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  • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
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  • C12N2510/00—Genetically modified cells

Definitions

• the present invention is in the field of therapeutic antibodies for the treatment or prevention of hematopoietic tumors of CD4 + CD56 + phenotype or inflammatory diseases, especially autoimmune, involving plasmacytoid dendritic cells (pDC). It relates to 5 monoclonal antibodies directed against the CD303 antigen, as well as their functional fragments and derivatives, the nucleic acids encoding the heavy chain and / or the light chain of the antibody, expression vectors, host cells, animals non-human transgenic or transgenic plants expressing these antibodies, as well as the therapeutic uses of these antibodies.
• DC Dendritic cells
• APC antigen-presenting immune system
• Dendritic cells have two main functions:
• DCs are able to differentiate into different subpopulations, depending on the stimuli they receive.
• DC cells There are three main types of DC cells: conventional DC cells, plasmacytoid DC cells (called “pDCs”) and inflammatory DC cells.
• DCs present self or non-self antigens in lymphoid organs or in the periphery.
• Inflammatory DCs probably derived from blood monocytes, only appear when stimulated by inflammation or infection.
• Plasmacytoid DCs pDCs
• pDCs Plasmacytoid DCs
• IFN class I interferons
• DCPs can cause haematopoietic tumors in which they acquire an additional marker (CD56).
• BPDCN blast plasmacytoid dendritic cells
• CD4 + CD56 + hematopoietic tumors
• anti-BDCA-2 antibodies that is to say anti-CD303
• the pDCs are also involved in certain inflammatory diseases, and in particular in certain autoimmune diseases, in particular through their secretion of type I IFN.
• Treatment relies on the use of anti-IFNa antibodies.
• this treatment leads to systemic neutralization of IFNa, potentially increasing the risk of opportunistic infections. It has therefore also been proposed in WO01 / 365487 and WO2012 / 080642 to use anti-BDCA-2 antibodies (anti-CD303, and in particular the AC144 antibody) for the treatment of autoimmune diseases, by eliminating the pDCs responsible for the local inflammation.
• WO201 / 09339 discloses humanized anti-CD303 antibodies, defined by the sequences of their heavy and light chains, and in particular the BIIB059 antibody, which is the one having the best affinity for the CD303 antigen.
• the BIIB059 antibody was produced in CHO and characterized for certain properties.
• the inventors have generated five chimeric monoclonal antibodies having such properties. At least two of these chimeric antibodies (122A2 and 102E9) have an ectodomain binding capacity of the human CD303 antigen greater than that of the BIIB059 antibody described in WO2014 / 09339 (in particular 122A2). In addition, humanized antibodies derived from these two chimeric antibodies were generated and characterized.
• humanized antibodies can be produced with a higher productivity than the original chimeric antibodies and, among those derived from the 122A2 chimeric antibody, some have an ectodomain binding capacity of the human CD303 antigen still greater than that of the chimeric antibody of origin and therefore much greater than that of the antibody BIIB059 described in WO2014 / 09339.
• the chimeric and humanized antibodies produced in YB2 / 0 further possess high affinity for the Fc ⁇ RIIIa receptor (CD16a), and are capable of inducing strong ADCC responses, even at low antigenic density.
• the antibodies further possess CDC activity and are capable of inhibiting the secretion of IFN- ⁇ and TNF- ⁇ .
• the present invention therefore relates to a monoclonal antibody directed against the ectodomain of the human CD303 antigen (SEQ ID NO: 130), or a functional fragment or a derivative thereof, characterized in that:
• the constant regions of the light and heavy chains are constant regions from a non-murine species.
• the present invention also relates to a monoclonal antibody directed against the ectodomain of the human CD303 antigen (SEQ ID NO: 130), or a functional fragment or a derivative thereof, characterized in that:
• the constant regions of the light and heavy chains are constant regions from a non-murine species.
• the present invention also relates to a nucleic acid encoding the heavy chain and / or the light chain of an antibody, functional fragment or derivative thereof according to the invention.
• the present invention also relates to a vector comprising a nucleic acid according to the invention.
• the present invention also relates to a host cell, a transgenic non-human animal or a transgenic plant comprising at least one nucleic acid according to the invention or a vector according to the invention.
• the present invention also relates to an antibody, functional fragment or derivative thereof according to the invention, for use as a medicament.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of tumors hematopoietic agents expressing the CD303 antigen or in the treatment or prevention of inflammatory diseases, including autoimmune diseases.
• FIG. 1 Antigen binding of antibodies according to the invention.
