88 89 91 GLT) VAL (90) 100 101 102 LET) VAL LLE 0 11ll 11 41 11~3 E LYS PHE PRO LE 121 123 ILE ASN4 132 ILE LEU 133 LYS 125 MET 134 ALA, 135 acid~ sequences characterized in that they have been isolated from the amino acid sequence to Claim 14 and in that they correspond to the peptide formulae: Asn Ala Phe Tyr Glu Ile Ile Phe Val Ile Ser Gly Asn Met Gly Tyr Tyr Asp Val Gin Giu Gln Ser His Gly Ser Ser Aia Val Ile Glu Asn Glu Phe Phiv Ile Ile His Asn Thr Phe Leu Glu Glu -Glu -Gly -Ser -Phe Arg Thr Val Phe Arc -Gly Glu- -Ile Sle Lys -Asp Gin -Phe -Gin- -Asn -Ser- according following Ala -Arg Gin Al~Ja- Gin -Asn Giu Glu- 33 Phe Arzg Ile Ile Leu Pro Asp Gly Ser Glu lie His Phe Pro Asn Asn Arg (11) Gly Val Pro Ser Ile Asp Ser Tlr Asn Phe Ala Ile Asn Val Gly Asn Ser Ala Glu Asp Leu Ala Leu His Ile Asn Pro Arg. 17. Amnino acid sequence according to any one of Claims 1 toAll, characterized in that it has a /3-pleated sheet structure and in that these sheets are at least in number, in positions of amino acids common to lectinO of diverse origin. 18. Amino acid sequence according to any one of Claims 1 to 17, characterized in that it contains at least two cysteine or half'-cystine residues which are in positions 44 and 62. 19. Amino acid sequence according to any one of Claims 1 to 18, characterized in that it contains a terminal peptide blocked on the nitrogen, which is composed of an N-acetylated Ser-Met sequence. 2 -Proooos for the purifiation of loetinz bini- -D-galactcside according to any one of Claims 1 19, starting from tissues of vertebrate anim s and in particular the electric eel and of human gans, such as, in particular, the placenta, b omogenization and fractionation of the animal t' sues, followed by isola- tion by affinity chromat aphy with the aid of a matrix of lactosyl-Sepharo which process is characterized in that the elut' buffer used to isolate the lectins consists a phosphate-buffered saline solution of pH 7 to which lactose and 2-mercaptoethanol have been 24. 0 Process for the determination of the sequences or sequencing of amino acids corresponding to part of the sequence of lectins binding p-D-galactoside according to any one of Claims 1 to 19, characterized by the use of an automatic degradation method by coupling phenyl Oat,) isothiocyanate with the terminal nitrogen of the peptide Sto be sequenced, cleavage of" the said amino-terminal I ;i i 34 residue by cyclization in an acid medium and conversion of the thiazolinom (sic) derivative formed to a phenyl- thiohydantoin derivative, which degradation method is carried out in the presence of polybrene. .l Process for obtaining, by synthesis, amino acid sequences corresponding to part of the sequence of lectins binding p-D-galactoside, characterized in that the said synthesis is carried out using a method derived from the method of EERGMAN and ZERVAS and using protec- tive groups to protect the reactive function. of the amino acids to be coupled, and appropriate coupling methods for coupling the protected amino acids with one another in order to form the sequences sought according to any of Claims 1 to 19. Anti-human placental lectin polyclonal anti- bodies, characterized in that