EP0781337A1

EP0781337A1 - Humanised antibody to integrin chain beta-1

Info

Publication number: EP0781337A1
Application number: EP95930574A
Authority: EP
Inventors: Christelle Margaritte; Marie-Alix San. Francisco Gen. Hospital POUL; Marie-Paule Lefranc; Florence Cervoni Mary; Alain Bernard
Original assignee: Proteine Performance SA; Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Proteine Performance SA; Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 1994-09-12
Filing date: 1995-09-12
Publication date: 1997-07-02
Also published as: FR2724393A1; WO1996008564A1; CA2199749A1

Abstract

Variable domains of a non-human immunoglobulin are humanised by selecting DNA sequences coding for the variable domains of a human immunoglobulin wherein the amino acid sequence of the framework regions of variable domains VH and VL is 70-95 % homologous with the amino acid sequence of the framework regions of variable domains VH and VL of non-human immunoglobulin, and wherein hypervariable loops H1 and H2 or L1, L2 and L3 have the same kind of canonical structure as hypervariable loops H1 and H2 or L1, L2 and L3, respectively, of non-human immunoglobulin, and by replacing the oligonucleotide sequences coding for the CDRs of human immunoglobulin with oligonucleotide sequences coding for the corresponding CDRs of non-human immunoglobulin. The cloning, in a baculovirus, of DNA fragments that code for said humanised variable domains, and the expression of the resulting humanised recombinant antibody, are also disclosed. Murine monoclonal antibody K20 has thus been humanised and expressed according to the Invention.

Description

HUMANIZED ANTIBODIES DIRECTED AGAINST THE BETA 1 CHAIN OF INTEGRINS

The present invention relates to obtaining, in insect cells, a humanized recombinant monoclonal antibody.

The monoclonal antibody K20 is a murine antibody which recognizes the β1 (CD29) subunit of integrins. In vi tro experiments have shown that K20 can inhibit the activation and proliferation of peripheral T lymphocytes induced by an anti-CD3 antibody [GROUX et al., Nature, 339, 152-154 (1989); TICCHIONI et al., J. Immunol. , 151, 119-127 (1993)]. It could therefore be considered using this antibody as part of immunosuppressive therapy.

However, the use of murine antibodies in human therapy is very limited, since repeated injections of non-human antibodies induce in patients an immune response which prevents long-term treatment.

In order to avoid this drawback, it has been proposed to make recombinant antibodies by genetic engineering, in which as much of the molecule as possible is derived from a gene of human origin.

So-called chimeric antibodies were obtained in which only the variable parts are of non-human origin.

To further reduce the risks of immune response, there is an increasing search at present for obtaining so-called humanized antibodies, the sequences of the variable parts of which are not directly involved in the recognition of the antigen are replaced by sequences of human origin. The humanization of a non-human antibody therefore involves determining with the greatest precision possible what are the amino acid residues of the non-human antibody which are involved in the recognition of the antigen (and which must be preserved in the humanized antibody), and which ones can, on the contrary, be replaced to reproduce the sequences of a human antibody, without affecting or affecting as little as possible, the recognition of the antigen.

The recognition of the antigen involves in particular the regions of the variable parts called CDR (regions determining the complementarity), but it was also noted that the positioning of certain amino acid residues of the framework regions (FR regions) could also influence conformation of the antigenic site.

The aim of the present invention is to humanize the murine antibody K20, and to express the humanized antibody thus obtained in an appropriate vector / host system. To humanize the antibody K20, the amino acid sequence of the VJJ and V _L variable regions of this antibody was first determined, which makes it possible to locate the CDRs. Conventionally, in fact, the humanization of an antibody is carried out by grafting its CDRs in place of the CDRs of a human antibody.

The inventors have had the idea of determining in addition, from the sequence of the variable regions of K20, the type of canonical structure of the hypervariable loops L1, L2, L3, H1 and H2. The canonical structures, which have been observed for most of the hypervariable loops (with the exception of the H3 loop), and which are conserved between different species, are determined by a small number of key residues from the FR regions and the CDRs: different types of canonical structures were defined, according to the size of the loop, and the nature of amino acid residues it comprises [CHOTIA and LESK, J. Mol. Biol., 196 (4), p 901, (1987); CHOTIA et al., Nature, 342, p 877, (1989), • CHOTIA et al., J. Mol. Biol. , 227 (3), p799, (1992)]. The inventors then selected human V _H and V _{L regions} whose amino acid sequence has a high homology (preferably between 74 and 95% homology) with the sequence of the Vfj and VL regions of the murine antibody K20, and whose hypervariable loops L1, L2, L3, Hl and H2 have the same type of canonical structure as the corresponding hypervariable loops of said murine antibody.

In the human Vg and V regions thus selected, the inventors then replaced the CDRs with the corresponding CDRs of the murine antibody K20, in order to obtain the _VH and _{VL regions} of the humanized antibody.

The process described above can be generalized to the humanization of the variable regions of any monoclonal antibody of non-human origin, in particular of murine origin.

The subject of the present invention is a method for humanizing the variable region Vy and / or the variable region V _j _, of a non-human immunoglobulin, which method is characterized in that it comprises at least the following steps :

a step in which a DNA sequence coding for the variable region V _∑ j or for the variable region V _L of a human immunoglobulin is selected, the amino acid sequence of the FR regions of said VJJ region or of said region Human VL having respectively between 70 and 95% homology with the amino acid sequence of the FR regions of the Vpj region or of the V _L region of the non-human immunoglobulin, and the hypervariable loops Hl and H2 of said region V Human _H or the hypervariable loops L1, L2 and L3 of said region Human VL with the same type of canonical structure as the H1 and H2 hypervariable loops of the V _H region or the L1, L2 and L3 hypervariable loops of the V _L region of non-human immunoglobulin, respectively - a step where proceeds to replace the oligonucleotide sequences coding for the CDRs of said human Vβ or V _{L region} , by the oligonucleotide sequences coding for the corresponding CDRs of the VJJ or VL region of non-human immunoglobulin. At the end of this step, a sequence coding for the Vy or VL region of humanized immunoglobulin is obtained.

The process according to the invention can be completed by the point mutation (replacement, addition or deletion of a codon) of one or more oligonucleotide sequences coding for one or more amino acids located in the FRs of the VJJ or V _{L region.} .

Such a mutation can for example be carried out in the case where the amino acid sequence of the V _H region or of the human V _L region which has been selected for its homology with the non-human VJJ or VL region, present, at a determined position, an "unusual" amino acid for the variability subgroup to which it belongs. It may then be appropriate to replace this amino acid with a more representative amino acid. In general, we will try to get as close as possible to the consensus sequences of the FRs of the most homologous human subgroup, in order to further reduce the immunogenicity of the humanized antibody.

It may also be advisable to carry out such a mutation when it is desired to increase the affinity of the humanized antibody for the antigen, - in this case we will seek to approximate the canonical structure of the hypervariable loops of the parental non-human immunoglobulin. The present invention also relates to a DNA fragment, coding for the variable region V _H or for the variable region VL of a humanized antibody, and characterized in that it is capable of being obtained by the method according to the invention, as defined above. A DNA fragment coding for the V _H variable region or for the VL variable region of a humanized antibody according to the invention can then be fused with a DNA fragment coding for the constant region of an H or L chain. human, in order to express the complete H and L chains obtained in this way.

According to a preferred embodiment of the present invention, said humanized antibody is obtained from the murine antibody K20. In the following, a humanized antibody obtained by the process according to the invention will be designated by the abbreviation Hu-Ac, and more particularly, the humanized antibody derived from the antibody K20, by the abbreviation Hu-K20.