• A Mean fluorescence intensity (MFI) of Jurkat Fc Gamma-CD303 cells (expressing approximately 25,000-35,000 CD303 molecules / cell) labeled with the antibodies according to the invention, at different antibody concentrations (shown in logarithmic units) .
• B Mean fluorescence intensity (MFI) of CAL-1 cells (cell line of BPDCN patients) labeled with the chimeric antibodies according to the invention, at different antibody concentrations (shown in logarithmic units).
• C Mean fluorescence intensity (MFI) of CAL-1 cells (cell line of BPDCN patients expressing approximately 3000-6000 CD303 molecules / cell) labeled with one of the chimeric antibodies 122A2 and 102E9, with the humanized antibody BIIB059 or by an irrelevant antibody.
• D Mean fluorescence intensity (MFI) of CAL-1 cells transfected with a CD303 expression vector and selected for their high CD303 expression, approximately 40,000-50000 CD303 molecules / cell, labeled with one of the chimeric antibodies 122A2 and 102E9, by the humanized antibody BIIB059 or by an irrelevant antibody.
• FIG. 3 Fc ⁇ RIIIa receptor binding (CD16a).
• A The CD16 receptor binding of the antibodies according to the invention was studied in a competitive experiment using murine anti-CD16 3G8 antibody coupled to phycoerythrin. The binding of anti-CD16 3G8 to CD16 (MFI fluorescence values) is measured as a function of the increasing added concentration of the antibodies according to the invention (g / ml).
• FIG. 4 ADCC activities induced by the chimeric antibodies of the invention and the humanized antibody BIIB059 against Jurkat Fc Gamma-CD303 (A) cells or CAL-1 (B) cells.
• A The percentage of lysis by ADCC (% lysis, as defined in Example 2)
• Jurkat target cells Gamma-CD303-Fc chain induced by the chimeric antibodies according to the invention is represented as a function of the antibody concentration (ng / mL, logarithmic scale).
• FIG. 6 Inhibition induced by the antibodies of the invention of the secretion of TNF- ⁇ by the purified pDCs activated with CpG, in the presence of the chimeric antibodies (10 g / ml) of the invention and a chimeric antibody control relevant.
• FIG. 7 CDC activities induced by the antibodies of the invention vis-à-vis the Jurkat Fc Gamma-CD303 chain cells.
• the percentage of CDC lysis (% lysis, as defined in Example 2) of Jurkat target cells chain Fc Gamma-CD303 induced by the chimeric antibodies according to the invention is represented as a function of the antibody concentration (ng / mL logarithmic scale).
• FIG. 1 Vacuum pCEP4 vector maps (A) and heavy or light chain expression vectors of humanized antibodies 122A2 (B-H) and 102E9 (I-N).
• Figure 9 Antigen binding of humanized antibodies derived from antibody 102E9. OD at 450 nm as a function of the concentration of the humanized antibodies derived from the antibody 102E9 tested at 1.25; 2.5; 5; 10 and 20 ng / ml, accompanied by controls: the supernatant alone (mock) and chimeric 102E9 antibody.
• A Humanized antibodies 102E9H5, 102E9H6, 102E9H7, 102E9H9, 102E9H10 and 102E9H11.
• B Humanized antibodies 102E9H13, 102E9H1, and 102E9H15.
• Figure 10 Antigen binding of humanized antibodies derived from antibody 102E9 to 5 ng / ml antibody. Comparison of relative affinities of humanized antibodies derived from 102E9 with respect to ectodomain CD303, the binding of the chimeric antibody being at 100%. Antibodies with a binding close to that of the chimeric antibody (relative affinity of at least 70%) are reported as *.
• Figure 1 Antigen binding of humanized antibodies derived from the antibody
• FIG. 12 Antigen binding of humanized antibodies derived from antibody 102E9 to 5 ng / ml of antibody. Comparison of relative affinities of humanized antibodies derived from 102E9 with respect to ectodomain CD303, the binding of the chimeric antibody being at 100%. Antibodies with a better binding to that of the chimeric antibody (relative affinity> 100%) are reported as *.
• Figure 14 Inhibition of IFN- ⁇ secretion by chimeric antibody 122A2 and humanized antibodies derived from 122A2.
• FIG. 15 Maps of heavy or light chain expression vectors of humanized antibodies 122A2H5 (A), 122A2H7 (B), 122A2H9 (C) and 122A2H10 (D), for production in YB2 / 0.
• the present invention relates to a monoclonal antibody directed against the ectodomain of the human CD303 antigen (SEQ ID NO: 130), or a functional fragment or a derivative thereof, characterized in that:
• the constant regions of the light and heavy chains are constant regions from a non-murine species.