they escantially consist of sera obtained fron blood of animals immunized by injec- tion of lectin according to any one of Claims 1 to 15 and 17 to 19, prified U<-Ma Anti-human placental lectin monoclonal anti- bodies, characterized in that they consist of clones isolated from hybridomas obtained by fusion of appropri- ate tumour cells, in particular SP 2-0 or NS-1, with spleen cells of mice immunized against the lectin of human placenta, according to any one of Claims 1 to and 17 to 19. pp-i t-- 4-.A Agent for the diagnosis of tumour conditions, characterized in that it comprises polyclonal antibodies according to Claim 23. and/or monoclonal antibodies amqording to Claiml*. aI,-S Therapeutic agent characterized in that it comprises a lectin according to Claim 1 or Claim 7, optionally in combination with at least one pharmacouti- cally acceptable excipient. 2 t6. Therapeutic agent characterized in that it comprises polyclonal antibodies according to claim 23 /Q and/or monoclonal antibodies according to Claim 24, optionally in combination with toxins and/or enzymes 35 directed in the same way as the lectin. 0~y FIL1' I I Q\ SE&R tiASN GLY VAL. VAL ASP GLU ARG 111E SER H ASfI VAL GLv Asii SER iu.. GLU ASP LEu ALA LEu His b's ALA GiLY GIN AsN LEu 1THR -3_ 2O~3 VL Y's GLV VAL PRO SER ILE ASP SER lfiA AsN Pit ALA ILE ILE ASN v _-w Pao ARG PtiE Asp ALA (J0 His GLY Asp GIN GIN ALA VAL VAL 15 VAL AsNi SER P* Gou GLY 6LY Aqaj(Th)GLY(THRI)GLU GWNARG)IU4J GLY GLV PHIE PRO P1* Icys Gui GLv Guu Asp PHE LYS ILE GIN ILE TH iPlIE ASu T SER Gcu CLu Pvt ARG ILE ~2 2 100 ILE L~u Peo AsP GLv SEA (Lu ALE o -P -19 0 110 r -120 HIS PHt PRO ASN AsN ARG TYR 111E His PHE (Lu GLv (Lu A A G ILE TYR SEA 'OLE (Lu ILE LYS 110 '2' bh wo 89/005812 P[F8/07 0 IN~JECTION ABSoRBANCE AT 206 N NJ F.E F- IC I~r I. 0 E.K 0.A.H.G F.O G. G.H. 1.I. L P. D. G. S. F.1. H. F. P. N. N. R- Mr* wo 89/0058 1 WO 8900581P CTIFR 88/00370 3 /8 Chick Eel J"wm. ho,. PtL7 L CTS CLA CLY PRO VAL CYS JEMLC L Z 4 ~c CL -TL 15CR KEY ASH CLY VAL VAL ALP GLJ A.A jL7 SLJ, PI4C jT 2 ALA CLY Eja ASA T Y VAZL SEPI 01) E CLU VAL LYS I'AS P MZ'k ILY PO LY ChickQ "e*r ?IUJY.4J. 100 AJOG LLV TNA VAL LYS OLT ILLC ILL JALA PRO IASH IALA LYS SCR ,DIC VAL [~JASN LEU TIIR VAL LYS CLY VAL PRO SCA ILEIL APISC T~ik ASH I M A LA 5C1 TlA~JI ILL M CLY CL VAL ALA PASP LA LYSl ASP PMC VAL Chick ML AS1J LLU CLY LY'S ASP SEA I A 1135 LCV Eel LC A F DCLI A5N~ SL14 ALA CLU AS5P LEU LLV ASOi LLLJ GL LYS ASP SEA ASH ASH LCU ui.I'a~. LE AS14 LC CLI LT LAPE VI ASH I' LCU LLU 113S PHI5: ASH PRO LCU HISFI~ASH PRO AMCO LEU MIS P14C ASU PAO 'k J LEU LEU HIS PSIL ASN Pf40 A I to Chick 1 mL ASP ALA HIS CLY ASP VAL E lLCU 1LEC VAL CYS ASH SEA LYS LY'S MET~ Eel (Phl ASP ALA 1115 CLY ASP GLN4 CLN ALA VAL VALW7ALASJ SE PHlL CLN CLY I1.. ME AJ7 HALA HIS CLI ASP, ALA ASN 7141 ILI VAL crs Ano lift ASP. LYI 0 L", 8 C Chick CLU CLV T"J CLY Imm CLU CLN A14C CLU VlS AL Phit PAO0 PHlL CLN LIS CLY Ee "L S P. CLY 1711R)CGLU CLN A14C CLU I IL PSIC PAO PHlL LI CLY HUm, p, CLI' ALA T kP GLY 7111 CLV CLN AA(; CLV ALA VAL PIIL PAO PRlE CLN P10 CLI Wmp CL.1 ALA TAP CLI )I C-L) C L LV A HTYPE PC Hum, hItp.I S Cx ASH T KP CLI CZ LU LM C~l ASC L ASP HIS5 L&U