The present invention relates to a DNA fragment, characterized in that it is chosen from the group consisting of:

a sequence coding for the variable domain of the light chain of the Hu-Ac antibody, fused to a sequence coding for the constant domain of the light chain of a human immunoglobulin; or - a sequence coding for the variable domain of the heavy chain of the Hu-Ac antibody, fused to a sequence coding for the constant domain of the heavy chain of a human immunoglobulin.

To express a humanized antibody in accordance with the invention, use will preferably be made of an expression system which has also been developed by the inventors, which uses an expression vector derived from a baculovirus, and makes it possible to produce both the heavy chain (H) and the light chain (L) of a given antibody in an insect cell. The present invention also relates to an expression cassette comprising a recombinant DNA sequence constituted by a sequence coding for the variable region of the light chain of the Hu-Ac antibody fused to a sequence coding for the constant domain of the light chain of a human immunoglobulin; or by a sequence coding for the variable region of the heavy chain of the Hu-Ac antibody fused to a sequence coding for the constant domain of the light chain of a human immunoglobulin, which recombinant DNA sequence is placed under transcriptional control. an appropriate promoter.

According to a preferred embodiment of the present invention, said promoter is a baculovirus promoter.

By way of example of baculovirus promoters which can be used for the implementation of the present invention, mention will be made of polyhedrin and P10 baculovirus promoters AcMNPV or S1MNPV, or derivatives of baculovirus promoters, constituted by synthetic promoters or recombinants, obtained from a baculovirus promoter, and functional in insect cells, such as for example that described by WANG et al, [Gene, 100, 131-137, (1991)]. An expression cassette according to this embodiment comprises for example.

- a baculovirus promoter, as defined above; a sequence coding for a secretory signal peptide

a DNA sequence coding for the H chain or the L chain of an HU-Ac antibody, as defined above.

A large number of sequences coding for signal peptides functional in insect cells can be used for the implementation of the present Invention. By way of nonlimiting example, mention will be made of the sequences coding for the signal peptides of Drosophila acetylcholinesterase, of ovine trophoblastin, as well as the sequences coding for the signal peptides of the H and L chains of murine or human immunoglobulins, etc.

Each of the cassettes according to the invention allows the expression, either of the light chain or of the heavy chain, of an Hu-Ac antibody, having the specificity of the parental monoclonal antibody.

The sequence coding for the constant domain of the light chain can be chosen from the sequences coding for the constant domains of the light chains kappa (K) and lambda (λ). The sequence coding for the constant domain of the heavy chain can be chosen from the sequences coding for the constant domains of the heavy chains γl, γ2, γ3, γ4, α, ε, andμ. It is thus possible to obtain a recombinant antibody belonging to the immunoglobulin class (IgG1, lgG2, IgG3, IgG4, IgA, IgE, IgM) which is desired.

The present invention also relates to recombinant vectors, carrying at least one expression cassette as defined above, - in this context, the present invention includes in particular recombinant baculoviruses allowing the expression of the antibody Hu- Ac, as well as transfer plasmids allowing the construction of said recombinant baculoviruses. The transfer plasmids in accordance with the invention carry an insert comprising: an expression cassette as defined above, and on either side of this cassette, baculovirus sequences homologous to those of the regions flanking the portion of the viral genome to replace which one wishes to insert said cassette. g

According to a preferred embodiment of the transfer plasmids in accordance with the present invention, said baculovirus sequences are homologous to those of the regions flanking the p10O gene, or homologous to those of the regions flanking the polyhedrin gene.

According to a particularly advantageous arrangement of this embodiment, the expression cassette containing the gene coding for the light chain of the Hu-Ac antibody is flanked by the regions surrounding the polyhedrin gene in wild baculovirus, and the cassette d The expression carrying the gene coding for the heavy chain of the Hu-Ac antibody is flanked by the regions surrounding the PlO gene in the wild baculovirus.

Schematically, the construction of the transfer plasmids in accordance with the invention is done by inserting into a plasmid capable of replicating in a bacterial host (in general E. coli), the region of the baculovirus gene (for example plO or polyhedrin, or any other baculovirus locus) in place of which it is desired to insert the genes coding for the H or L chains of immunoglobulin. In this region, the coding sequence of the baculovirus gene (and possibly the promoter sequence of said gene) is replaced by the sequence coding for the immunoglobulin chain to be expressed (and optionally by the promoter sequence under whose control it is desired to express this chain. immunoglobulin, if it is for example a "derivative" promoter). The transfer plasmid thus obtained therefore contains an insert comprising a heterologous sequence (sequence of a chain of the Hu-Ac antibody) flanked by baculovirus sequences.

To allow the simultaneous expression of the heavy chain (H chain) and the light chain (L chain) and their reassociation to form the molecule of recombinant antibodies, the inventors inserted the two cassettes on the same expression vector. They thus obtained a recombinant double baculovirus in which the coding sequence of each of the H and L chains is under the control of a strong promoter.

A recombinant baculovirus in accordance with the invention, allowing the expression of the Hu-Ac antibody, can be constructed according to the following principle:

- One prepares separately two transfer plasmids, as defined above: one for the H chain, and one for the L chain.

The insect cells are then cotransfected with the DNA of the transfer vectors thus produced and the DNA of the baculovirus. This cotransfection is carried out in two stages:

The transfer plasmid containing the expression cassette for the light chain gene flanked by the regions surrounding the polyhedrin gene in wild baculovirus, is used, with the wild baculovirus DNA AcMNPV, to cotransfect insect cells in culture.

By homologous recombination between the viral DNA and the plasmid, the light chain coding sequences of the recombinant immunoglobulin are transferred into the viral genome.

After replication of the viral DNA in the transfected cells, the recombinant baculoviruses which have integrated the light chain sequence of the recombinant immunoglobulin are selected. These recombinant baculoviruses are selected according to two criteria: their inability to produce polyhedra and their capacity to express the light chain.

In a second step, the cells are cotransfected with the DNA of the recombinant baculovirus obtained at the end of the first step, and with that of the transfer plasmid containing the expression cassette. carrying the gene coding for the heavy chain of the recombinant Hu-Ac antibody flanked by the regions surrounding the PlO gene of baculovirus. By homologous recombination, as before, the heavy chain gene is transferred into the viral DNA.

One then selects the double-recombinant viruses which are capable of simultaneously producing a heavy chain and a light chain of immunoglobulin. The detection of the heavy chain and the light chain of immunoglobulin is carried out by ELISA.

The subject of the present invention is a recombinant baculovirus, characterized in that it comprises at least one expression cassette, as defined above, comprising a sequence coding all or part of the H chain or a sequence coding for all or part of the L chain of the Hu-Ac antibody, which sequence is placed under transcriptional control of a strong baculovirus promoter.

According to one embodiment of a recombinant baculovirus according to the invention, it comprises an expression cassette comprising the sequence coding for the H chain and an expression cassette comprising the sequence coding for the L chain, of the antibody Hu -Ac.

According to a preferred arrangement of this embodiment, the promoter controlling the transcription of the sequence coding for the L chain of the Hu-Ac antibody, and the promoter controlling the transcription of the sequence coding for the H chain of the Hu antibody -Ac, are two different promoters. According to an advantageous modality of this provision, one of said promoters is located at the location occupied in the wild baculovirus, by the polyhedrin promoter and the other is located at the location occupied in the wild baculovirus, by the promoter of PlO. According to yet another advantageous modality of this arrangement, one of the promoters is the promoter of polyhedrin or one of its derivatives, and the other is the promoter of plO or one of its derivatives. Advantageously, the promoter controlling the transcription of the sequence encoding the light chain of the Hu-Ac antibody is the promoter of polyhedrin or one of its derivatives, and the promoter controlling the transcription of the sequence encoding the heavy chain of the Hu-Ac antibody is the promoter of PlO or one of its derivatives.