• the heavy chains comprise three CDR-H (heavy chain CDR according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences
• the light chains comprise three CDR-Ls (light chain CDR according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences:
• CDR1 -H-family 1 SEQ ID NO: 1, CDR2-H-family 1: SEQ ID NO: 2, CDR3-H-family 1: SEQ ID NO: 3, CDR1-L-family 1: SEQ ID NO: 4, CDR2-L-family 1: SEQ ID NO: 5, CDR3-L-family 1: SEQ ID NO: 6; or ii) CDR1 -H-family 2: SEQ ID NO: 7, CDR2-H-family 2: SEQ ID NO: 8, CDR3-H-family 2: SEQ ID NO: 9, CDR1-L-family 2: SEQ ID NO: 10, CDR2-L-family 2: SEQ ID NO: 11, CDR3-L-family 2: SEQ ID NO: 12.
• the heavy chains of an antibody, functional fragment or derivative thereof according to the invention comprise three CDR-H (heavy chain CDR according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences, and the light chains include three CDR-Ls (light chain CDRs according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% of identity with the following sequences:
• CDR1 -H-122A2 SEQ ID NO: 13
• CDR2-H-122A2 SEQ ID NO: 14
• CDR3-H-122A2 SEQ ID NO: 15
• CDR1 -L-122A2 SEQ ID NO: 16
• CDR2-L-122A2 SEQ ID NO: 17
• CDR3-L-122A2 SEQ ID NO: 18;
• CDR1 -H-102E9 SEQ ID NO: 19
• CDR2-H-102E9 SEQ ID NO: 20
• CDR1 -H-104Cl2 SEQ ID NO: 25, CDR2-H-104Cl2: SEQ ID NO: 26, CDR3-H-104Cl2: SEQ ID NO: 27, CDR1 -L-104Cl2: SEQ ID NO: 28, CDR2-L-104C12: SEQ ID NO: 29, CDR3-L-104C12: SEQ ID NO: 30;
• CDR1 -H-1 14D1 1 SEQ ID NO: 31, CDR2-H-1 14D11: SEQ ID NO: 32, CDR3-H-1 14D1 1: SEQ ID NO: 33, CDR1 -L-1 14D11: SEQ ID NO: 34, CDR2-L-1 14D1 1: SEQ ID NO: 35, CDR3-L-1 14D1 1: SEQ ID NO: 36; or v) CDR1 -H-104E10: SEQ ID NO: 37, CDR2-H-104E10: SEQ ID NO: 38, CDR3-H-104E10: SEQ ID NO: 39, CDR1 -L-104E10: SEQ ID NO: 40 , CDR2-L-104E10:
• SEQ ID NO: 41 CDR3-L-104E10: SEQ ID NO: 42.
• the heavy chains of an antibody, functional fragment or derivative thereof according to the invention comprise a variable region having a sequence chosen from SEQ ID NO: 43 to 47 or a sequence having at least 80% identity with one of SEQ ID NO: 43 to 47.
• the light chains of an antibody, functional fragment or derivative thereof according to the invention comprise a variable region having a sequence selected from SEQ ID NO: 48 to 52 or a sequence having at least 80% d identity with one of SEQ ID NO: 48-52.
• the antibody, functional fragment or derivative thereof according to the invention has heavy and light chains whose variable regions have the following amino acid sequences or sequences having at least 80% d identity with the following sequences:
• 122A2 antibody heavy chain: SEQ ID NO: 43, light chain: SEQ ID NO: 48, ii) 102E9 antibody: heavy chain: SEQ ID NO: 44, light chain: SEQ ID NO: 49, iii) 104C12 antibody heavy chain: SEQ ID NO: 45 light chain: SEQ ID NO: 50, iv) 114D11 antibody: heavy chain: SEQ ID NO: 46, light chain: SEQ ID NO: 51, or
• Antibody 104E10 heavy chain: SEQ ID NO: 47, light chain: SEQ ID NO: 52.
• VH-122A2 SNYNQKFKGKATMTVDKSSTTAYMELARLTSEDSAIYYCARNGNFYVMDYWGQGTSV
• VL-122A2 SRFSGSGSGTDYSLTISNLDQEDIATYFCQQGNTLPWTFGGGTKLEIK
• VL-104C12 SRFSGSGSGTDYSLTVRNLEQEDIGTYFCQQGKTLPWTFGGGTKLEIR (SEQ ID NO
• VL-104E10 RFSGSGSGTSYSLTISRMEAEDAATYYCQQRRSYPYTFGGGTKLEIK (SEQ ID NO:
• the human antigen CD303 is a member of the 4 th C domain C-type lectin family and is also called CLEC4 ( "C-type lectin domain family-4, member C");DLEC;HECL;BDCA-2;CLECSF7;CLECSF11; or PRO34150 (see the EnterGene site for the CLEC4 gene).