CYS Pl PHC CR LT Num~op 2 TA RP OLT Tp11G CLV SALG AJ IC PRO ALA C~FH PiiC ICLr PRO CLI' F IG-3. WO 89/00581 PCTIFR88M0370 3'/18 Chick Eel3 Hum, P, HumI. r) P. I ALA PIK0I CLLI ASP 157 VA;.. FLY LZS 11 LC r.. AL-A CL V ILE 7"111 PtIC 5R ILE AS?;III,:) SCk ALCP LCU THiA 1LE Trit PHl nS? SEP CLU CLU PliC X1Mr I LC THAi VP1E AS? GLN ALA ASHF ELU A C4.LU VAL. IM VA L TH CGLI SEA ASP LY'S PD4C LYS 7 1 [jILE TH~~A L CIS LE VIA PtE ASP CLJ4 ALA ASP[FL C U T AEIL Chick Eel mtfi hp. 4 I4 PAO ASP GLY HSCI C H H LEV) PkO ASP C;LY HIS CLI) PtIL LYS PNC P RO PRO $P0o P SO PRO ASH XHC LCU CLY jE UC4 ASH t- ASN(A)_TY~t J r, ASH XA LCU A5t4 LI.:I ASH LEU ASS LCJ ASH4 A.FiC LCU LHI ASt? ARC LCUAS Chick sc~VAL ?IiC ASP [3 ?HE ASP ThA "I LYAP PH CLTHR LEU ARC 5 E VAL Eel "ItS PhEk <;Lt CLY GLI) ALAAC T TYA SEP.I )ILE CLUF ILE LYS Hum~, P, GLI) AL.A tLf ASN LCIJ MET ALA ALA ASSP GLr ASP PNE LYS ILE LYS 't L LI ALA ILC A~ TYk Mu~r~p~I 5 L~~fYA~ IL AG LY' LP1 ASN ET SLY SEA PI(L LI ALA ILE ASO TYh4 MEt ALA ALA ASIP CLY ALPI' FHC LYS t.Lr LIS JVAL AhMj Chick SIA U.LU Eel W4uti, Wuai p NLufther I LYS 2 L YS CL' Huim.hvp 2 CLVLQ F1G-3EN BETA SHEETS HYDROPATHY 0 .Qi0 0 0-C 0 0 C> tri 0 rnI OLEWR»All.Dd I OW/69 0 At PCr/FM/003-70 5.20-.. 4. 5 5 1 -9$ FIG- wo 89/WSS I WO 8900581PCTJFRSS/00370 6/8 FIG. 6 HPLI 4HP LIZ2 0.27 mg4r -WHPLY 3 0OA rri allPLY 4 1.42 m In c4HPL7 5 .54n n6 d HPLYZ d HP L:Tt P Ly 4 dHLY 3 5 2 dition ‘112 3 REPLACEMENT 1 SE8ET 1’1 :VO 89/0″081 PCTFRs/00oo370 7/8 ke,. I do Iii;’ I I Hybridization of AA 31 cell DNA, digested with EcoRI, and phage A DNA, digested with HindIII, with the 3 2 P-labelled P38 probe. The stringency of the washes decreases from left to right. P38 specific activity: 3 x 105 cpm/pmole 1 phage A DNA 10 pg/well 2 A4 3 1 DNA 10 pg/well WO 89/00581 PCTIFRSS/00370 8 /8 WL TQ» A*.4tL I j. NMS 44, O NR,.oHPL .M4 FIG_. 6 INTERNATIONAL SEARCH REPORT International Application No PCT/FR88/00370 1. CLASSIFICATION OF SUBJECT MATTER (if sevaral classifcation symbcls apply, Indicate all) According to International Patent Classficatlon (IPC) or to b6th National Classification and IPC SC 07 K 13/00;7/06;7/08;7/10;C 07 K 3/20;A 61 K 39/395; Int.Ci :G 01 N 33/577;//C 07 K 15/06;C 07 K 15/08 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System I Classification Symbols nt.cl4 G 07 K 13/00;7/00;C 07 K 3./00;A 61 K 39/00; G 01 N 33/00;C 07 K 15/00; A 61 K 37/00; 35/00 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched ill, DOCUMENTS CONSIDEKI TO BE RELEVANT’ Category I Citation of Documei 11 with indication, where appropriate, of the relevant passages 12 Relevant to Claim No, 13 X 71st Annual Neeting of the Federation of 1-8 American Societies for Experimental Bio- logy»,Washington,D.C.29 March 02 April 1987;Federation Proceedings,Vol.46,No 3 A.D.Strosberg et al.:»Extensive structur- al homology between lactose binding lec- tins from fish,birds,and iMammals»,page 947; see the whole article X Biological Abstracts,Vol.79,No.. ,1985,abstr- 26 act No.622 Philadelphia,US;J. H’irabayashi et al.:»Human placenta beta- galactoside- binding lectin:Purification and some pro perties»,& Biochem Biophys Res.Commun 122 (3),938-944,1984,see abstract A Proc.Natl.Acad.Sci.USA,Vol.83,October 1986, The National Academy of Sciences (US) M.A.Gitt et al.:»Evidence that a human soluble beta-glactoside-binding lectin is encoded by a family of genes»,pages