According to another arrangement of this embodiment, the sequence coding for the signal peptide associated with the L chain of the Hu-Ac antibody, and the sequence coding for the signal peptide associated with the H chain of the Hu-Ac antibody , are two different sequences.

A recombinant baculovirus conforming to the invention was deposited on September 9, 1994, with the C.N.C.M. (National Collection of Cultures of Microorganisms, held by the Institut Pasteur, 25 rue du Docteur ROUX, Paris), under the number 1-1476.

The present invention also encompasses insect cells infected with a recombinant baculovirus according to the invention.

The expression and production of the recombinant monoclonal antibody Hu-Ac is obtained in vitro in insect cells according to the invention.

Infection of the cells with a double recombinant baculovirus in accordance with the invention results in the simultaneous production of the H and L chains. These chains come together to reconstitute the Hu-Ac antibody which is subsequently secreted in the culture medium. .

The present invention also relates to a process for the preparation of a humanized recombinant antibody, characterized in that it comprises a step at during which insect cells infected with a recombinant baculovirus according to the invention are cultivated, and a step during which said antibody is obtained from the culture medium. A humanized recombinant antibody in accordance with the invention can be used for diagnosis, or for therapy. A particularly advantageous use, in the case of the humanized antibody K20, relates to obtaining immunosuppressive drugs. The present invention will be better understood with the aid of the additional description which follows, which refers to examples of obtaining, in accordance with the invention, the humanized recombinant antibody Hu-K20, conforming in a recombinant baculovirus. It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention of which they do not in any way constitute a limitation. Example 1. Cloning and sequencing of the variable parts of the heavy chain and of the light chain of the antibody K20.

MAb K20 is a murine antibody produced by a hybridoma. It is directed against the β subunit of the CD29 receptor of lymphocytes [BOUMSELL et al. , J. Exp. Med. 152, p. 229 (1980)].

The total RNA of the hybridoma producing the monoclonal antibody K20 was extracted using the "RNA extraction Kit" from PHARMACIA. The cDNA is synthesized from 10 μg of RNA by reverse transcription (First-Strand cDNA Synthesis kit; PHARMACIA).

The variable parts of the heavy chains (VJJ) and light chains (VL) are amplified by PCR (polymerase chain amplification) from 1 μg of single-stranded cDNA, using Taq DNA polymerase. The amplification products are purified by electrophoresis on 1.2% agarose gel to separate them. oligonucleotides; the band corresponding to the desired product is excised from the gel, and the DNA is eluted by centrifugation (microcentrifuge SPIN-X, COSTAR, Cambridge, MA), then extracted with phenol / chloroform and precipitated with ethanol,

1. Cloning of the variable region of the light chain K of K20:

The reverse transcription was carried out using the primer VKIFOR: * VK1F0R (SEQ ID NO: 1):

5 '- GGT AGA TCT CAG CTT GGT CCC 3'

This primer is complementary to the consensus sequence at the 3 'end of the VK variable region of the murine genes, and has a BglII site (underlined). The cDNA obtained was amplified by PCR using on the one hand the primer VKIFOR above, and on the other hand the primer VKIBAC: * VK1BAC (SEQ ID NO: 2): 5'- GAC ATT CAG CTG ACC CAG TCT CCA -3 * (PvuII site underlined).

These primers have been described by ORLANDI et al. [Proc. Natl. Acad. Sci. USA, 86, p 3833, (1989)].

The amplification product was recovered by electrophoresis on agarose gel, extraction with phenol / chloroform and precipitation with ethanol, then digested with PvuII and BglII. and the fragment obtained was cloned into phage M13VKPCR1 (ORLANDI et al., cited above), previously digested with PvuII and Bell, to give phage M13VK.K20PCR1 (FIG. 1A). 2- Clonacre of the variable region of the heavy chain yl of K20:

Reverse transcription on the total RNA extracted from the K20-producing hybridoma was carried out using the primer VH1FOR. This same primer, and the primer VH1BAC were subsequently used to amplify the V ^ region from the cDNA: * VH1F0R (SEQ ID NO: 3):

5'-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CCA G-3 '(BstEII site underlined). * VH1BAC (SEQ ID NO: 4): 5'-AG GT (C / G) (A / C) A (A / G) CTG CAG (C / G) AG TC (A / T) GG-3 '( Pstl site underlined).

These primers have been described by ORLANDI et al. (publication cited above).

The amplification product was purified as described above, then digested with BstEII and Pstl. and the fragment obtained was cloned into phage M13VHPCR1

(ORLANDI et al., Publication cited above), previously digested with BstEII and Pstl. to give phage

M13VHK20PCR1. FIG. 1A represents the stages of the cloning of the V _κ and V _H sequences of K20. Phages M13VK.K20PCR1 and

M13VHK20PCR1 include, in addition to the K20 sequences, an Ig H chain promoter (P), and the leader sequence

(L) of the mouse V _H V47 gene. The nucleotide sequence of the K20 inserts of phages M13VKK20PCR1 and M13VHK20PCR1 has been determined.

The sequence of the insert coding for the variable region V _κ of the light chain of the antibody K20 is represented in FIG. 2 A, and identified on the list of sequences in the appendix under the number SEQ ID NO: 5.

The sequence of the insert coding for the VJJ variable region of the heavy chain of the antibody K20 is represented in FIG. 2B, and identified on the list of sequences in the appendix under the number SEQ ID NO .- 6. The locations CDRs, as well as those of the primers which have been used, and those of the restriction sites which have allowed cloning are indicated in these Figures 2A and 2B. Example 2. Humanization of the variable parts of the heavy chain and the light chain of the antibody K20.

FIG. 1B represents the stages of humanization of the variable regions V _κ and Vg of K20. 1. Humanization of the variable region of the light chain K of K20:

M13VKPCR1 contains a V _κ gene of mouse, which comprises the framework regions (FRs) of the K protein REI human myeloma, and the complementarity determining regions (CDRs) of the mouse monoclonal antibody D1.3 [Verhoeyen et al. , Science, 239, p 1534

(1988)].

The framework regions of this V _gène gene were used to humanize the V _κ gene of K20. 3 oligonucleotides, VKMUTCDR1, VKMUTCDR2, and

VKMUTCDR3, corresponding respectively to CDRs 1, 2, and 3 of the chain V _κ of K20 were built, in order to replace the CDRs V _κ of D1.3. These 3 oligonucleotides also include 12 additional nucleotides at the 5 'end and at least 12 additional nucleotides at the 3' end, including a C or G terminal, and complementary to the immediate vicinity of the CDR to be replaced.

The mutagenesis was carried out according to the protocol recommended by KUNDLE [Proc. Natl. Acad. Sci. USA, 82, p 488 (1985)]

The phage resulting from the replacement in M13VKPCR1, of CDRs V _κ of D1.3 by the CDRs of V _κ of K20, is called M13HuVKK20PCRl

2. Humanization of the variable region of the heavy chain of K20:

The framework regions of the human V _H gene F1 (EMBL accession number X17675) were used to humanize the VJJ gene of K20. The Vg FI gene was amplified by PCR from the vector M13mpl8VHFlRF. [MILILI et al., Mol. Immunol. , 28, p 753 (1991)], using the primers VH1BAK and VH1FOR described above. The amplification product was purified as described above, then digested with BstEII and Pstl. and the fragment obtained was cloned into phage M13VHPCR1.