• antibody or “immunoglobulin” is meant a molecule comprising at least one binding domain to a given antigen and a constant domain comprising a Fc fragment capable of binding to FcR receptors.
• an antibody is composed of 4 polypeptide chains: 2 heavy chains and 2 light chains linked together by a variable number of disulfide bridges providing flexibility to the molecule.
• Each light chain consists of a constant domain (CL) and a variable domain (VL); the heavy chains being composed of a variable domain (VH) and 3 or 4 constant domains (CH1 to CH3 or CH1 to CH4) according to the isotype of the antibody.
• VH variable domain
• CH1 to CH3 or CH1 to CH4 constant domains
• antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (VH) and a constant region.
• Variable domains are involved in antigen recognition, while constant domains are involved in the biological, pharmacokinetic and effector properties of the antibody.
• variable region differs from one antibody to another.
• the genes coding for the heavy and light chains of the antibodies are generated by recombination of respectively three and two distinct gene segments called VH, DH and JH-CH for the heavy chain and VL and JL-CL for the light chain.
• the CH and CL segments do not participate in the recombination and form the constant regions of the heavy and light chains respectively.
• the recombinations of the VH-DH-JH and VL-JL segments form the variable regions of the heavy and light chains, respectively.
• the VH and VL regions each have 3 hyper-variable areas or complementarity determining regions (CDRs), called CDR1, CDR2 and CDR3, the CDR3 region being the most variable since it is located at the recombination zone.
• CDRs complementarity determining regions
• These three CDR regions, and particularly the CDR3 region are in the part of the antibody that will be in contact with the antigen and are therefore very important for antigen recognition.
• the antibodies that retain the three CDR regions and each of the heavy and light chains of an antibody retain for the most part the antigenic specificity of the original antibody.
• an antibody retaining only one of the CDRs, and in particular CDR3 also retains the specificity of the original antibody.
• the CDR1, CDR2 and CDR3 regions are each preceded by the FR1, FR2 and FR3 regions respectively, corresponding to the framework regions (FR region framework) which vary at least from one VH or VL segment to another.
• the CDR3 region is also followed by an FR4 framework region.
• the CDRs of an antibody are defined from the amino acid sequence of its heavy and light chains with respect to criteria known to those skilled in the art.
• Various methods for determining CDRs have been proposed, and the portion of the amino acid sequence of a heavy or light chain variable region of an antibody defined as a CDR varies depending on the method chosen.
• the first method of determination is that proposed by Kabat et al (Kabat et al. 1991).
• CDRs are defined by looking for the amino acids responsible for antigen binding of the antibody.
• a 2nd method was proposed by IMGT, this time based on the determination of the hypervariable regions.
• the constant domains are characterized by an amino acid sequence very close to one antibody to another, characteristic of the species and the isotype, with possibly somatic mutations.
• the Fc fragment is naturally composed of the constant region of the heavy chain excluding the CH1 domain, that is to say the lower hinge region and the constant CH2 and CH3 or CH2 to CH4 domains (depending on the isotype ).
• the complete Fc fragment is composed of the C-terminal portion of the heavy chain from the cysteine residue at position 226 (C226), the numbering of amino acid residues in the Fc fragment being throughout the present invention. description that of the EU index described in Edelman et al. 1969 and Kabat et al. 1991.
• the corresponding Fc fragments of other types of immunoglobulins can be easily identified by those skilled in the art by sequence alignments.
• the Fc fragment is glycosylated at the CH2 domain with the presence, on each of the 2 heavy chains, of an N-glycan linked to the asparagine residue at position 297 (Asn 297).
• binding domains located in the Fc, are important for the biological properties of the antibody:
• FcRn receptor binding domain involved in the pharmacokinetic properties (in vivo half-life) of the antibody:
• FcR receptor binding domain involved in phagocytosis or ADCC responses (for "antibody-dependent cellular cytotoxicity): located in the CH2 domain.
• the Fc fragment of an antibody may be natural, as defined above, or may have been modified in various ways, provided that it comprises a FcR receptor binding domain (Fc ⁇ R receptors for IgG) functional, and preferably a functional FcRn receptor binding domain.
• the modifications may include the deletion of certain portions of the Fc fragment, provided that it contains a FcR receptor binding domain (Fc ⁇ R receptors for IgG). functional, and preferably a functional FcRn receptor binding domain.