7603-7607; cited in the application SSpecial categories of cited documental Io later document published after the International filing date document defiing the genera state of the art which is not o priority date and not n onfit with the application but considered to aeof part r relevance cited to understand the principle or theory underlying the earlier document ut published orn or after the International document of particular relevance the claimed Invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on prlority claim(s) or involve an Inventive step which is cited to establish the publication date of another document of particular relevance!’ the claimed Invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu. other means ments, such combination being obvious to a person skilled document published prior to the International fling date but In the art, later than the priority date claimed document member of the same patent family IV, CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report October 1988(05.10.88) 04 November 1988(04.11.88) International Searching Authority Sgnature of Authorized Officer European Patent Office Form PCT/ISA2t0 (second sheet) (January 198) Intomralonl Application No. PCT/FR 88/00370 I’l. DOCUMENTS CONSIDERED TO 01 RELEVANT (CONTINUED FROM THE SISCOND SIIJE catagovy Citon f Doawnut, with dton, wireapropWth o b Pe elent g Riwnt to Clalm No 2 Chemical Abstracts,Vol.107,1987 (Columbus Ohio,US) C.Southan et al.;»Amino acid sequence of beta-galactoside-binding bovine heart lectin.Member of a novel class of vertebrate proteins»,see page 277,abstract No.73017y Febs Lett.1987, 214(2),301-4 Form PCTIISAm1 (wito Shet) (4usuy i1) IL U IC RAPPORT DE RECHERCHE INTERN ATIONALSc Osmtinds Internationale N1 PCT/FR 8 8/ 00 37 0 1, CLASSEMIN? OR L’INVUP4TION (si Piulour symboles do classification sont spplicablos, lot Indiquor tous)I Solon Is classification Internatioafl@ des brovets (CIS) ou A Is loisolnIclsicaon atnl.elCB 4 C 07 K 13/00; 7/06; 7/08 7/ 10; C 07 K 3/20 A 61 K39/395; CIB G 01 N 33/577; 07 K 15/06; C 07 K 15/08 1 OMAINES SUR LISQUILS LA MECHERCHK A POR011T9 Documentation minimal. consultio I Syt.16me do classification Symboles do classification 4 C 07 K 13/00; 7/00; C 07 K 3/00; A 61 K 39/00; CIB G 01 N 33/00; C 07 K 15/00; A 61 K 37/00; 35/00 Documentation coniut~o suite quo as documentation minimal. dens Is mosuro 04~ do tlis documents font Part!@ des domain., our Iosqials Ia rechercheo a Porti III, OOCUMINTS C0JNSID111RIs COMME PIRTINP4T1.4 Catigorio Identification des documents dIl»~ ay~c Indication, I n~cossagro, N, dos rovandlcations dos passages perignonts Is visi 2; X 71st Annual Meeting of the Federation 1-8 of American Societies for Experimnental Biology», Washington, D.C. 29 mars 2 avril 1987; Federation Proceedings, vol. 46, no. 3 A.D. Strosberg et «Extensive structural homology between lactose binding lectins from fish, birds, and mammxals», page 947, voir 3.article en entier X Biological Abstracts, vol. 