3 oligonucleotides, VHMUTCDR1, VHMUTCDR2, and VHMUTCDR3, corresponding respectively to CDRs 1, 2, and 3 of the VJJ chain of K20, were constructed, in order to replace the CDRs V _H of F1 by those of K20.

Mutagenesis was carried out as described above, and the resulting phage is named M13HuVHK20PCR1. The reverse complementary sequences to those of the oligonucleotides VKMUTCDR1, VKMUTCDR2, VKMUTCDR3, VHMUTCDR1, VHMUTCDR2, and VHMUTCDR3, used for humanization of the variable regions of the heavy and light chains are represented in FIG. 3, and are also identified in the sequence list attached under the respective numbers SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

In FIG. 3, the nucleotides corresponding to the CDRs of the humanized genes are underlined; the nucleotides initially existing in the VKREI / D1.3 gene and in the VHF1 gene are in capital letters; nucleotides changed by mutagenesis are in lowercase letters; the locations of the nucleotides deleted by mutagenesis of the initial genes are represented by dashes. Example 3 Construction of Transfer Plasmids Carrying Humanized Variable K20 Sequences

A) TRANSFER PLASMID FOR THE KAPPA LIGHT CHAIN: 1) Obtaining the plasmid pBO a- Plasmid pGmAcll6T:

This vector is derived from the plasmid pGmAcll5T [ROYER et al., J. Virol., 66, 3230-3235, (1992)], itself derived from the plasmid pAcl [CHAABIHI et al. , J. Virol., 67, 2664-2671 (1993)] containing the EcoRI-I fragment of the autographa californica nuclear polyhedrosis baculovirus (AcMNPV), and therefore the polyhedrin gene, and the sequences flanking said gene. To obtain pGmAcll6T, the plasmid pGmAcllST was deleted from a 1900 bp fragment going from an EcoRI site located upstream of the polyhedrin gene to an Xhol site located 1900 bp downstream of this E≤≤RI site.

5 μg of the plasmid pGmAcllST were digested for 2 hours at 37 ° C. with 15 units of Xhol enzyme (BOEHRINGER), in a reaction volume of 50 μl and under the conditions recommended by the supplier. After extraction with phenol / chloroform, the plasmid DNA was precipitated with alcohol. This DNA was then partially cut by EcoRI (BOEHRINGER) in a reaction volume of 50 μl in the presence of 0.5 unit of enzyme. The incubation was carried out at 37 ° C for 20 minutes. After a new phenol / chloroform extraction, the ends generated by the Xhol and EcoRI cleavages were made blunt by the Klenow enzyme (BIOLABS) in the presence of the 4 dNTPs, according to the protocol recommended by the supplier. The plasmid DNA was finally precipitated with alcohol and incubated with the ligase of phage T4 (BOEHRINGER) under the conditions recommended by the supplier.

Competent E. coli bacteria have been transformed by part of the ligation mixture; the screening of the colonies resulting from this transformation has allowed to select the plasmid pGmAcll6T. This plasmid contains a BglII site downstream of the polyhedrin promoter. b- Signal peptide: The coding sequence of this peptide is that of the signal peptide of a mouse V _H gene [NEUBERGER MS, EMBO J., 2, 1373-1378, (1983)].

This sequence was chemically synthesized in the form of two complementary oligonucleotides having ends allowing the insertion of the duplex into a BglII site. For pairing, 15 μg of each of the two oligonucleotides are incubated in 50 μl of buffer (1 mM Tris pH 7.5, 0.1 mM EDTA), for 5 minutes in a water bath at 70 ° C. The bath is then left to cool to room temperature (22 to 25 ° C). The pairing product is used directly in the ligation reactions with the plasmid pGmAcll6T previously cut with BglII.

The ligation conditions are as follows:

1 μg of the plasmid pGmAcll6T cut with BglII; 1 μg of the double-stranded oligonucleotide carrying the sequence coding for the signal peptide; 2 μl of 10X ligase buffer (BOEHRINGER); distilled water qs 19 μl; 1 unit (Îμl) of ligase (BOEHRINGER). Incubation is carried out at 22 ° C for 2 hours; the ligation product is used to transform competent E. coli bacteria, c- CK constant region: The coding sequence of the constant region of the human light chain K was amplified by PCR using as template B cell lymphocyte cDNA humans. Human lymphocytes (approximately 5 × 10 ⁸ ) were prepared from 200 ml of blood using HISTOPAQUE® (SIGMA). Total RNA was extracted from these lymphocytes using a PHARMACIA kit (RNA extraction kit). The first strand of cDNA was prepared from total RNA using the "First-Strand cDNA synthesis kit" from PHARMACIA.

The primers used to amplify the cDNA CK are the following: * HuCKBAC (SEQ ID NO: 13): 5 '-AG CTC GAG ATC AAA CGG-3' (the Xhol site is underlined).

This primer corresponds to a consensus sequence in 3 ′ of the sequences coding for the variable domains of the light chains of mouse immunoglobulins (JK), and contains a site of cleavage by Xhol. * HuCKFOR (SEQ ID NO: 14): 5 '-GAA GAT CTA ACA CTC TCC GCG GTT GAA G-3 * (the BglII site is underlined). This primer is complementary to the end

3 ′ of the human CK genes and provides a BglII site downstream of the TAG stop codon.

Amplification with the primers HUCKBAC and HUCKFOR made it possible to obtain a fragment of approximately 340 bp containing the entire CK region framed by the Xhol and BglII sites.

The amplification product was digested with BσlII and Xhol before being cloned into the Xhol-BglII sites of the plasmid pGmAc carrying the sequence coding for the signal peptide.

The composition of the ligation mixture is as follows: l μg of the plasmid pGmAc cut with Xhol and

BglII, - 200 ng of the CK fragment amplified and digested with BglII and Xhol, 2 μl of 10X ligase buffer (BOEHRINGER), distilled water q.s.p. 19 μl, 1 unit (lμl) of ligase

(BOEHRINGER).

Incubation is carried out at 22 ° C for 2 hours; the ligation product is used to transform competent E. coli bacteria.

The plasmid obtained is called pBOc. 2) Cloning of the humanized variable region of the K chain of K20

The cloning of the humanized variable region of the light chain K of K20 was carried out as follows:

The humanized V _κ region of K20 was amplified by PCR using: as template, the DNA of the phage M13HuVKK20PCRl described in Example 2 above, - - an OPP-SoVK primer (3 '), the sequence of which is the next one:

* OPP-SθVK (3 ') (SEQ ID NO: 15):

5 '-ACG TTT CTC GAG CYT GGT SCC - 3' (Xhol site underlined) Y represents C or T, and S represents C or G

- a primer VKK20Hu (5 '), the sequence of which is as follows:

* VKK20Hu (5 ') (SEQ ID NO: 16): 5' - GAC ATC GAG CTC ACC CAG- 3 '(Sacl site underlined)

These primers provide the Xhol and Ssal £ restriction sites respectively.

Amplification was performed in 30 successive incubation cycles at 95 ° C for 30 seconds, 40 ° C for 30 seconds, and 72 ° C for 30 seconds, then was followed by incubation at 72 ° C for 10 seconds. minutes.

After amplification of the V _κ region, digestion with the Sacl and Xhol enzymes of the amplification product was carried out and the fragment obtained was inserted between the Sacl and Xhol sites of the plasmid pBLUESCRIPT IIKS; the ligation product is used to transform competent E. coli bacteria.

A few clones are selected, and sequenced for verification. The plasmid DNA of the selected clone is prepared, digested with Sac1 and Xhol, and the Sacl-Xhol fragment of 303 bp is purified.

The SacI-Xhol fragment is introduced into pBCK. The composition of the ligation mixture is as follows:

2 μl of 10X buffer for T4-DNA ligase (BOEHRINGER); 100 ng of the SacI-Xhol fragment. 1 μl of the plasmid pBO cut with Sacl and Xhol; 1 μl of ligase (1 unit), and distilled water q.s.p. 20 μl.

The incubation is carried out at 16 ° C for 8 hours then the ligation product is used to transform competent E. coli bacteria.

A clone containing the plasmid pBCK-VκK20hu is selected, which will be used as a transfer vector.

B) TRANSFER PLASMID FOR HEAVY CHAIN

1) Obtaining the plasmid pBCvl a- Transfer plasmid: The plasmid pGml6 [BLANC et al, Virology, 192,

651-654, (1993)] is derived from a plasmid into which the EcoRI-P fragment of the baculovirus AcMNPV containing the plO gene has been cloned. Almost all of the coding sequence has been deleted and replaced by a BglII site allowing the insertion of sequences to be expressed under the control of the p10 promoter. b- Signal peptide: The coding sequence of this peptide is that of a mouse V _H gene (NEUBERGER MS., 1983. EMBO J, 2, 1373-1378). It has been chemically synthesized in the form of complementary strands, so that it can be inserted into a BglII site. The pairing and ligation conditions are identical to those used for the light chain. c- Human constant regions (Cγl): The cDNA of the coding sequence of the human Cγl region was amplified by PCR using the following primers: * HuCγlBAC (SEQ ID NO: 17):

5 'CAA GGT ACC ACG GTC ACC GTC TCC - 3' (Kpnl site underlined).

This primer includes a Kpnl site. * HuCγlFOR (SEQ ID NO: 18): 5 '-GAAGATC TCA TTT ACC CGG AGA CAG GGA G-3' (BglII site underlined).

The primer makes it possible to reconstitute, after amplification, a BglII site downstream of the stop codon.

The template used to amplify the human Cγl region is the same mixture of cDNAs as that used for the amplification of the CK sequences.

The amplification product was sequenced and cloned into the transfer vector pGml6 carrying the sequence coding for the signal peptide. The construction obtained was called pBCγl.

2) Cloning of the humanized variable region of the heavy chain of K20

The cloning of the humanized VJJ region of the heavy chain of K20 was carried out as follows:

The humanized VJJ variable region of K20 was amplified by PCR using:

- As a template, the DNA of phage M13HuVHK20PCRl described in Example 2 above; - the primer VH1BAC below (SEQ ID NO: 19)

5'-AGG TSM ARC TGC AGS AGT WCG GG-3 '(Pstl site underlined)

S = C OR G; M = A OR C; R = A OR G; W = A OR T - the VHFOR primer below (SEQ ID NO: 20):

5'-TGA GGA GAC GGT GAC CGT GGT ACC TTG GCC CCA G-3 ' (Bs_ £ EII and Kpnl sites underlined).

The VHFOR primer differs from the VH1FOR primer in that a C has been replaced by an A to create a site

Kpnl. These primers provide the Pstl and Kpnl enzyme restriction sites respectively.

After amplification, a Pstl-Kpnl digestion of the amplification fragment obtained was carried out and the fragment obtained was inserted between the Pstl and Kpnl sites of the plasmid pBLUESCRIPT IIKS; the ligation product is used to transform competent E. coli bacteria.

A few clones are selected, and verified by sequencing. The plasmid DNA of the selected clone is prepared, digested with Pstl and Kpn11. and the Pstl- fragment

Kpnl of 327 bp is purified.

The Pstl-Kpnl fragment is introduced into pBCγl to give the loaded plasmid pBCγi-VHK20hu. Example 4: Construction of a recombinant baculovirus producing the humanized K20 antibody a- Insertion of the heavy chain:

The loaded plasmid pBCγi-VHK20hu was used in cotransfection with the DNA of a modified baculovirus called AcSLplO [CHAABIHI et al., J. Virol., 67, 2664-2671

(1993)], which lacks the polyhedrin gene

(promoter + coding sequence), but carries the coding sequence of the polyhedrin under the control of the PlO promoter, in the natural PlO locus. As this virus produces polyhedra in infected cells, recombination at the PlO locus can thus be easily detected. The cotransfection conditions are as follows: 500 ng of viral DNA are mixed with 5 μg of plasmid DNA and 40 μl of DOTAP solution (BOEHRINGER) in 3 ml of culture medium without serum for insect cells. This mixture is used to cover 4 x 10 ⁶ Sf9 cells (ATCC35CRL 1711); after 4 hours of contact, the cotransfection mixture is replaced with 4 ml of complete medium and the incubation is carried out at 27 ° C. for 5 days. Following the cotransfection, the virus producing the heavy chain of the humanized K20 antibody under the control of the PlO promoter was purified by the lysis plaque technique. This virus was called bPPl- HK20hu. b- Insertion of the light chain:

The loaded plasmid pBCκ-VκK20hu was used in cotransfection with the DNA of the modified baculovirus bPPl-HK20hu.

The double recombinants were selected by the technique of limiting dilution, associated with ELISA.

After cotransfection, a range of dilutions of the infectious supernatant is made, and each dilution is used for infection of insect cells. Three days after infection, the supernatants are tested by ELISA to find the presence of correctly assembled human-type antibodies. The supernatants from the most positive wells for the highest dilutions are in turn diluted and used to infect other cultures; after a few dilution / infection cycles, the supernatants enriched in baculovirus producing the whole antibody are spread on a cell carpet, and cloned by the lysis plaque method. A clone named Acl0HK20hu-33LK20hu (111) was selected. This clone was deposited with the CNCM, on September 9, 1994, under the number 1-1476. Example 5 - Production and purification of the humanized antibody K20 (Hu-K20):

The double reco hiding virus AclOHK20hu-

33LK20hu (III) was amplified by a series of passages on cultured insect cells. The viral stock was then used to infect a culture shaken in a spinner (500 ml of culture with 10 ⁶ cells per ml).

After 72 hours of infection, the culture is harvested and centrifuged at 1000 g to clarify the supernatant. The latter was concentrated to 1/3 of its initial volume by centrifugation through a membrane having a cutoff threshold of 30 kDa (CENTRIPEP 30, Amicon) (1st centrifugation: 1000g, 20 ° C, 30 minutes; elimination of the filtrate; 2nd centrifugation: 1000g, 20 ° C, 20 minutes).

The solution was equilibrated in a protein A binding buffer, by dilution in this buffer followed by a new concentration by centrifugation (dilution buffer: Glycine 1.5M, 3M NaCl; pH 8.9). The balanced solution is then passed through a protein A column, which is itself balanced in the same buffer as the Hu-K20 solution. After rinsing the column, the antibody is eluted with an elution buffer (0.1M acetate, 0.5M NaCl; pH3). The elution is followed by measuring the OD at 280 nm. The fractions containing the antibody were mixed and concentrated by centrifugation through a membrane having a cutoff threshold of 10 kDa

(CENTRIPEP 10, AMICON). The concentrated solution is diluted with PBS and then reconcentrated in the same way. The antibody solution obtained is stored at + 4 ° C in PBS (137 mM NaCl; 2.7 mM KC1; 4.3 mM Na ₂ HPO ₄ -7H ₂ 0; 1.4 mM KH ₂ P0 ₄ .