79, no. l, 26 1985, r~sUni6 no, 622 Philadelpia, US; J, I-irabayashi et al,: «Human placenta beta-galacto- id-binding lectin: Pu.irif~catioa and some properties», Biochem Biophys Res, Commun 122(0), 938-944, 1984, voir resume *Cal~gorles sp6ciales do documents cit~o, mi To document Ult~roUr pUbII4 postiourm*M4ntds diOd~ a A douenol dfinhson l~lt gn~ra do10 tchnouononinternational Cu A Is date do Pflori t~ -01 npofnant Pas *A cn dhr isw commo aa~c in4ro o Is porun n iqo no I141. do Is technique Continent, mois citi pout comptendro coriold~~~~~~~~til compaI41(netPnnn ,rincipo ou Ia thiorio constituent Is bass do iInvonion oa document ant~rlour, maie oubli k 14 dais do d4p~t inttrI44 a o document Porticulrnmonl porlinont: ‘1invonlln tovondi. lionl o apt. ctto atequis no pout Acre tofliditA conmo nouvoll 6wJ Comma 00L documeont pouvont )slot un douto our un. rovendicalion do impiiquant un. activili invontivo priorito ou citA Pout w~ormr i a ota do pubic*lc ~O ayodcmn aiojlrmn o1nn~Inolinl~n sute citation ou Pour une ralson optclsle (tells q ‘ndiqui.) diquit no pout &(to Consid~ro comma implioujant un. *On document 0t r~firant A uno divulgation oral*,.A un usage, A Ictinilt InventIwo loroque 10 ldocUmont ort soci i Aun ou uno exposition ou tout sutros movons PlusoIIts autros documons do m~mo nour, tie dombi. o F a doCumorit Publl6 event Is date do dm81t International, mao noso 6tant #yidtnts pout Un. porson’io du milior. poti rermont A 14 data do Pfiorlt# rovendiqumo is As document qi feit Pirtle do Ia mAin. famills do btovols IV, CERlIPICATION (Jai: A laquoilo Is reclirorIintormationslo a 416 offoctivomont 01054 octobre 1988 AdministtoliOri chatillo do is tochocho Internationale OFFIC EUROPEEV4 DES BREVETS rotmuiairs PCTIISA1210 (dousi~me fouillo) (Janivto isss) Date Wesp4dilton dU Present rapport Vo rochorcho Internatlionals ?I k~ *0it1t 1 ~2 boemandit Internationale N, PCT/F’R 88/00370 -2- (suiTE o’8 mrmaiEGNmemars imwOIuds SUR LA Ill. DOCUMINTS CONSIDints COMME !ERTIMNTa DEUXIBMK FIEUILLI) Cat~gorie’ lio dee dowuflts w avloe Wdicaiofi. 0 1, sare, W doe reveikation, I doe P41SM pmei»tnes OlI A Proc. Natl. Acad. Sci, USA, vol. 83, octobxe 1986, The National Academy of Sciences (US) M.A. Gitt et al.: «Evidence that a, human soluble beta-galactoside- binding lectin is encoded by a family of genes», pages 7603-7607 (cit6 dans 3.4 deniande) A Chemical. Abstracts, vol. 107, 1987 (Columbus, Ohio, US) C. Southan et al.: «Amino acid sequence of beta-ga lactos ide -bindingj bovIne heart lectia. Member of a novel cl~as s of vertebrate proteins», voir page 277, r~suni6 no. 73017y Vebs Lett. 1987, 214(2), 301-4 FwmWeaIre OCTAISAWOt (IOUl i dlannelle) (.J4wMWIN)

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Lectines fixing beta-d-galactoside

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SEQUENCES OF AMINO ACIDS REPRODUCING AT LEAST IN PART THE SEQUENCE OF ANIMAL AND HUMAN LECTINS, THEIR PROCESSES FOR OBTAINING THEIR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS

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Anti-galectin-1 monoclonal antibodies and fragments thereof

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