The quality of the antibody was checked by polyacrylamide-SDS gel electrophoresis, using a commercial human IgG1 (SIGMA) as control. This experiment showed that the antibody unreduced chimera (intact disulfide bridges) migrates at the same level as the control human antibody.

After treatment with dithiotreitol (DTT), the appearance of two bands corresponding to the heavy and light chains is observed, and migrating at the same level as the chains of the control human antibody also reduced to DTT.

To confirm the previous results, the proteins of the fractions comprising the antibody were transferred onto a nitrocellulose membrane, and the H and L chains were detected by antibodies specific for the human Cγl and CK region.

This experiment demonstrates that the antibody produced by insect cells is indeed made up of H and L chains, and that the constant parts of these chains are recognized by specific antibodies.

The selected virus AclOHK20hu-33LK20hu (lll) is multiplied on Sf9 insect cells in TGV2 medium

(2% fetal calf serum). The concentration of antibody produced and secreted is evaluated by ELISA; it is approximately 10 mg / l.

EXAMPLE 6 Immunofluorescence verification of the specificity of Hu-K20 produced by insect cells: The parental monoclonal antibody K20 is directed against the β1 subunit of human integrins

(CD29 receptor) located on the surface of lymphocytes.

The binding of K20 to the CD29 molecule inhibits the proliferation of activated human T4 lymphocytes [GROUX et al., Nature, 339, 152-154, (1989)]. The suppressive effect of K20 on T cell proliferation makes it possible to envisage the use of this antibody in the prevention of transplant rejection.

The binding of the antibody Hu-K20 produced in accordance with the invention, to the molecule CD29 was checked by immunofluorescence. Protocols experimental studies have been previously described [GROUX et al. Nature 339, 152-154 (1989); TICCHIONI et al. Journal of Immunology 151, 119-127 (1993)]. Immunofluorescence experiments: Human lymphocytes carrying the receptor

CD29 were fixed on a glass slide and then incubated in the presence of Hu-K20 produced in accordance with the invention, parental murine K20, or buffer alone.

After a series of rinses, either a fluorescent secondary antibody specific for Hu-K20 produced in accordance with the invention, or a fluorescent secondary antibody specific for parental murine K20, is added.

After a further rinse, the preparations were observed under a microscope to visualize the fluorescence. This shows that the Hu-K20 produced in accordance with the invention binds specifically to the lymphocytes carrying the CD29, as does the parental murine K20 positive control; no fluorescence was detected in the absence of the K20 antibodies, or on B lymphocyte cells not carrying the CD29 antigen. Example 7 Comparison of the Specificity of the Hu-K20 Produced by the Insect Cells, Compared to that of the Initial Mu-K20: MATERIAL and METHODS

The cell lines used are:

- the T JURKAT T6 E6-1 lymphocyte line from the American Tissue Cell Collection (ATCC),

- the line HPB-A11 (donation from C. ROZENFELD, Paris) line B RAJI (Burkitt's lymphoma) from the American Tissue Cell Collection (ATCC),

- line B 88/66 (gift from C. ROZENFELD, Paris) These lines are cultivated in RPMI 1640 medium

(AXCELL, France) supplemented with 2 mM L-glutamine (TECHGEN, France), 1 mM sodium pyruvate (TECHGEN), 50 mII / ml of penicillin and 50 μg / ml of streptomycin

(TECHGEN), and 5% fetal calf serum (GIBCO, Grande

Brittany) decomplemented by 30 minutes of heating at 56 ° C. The cells are kept in a humidified incubator at 37 ° C and 5% CO2.

2. T cells from peripheral blood.

The lymphocytes come from healthy voluntary donors (ARNAUD TZANCK Blood Transfusion Center, Saint Laurent du Var). The mononuclear cells are recovered after centrifugation on a Ficoll-Hypaque gradient (EUROBIO, France). Monocytes are removed by two one hour adhesion cycles on plastic at 37 ° C. Among the non-adherent cells thus obtained, residual monocytes, NK cells and B lymphocytes are eliminated by immunomagnetic selection using monoclonal antibodies directed against specific surface markers of these cell types and magnetic beads covered with goat anti-mouse immunoglobulin antibody (DYNABEADS, DYNAL, Norway). Lymphocyte purity is checked by checking the phenotype.

3. Thymocytes.

They are obtained from the thymus of children (4 months to 5 years) undergoing cardiological interventions at the Cardio-thoracic Center of Monaco. The residual erythrocytes are optionally removed by centrifugation on a Ficoll-Hypaque gradient.

4. Immunofluorescent labeling and flow cytometry. The cells (1x10 ^e ) are washed twice in PBS (140 mM NaCl, 3 mM KC1, Na ₂ HPθ4 6 M, KH Pθ4 l M, pH 7.4) 0.1% BSA (fraction V, BOEHRINGER), 0 , 1% NaN3. They are then incubated with a first antibody in a final volume of 100 μl for 30 min. at 4 ° C. After two washes, the cells are incubated in the same way with the second antibody, then at again washed twice. The cells are then fixed at the final concentration of 1x10 ⁶ , in 500 μl of PBS 1% formaldehyde. II. RESULTS 1. Determination of dose-response curves in flow cytometry and tissue distribution.

JURKAT E6-1, HPB-A11, RAJI, 88/66, thymocytes and human peripheral T lymphocytes cells are incubated with increasing concentrations (0.01 to 30 μg / ml) of each of the two murine and humanized K20 antibodies . After two washes, the cells are incubated with RAM-FITC (fluorescein conjugated F (ab ') 2 fragment of rabbit immunoglobulin to mouse immunoglobulin (DAKOPATTS, Denmark) at a dilution of 1/100 or RAHu- FITC (Fluorescein conjugated rabbit immunoglobulin to human IgA, IgG, IgM, DAKOPATTS, Denmark) or GAHu-FITC (fluorescein conjugated F (ab ') 2 fragment of goat immunoglobulin to human immunoglobulin (TEBU, France) diluted to 1/100. The results, which are illustrated by the

Figure 4 (4A to 4G), are as follows:

The two antibodies Mu-K20 and Hu-K20 bind to all the cell types tested with a saturating concentration close to 0.3 μg / ml, except on the line B 88/66 which does not express βl-integrin. The labeling observed only in the case of the humanized K20 antibody on the 88/66 cells is due to the fixation of GAHu-FITC which recognizes the surface immunoglobulins constitutively present on the surface of these B cells. This fixation is not observed in the case of Raji cells, since these were presaturated before labeling with rabbit immunoglobulins anti-human immunoglobulins. 2. Locking of the fixing of the murine K20. JURKAT E6-1 cells are incubated with increasing concentrations (from 0.01 to 30 μg / ml), murine (Mu-K20) or humanized (Hu-K20) antibody K20, or human IgG1 immunoglobulins (SIGMA, France). After washing, the cells are incubated with a saturated concentration of K20-FITC (IMMUNOTECH, France) of 8 μl in a final volume of 100 μl.

The results are illustrated in Figure 5, which shows that:

Humanized K20 clearly recognizes the same epitopes as murine K20 since it specifically blocks the fixation of murine K20. For each of the two antibodies Mu-K20 and Hu-K20, when the cells are incubated with a concentration of l μg / ml, it is noted that there are no longer any sites available for the fixation of the murine K20 coupled to the FITC. This is correlated with the value of the saturation concentration determined previously.

3. Relocation of the murine K20.

Jurkat E6-1 cells are incubated with a saturated concentration of K20-FITC (IMMUNOTECH, France) in a final volume of 100 μl. After washing, the cells are incubated with increasing concentrations (0.01 to 30 μg / ml) of each of the murine (Mu-K20) or humanized (Hu-K20) or human immunoglobulin IgG1 antibodies (SIGMA, France) .

The results are illustrated in FIG. 6, which shows that the murine and humanized K20 monoclonal antibodies displace the binding of the murine K20 coupled to the FITC, which is not observed for human immunoglobulins of the same subclass as the humanized antibody. . 50% of the displacement is reached for a concentration of Mu-K20 or Hu-K20 antibodies of the order of 30 μg / ml.

4. Competition of the Mu-K20 and Hu-K20 antibodies with the Mu-K20 antibody coupled to the FITC.

Jurkat E6-1 cells are incubated with

8 μl of K20-FITC at a saturated concentration (IMMUNOTECH, France), and increasing concentrations

(0.01 to 30 μg / ml) of murine or humanized K20 antibodies as well as human IgG1 immunoglobulins (Sigma, France). The results are illustrated in Figure 7, which shows that the competition curves observed for each of the antibodies are superimposable. The value of 50% competition is reached for antibody concentrations between 0.3 and 1 μg / ml. The IgGl antibody used as a control does not compete with K20-FITC even at the highest concentration of 30 μg / ml. Example 8 - Comparison of the biological activities of Mu-K20 and Hu-K20: 1. Action on the proliferation of T lymphocytes

Peripheral blood T cells come from healthy voluntary donors (ARNAUD TZANCK Blood Transfusion Center, Saint Laurent du Var). The mononuclear cells are recovered after centrifugation on a Ficoll-Hypaque gradient (EUROBIO, France). Monocytes are removed by two one hour adhesion cycles on plastic at 37 ° C. Among the non-adherent cells thus obtained, the residual monocytes, NK cells and B lymphocytes are eliminated by immunomagnetic selection using different monoclonal antibodies and magnetic beads coated with goat anti-mouse immunoglobulin antibody (DYNABEADS , DYNAL, Norway). Lymphocyte purity is controlled by phenotyping.

A CD3 monoclonal antibody (10 μg / ml) (X35, Dr MARTIN, Rennes) is immobilized on plastic in 96-well plates in which T lymphocytes are cultured at the rate of 50,000 cells per well in a final volume of 200 μl of RPMI 1640 , 10% SVF. Interleukin 2 (10 ng / ml) (R AND D SYSTEM, GB) and various dilutions of antibodies Mu-K20, Hu-K20 and human Ig controls are added to the wells. After 96 hours of culture at 37 ° C., 1 mCi of [ ³ H] thymidine is added per well. After 18 hours of incubation, the cellular DNA is trapped on filter paper using a semi-automatic sampler. The incorporation is measured in counts per minute by counting in liquid scintillation. The results are illustrated in Figure 8, which shows that the two antibodies Mu-K20 and Hu-K20 inhibit the proliferation of T lymphocytes from a low concentration of 1 ng / ml. For concentrations greater than 0.1 μg / ml which is the saturating concentration of the antibody, it is noted that the inhibition can decrease. 2. Measurement of adhesion to fibronectin.

Fibronectin and bovine albumin (50 μg / ml) (SIGMA) are immobilized in 96-well plates, where JURKAT cells are incubated beforehand loaded with a fluorescent probe, BCECF

(Boehringer), and labeled using K20 antibodies (10 μg / ml) and 4B4 antibody (COULTER CLONE), which is an anti-VLA4. After incubation for 2 hours at 37 ° C., the plates are washed 3 times with PBS, and the fluorescence measured on CITYFLUOR (MILLIPORE). The specific adhesion is calculated by taking into account the number of cells initially deposited in the well (150,000) and the non-specific binding to albumin. The results are illustrated in FIG. 9, which shows that the two antibodies Mu-K20 and Hu-K20 do not inhibit the binding to fibronectin, unlike the antibody 4B4.

The region of residues 207 to 218 of the integrin βi chain is critical for the binding of the antibody 4B4; This region is located between the two putative ligand binding sites (residues 120-182 and 220-231). For its part, the murine K20 antibody recognizes the carboxy-terminal region of β1 (residues 426-587) (TAKADA and PUZON, 1993, J. Biol. Chem. 17597-17601). 3. Measurement of the cytotoxicity induced by the complement.

After labeling with one or other of the two antibodies Mu-K20 or Hu-K20, the cells are incubated with rabbit complement for 50 min. at room temperature. After washing, the number of dead cells / number of living cells after staining with Trypan Blue is determined. The murine antibody D66 (CD2) is used as a positive control.

This experiment, the results of which are illustrated in FIG. 10, shows that the antibodies Mu-K20 and Hu-K20 do not induce the lysis of the cells in the presence of complement.

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(1) GENERAL INFORMATION:

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(ii) TITLE OF THE INVENTION: OBTAINING A HUMANIZED RECOMBINANT MONOCLONAL ANTIBODY FROM A MURIN MONOCLONAL ANTIBODY, ITS PRODUCTION IN INSECT CELLS, AND ITS USES.

(iii) NUMBER OF SEQUENCES: 20

(iv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GGTAGATCTC AGCTTGGTCC C 21

(2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GACATTCAGC TGACCCAGTC TCCA 24

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: TGAGGAGACG GTGACCGTGG TCCCTTGGCC CCAG 34

(2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: AGGTSMARCT GCAGSAGTCW GG 22

(2) INFORMATION FOR SEQ ID NO: 5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 325 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION:! .. 325

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:

GACATCCAGC TGACCCAGTC TCCATCCTCA CTGTCTGCAT CTCTGGGAGG CAAAGTCACC 60

ATCACTTGCA AGGCAAGCCA AGACATTAAC AAGTATATAG CTTGGTACCA ACACGAGCCT 120

GGAAAAGGTC CTAGGCTGCT CATACGTTAC ACATCAAAAC TAGAGTCAGG CATCCCATCA 180

AGGTTCAGTG GAAGTGGGTC TGGGAGAGAT TATTCCTTCA GCATCAGCAA CCTGGAGCCT 240

GAAGATATTG CAACTTATTA CTGTCTACAG TATTATAATC TTTGGACGTT CGGTGGAGGG 300 ACCAAGCTGG AGATCAAACG TAAGT 325

(2) INFORMATION FOR SEQ ID NO: 6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 349 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ix) CHARACTERISTIC:

(A) NAME / KEY: CDS

(B) LOCATION:! .. 349 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:

CAGGTCCAAC TGCAGGAGTC TGGGACTGAG CTCGTGAGGC CTGGGGCTTC AGTGAAGTTG 60

TCCTGCAAGG CTTCTGGCTA CACTTTCACT GACTACTATA TAAGCTGGGT GAAACAGAGG 120

CCTGGACAGG GACTTGAGTG GATTGCAAGG ATTTATCCTG GAAGTGGTAA TACTTTCTAC 180

AATGAGAAAT TCAAGGGCAA GGCCACACTG ACTGCAGAAA CGTCCTCCAA CACTGCCTAC 240

ATGCAGCTCA GCAGCCTGAC ATCTGAGGAC TCTGCTGTCT ATTTCTGTGC AATTTACTAC 300

GGTAGTGGTG ACTACTGGGG CCAAGGGACC ACGGTCACCG TCTCCTCAG 349 (2) INFORMATION FOR SEQ ID NO: 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 53 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: CCATCACCTG GAAGGCAAGC CAGGACATTA ACAAGTATAT AGCTTGGTAC CAG 53

(2) INFORMATION FOR SEQ ID NO: 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: CAAAGCTGAT CCGTTACACA TCAAAACTAG AGTCAGGTGT GCCAAGC 47

(2) INFORMATION FOR SEQ ID NO: 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 45 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:

CCTACTACTG CCTACAGTAT TATAATCTTT GGACGTTCGG CCAAG 45

(2) INFORMATION FOR SEQ ID NO: 10:

(i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 base pairs (B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GATACACCTT CACTGACTAC TATATAAGCT GGGTGCGCCA G 41

(2) INFORMATION FOR SEQ ID NO: 11:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: GAGTGGATGG GAAGGATTTA TCCTGGAAGT GGTAATACTT TCTACAATGA GAAATTCAAG 60 GGCAGAGTCA CC 72

(2) INFORMATION FOR SEQ ID NO: 12:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: TATTACTGTG CGATTTACTA CGGTAGTGGT GACTACTGGG GC 42

(2) INFORMATION FOR SEQ ID NO: 13:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: AGCTCGAGAT CAAACGG 17

(2) INFORMATION FOR SEQ ID NO: 14:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: GAAGATCTAA CACTCTCCGC GGTTGAAG 28

(2) INFORMATION FOR SEQ ID NO: 15:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: ACGTTTCTCG AGCYTGGTSC C 21

(2) INFORMATION FOR SEQ ID NO: 16:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: GACATCGAGC TCACCCAG 18

(2) INFORMATION FOR SEQ ID NO: 17:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: CAAGGTACCA CGGTCACCGT CTCC 24

(2) INFORMATION FOR SEQ ID NO: 18:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GAAGATCTCA TTTACCCGGA GACAGGGAG 29

(2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: AGGTSMARCT GCAGSAGT C GGG 23

(2) INFORMATION FOR SEQ ID NO: 20:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: Single

(D) CONFIGURATION: linear

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: TGAGGAGACG GTGACCGTGG TACCTTGGCC CCAG 34

Claims

1) Method for humanizing the VJJ variable region and / or the VL variable region of a non-human immunoglobulin, which method is characterized in that it comprises at least the following steps:

a step in which a DNA sequence coding for the variable region V _H or for the variable region V _L of a human immunoglobulin is selected, the amino acid sequence of the FR regions of said VJJ region or of said VL region human having respectively between 70 and 95% homology with the amino acid sequence of the FR regions of the VJJ region or of the V _L region of the non-human immunoglobulin, and the hypervariable loops Hl and H2 of said V _H region human or the hypervariable loops L1, L2 and L3 of said region V _L human having respectively the same type of canonical structure as the hypervariable loops Hl and H2 of the region VJJ or the hypervariable loops Ll, L2 and L3 of the region VL of 1 non-human immunoglobulin

a step in which the oligonucleotide sequences coding for the CDRs of said human VJJ or VL region are replaced by the oligonucleotide sequences coding for the corresponding CDRs of the Vy or VL region of the non-human immunoglobulin.

2) DNA fragment coding for the VJJ variable region or for the VL variable region of a humanized Hu-Ac antibody, characterized in that it is capable of being obtained by the method according to claim l. 3) DNA fragment according to claim 2, characterized in that it is capable of being obtained from the murine antibody K20, and code for the variable region VJJ or for the variable region VL of a humanized antibody referred to as Hu-K20. 4) Expression cassette characterized in that it comprises at least one DNA fragment according to any one of claims 2 or 3, placed under transcriptional control of an appropriate promoter. 5) Expression cassette according to claim 4, characterized in that it comprises the elements allowing the expression of the heavy chain or the light chain of a recombinant Hu-Ac antibody, said elements being constituted by: - a baculovirus promoter, under transcriptional control of which are placed:

- a sequence coding for a secretion signal peptide, ^• and

a sequence coding for the variable domain of the light chain of the Hu-Ac antibody and a sequence coding for the constant domain of the light chain of a human immunoglobulin; or

a sequence coding for the variable domain of the heavy chain of the Hu-Ac antibody and a sequence coding for the constant domain of the heavy chain of a human immunoglobulin.

6) Expression cassette according to any one of claims 4 or 5, characterized in that the baculovirus promoter is the promoter of polyhedrin or one of its derivatives, or else the promoter of plO or one of its derivatives.

7) Recombinant vector, characterized in that it comprises at least one DNA fragment according to any one of claims 2 or 3. 8) Recombinant vector according to claim

7, characterized in that it comprises at least one expression cassette according to any one of claims 4 to 6. 9) Recombinant vector according to claim 8, characterized in that it constitutes a transfer vector, carrying an insert comprising: an expression cassette according to any one of claims 4 to 6, and on either side of this cassette , baculovirus sequences homologous to those of the regions flanking the portion of the baculovirus genome in replacement of which it is desired to insert said cassette.

10) Transfer vector according to claim 9, characterized in that said baculovirus sequences are homologous to those of the regions flanking the p10O gene, or homologous to those of the regions flanking the polyedrine gene.

11) Transfer vector according to claim 10, characterized in that the expression cassette containing the gene coding for the light chain of the antibody Hu-Ac is flanked by the regions surrounding the polyhedrin gene in wild baculovirus, and the expression cassette carrying the gene coding for the heavy chain of the Hu-Ac antibody is flanked by the regions surrounding the PlO gene in the wild baculovirus.

12) Recombinant baculovirus characterized in that it comprises at least one expression cassette according to any one of claims 4 to 6.

13) Recombinant baculovirus according to claim 12, characterized in that it comprises an expression cassette comprising the sequence coding for the H chain of the Hu-Ac antibody, and an expression cassette comprising the sequence coding for the chain L of the antibody Hu-Ac.

14) Recombinant baculovirus according to claim 13, characterized in that the promoter controlling the transcription of the sequence coding for the L chain of the Hu-Ac antibody, and the promoter controlling the transcription of the sequence coding for the H chain of the Hu-Ac antibody, are two different promoters.

15) Recombinant baculovirus according to claim 14, characterized in that one of said promoters is located at the location occupied in the wild baculovirus, by the polyhedrin promoter and the other is located at the location occupied in the baculovirus wild, by the promoter of PlO.

16) Recombinant baculovirus according to any one of claims 12 to 15, characterized in that the transcription of the sequence coding for the light chain of the Hu-Ac antibody is under the control of the promoter of the polyhedrin or of one of its derivatives , and the transcription of the sequence encoding the heavy chain of the Hu-Ac antibody is under the control of the promoter of PlO or one of its derivatives.

17) Recombinant baculovirus according to any one of claims 12 to 16, characterized in that the sequence coding for the signal peptide associated with the L chain of the Hu-Ac antibody, and the sequence coding for the signal peptide associated with the H chain of the Hu-Ac antibody, are two different sequences.

18) Recombinant baculovirus according to claim 12, characterized in that it is the recombinant baculovirus AclOHK20hu-33LK20hu (III), deposited on September 9, 1994, with the C.N.C.M., under the number 1-1476.

19) Insect cells infected with a recombinant baculovirus according to any one of claims 12 to 18.

20) A method of preparing a humanized recombinant antibody, characterized in that it comprises a step during which insect cells are cultivated according to claim 19, and a step during which one obtains said antibody from the culture medium.