• the modifications may also include different amino acid substitutions that may affect the biological properties of the antibody, provided that it contains a functional FcR receptor binding domain, and preferably a functional FcRn receptor binding domain.
• the antibody when it is an IgG, it may comprise mutations intended to increase binding to the Fc ⁇ RIIIa receptor (CD16a), as described in WO00 / 42072, Shields et al-2001, Lazar et al. 2006, WO2004 / 029207, WO2004 / 063351, WO2004 / 074455. Mutations to increase FcRn receptor binding and thus in vivo half-life may also be present, as described for example in Shields et al-2001, Dall'Acqua et al. 2002, Hinton et al. 2004, Dall Acqua et al.
• mutants comprising mutations making it possible to increase the binding to the FcRn receptor and therefore the half-life in vivo are the mutants comprising the following combinations of mutations in their Fc fragment, described in WO2010 / 106180 :
• fragments or derivatives without Fc fragment of the antibody may be useful in the context of the invention, in particular for the treatment of inflammatory diseases, in particular autoimmune, involving pDC, the antibody according to the invention.
• the invention may also have a mutated Fc fragment lacking effector functions. Examples of mutations leading to a mutated Fc fragment lacking effector functions are the single deletions of amino acids in position 293 (Del293) or 294 (Del294) of the Fc fragment, where the numbering of the amino acids in the Fc fragment is that of the EU index of Kabat (WO2012 / 175751).
• “monoclonal antibody” or “monoclonal antibody composition” is meant a composition comprising antibody molecules having identical and unique antigenic specificity.
• the antibody molecules present in the composition are likely to vary in their post-translational modifications, and in particular in their glycosylation structures or their isoelectric point, but have all been coded by the same heavy chain sequences and light and therefore, before any post-translational modification, have the same protein sequence.
• Some protein sequence differences, related to post-translational modifications eg cleavage of C-terminal lysine of the heavy chain, asparagine deamidation of residues and / or isomerization of aspartate residues
• the percentages of identity to which reference is made in the context of the disclosure of the present invention are determined on the basis of an overall alignment of the sequences to be compared, that is to say on an alignment of the sequences taken in their entirety over the entire length using any algorithm well known to those skilled in the art such as the Needleman and Wunsch-1970 algorithm.
• This sequence comparison can be performed using any software well known to those skilled in the art, for example the needle software using the parameter "Gap open” equal to 10.0, the parameter “Gap extend” equal to 0.5 and a "Blosum 62" matrix.
• the needle software is for example available on the ebi.ac.uk world wide website under the name "Align”.
• the CDR or the variable region of an antibody according to the invention has an amino acid sequence which is not 100% identical to one of those described above and in the sequence listing (reference sequences ) but which has at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with at least such a reference sequence, it may have insertions, deletions or substitutions with respect to the reference sequence.
• the substitution is preferably carried out by an "equivalent" amino acid, that is to say any amino acid whose structure is close to that of the original amino acid and is so little likely to alter the biological activities of the antibody. Examples of such substitutions are shown in the following Table 4:
• the antibodies can be of several isotypes, depending on the nature of their constant region: the constant regions ⁇ , ⁇ , ⁇ , ⁇ and ⁇ correspond respectively to immunoglobulins IgG, IgA, IgM, IgE and IgD.
• the monoclonal antibody present in a composition used as a medicament in the context of the invention is of IgG isotype.
• this isotype shows an ability to generate ADCC ("Antibody- Dependent Cellular Cytotoxicity", or antibody-dependent cellular cytotoxicity) activity in the largest number of (human) individuals.
• the constant regions ⁇ comprise several subtypes: ⁇ 1, j2, j3, these three types of constant regions having the particularity of fixing the human complement, and j4, thus creating the lgG1, IgG2, IgG3, and IgG4 sub-isotypes.
• the antibody monoclonal present in a composition used as a medicament in the context of the invention is isotype IgG1 or IgG3, preferably IgG1.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously a chimeric or humanized antibody, in particular a chimeric antibody whose constant region of the heavy and light chains is of human origin.
• chimeric antibody an antibody which contains a naturally occurring variable (light chain and heavy chain) derived from an antibody of a given species in association with the constant light chain and heavy chain regions of an antibody of a heterologous species to said given species.
• the monoclonal antibody composition for use as a medicament according to the invention comprises a chimeric monoclonal antibody, it comprises human constant regions.
• a chimeric antibody can be prepared using genetic recombination techniques well known to those skilled in the art.
• the chimeric antibody may be made by cloning for the heavy chain and the light chain a recombinant DNA comprising a promoter and a sequence coding for the variable region of the non-human antibody, and a sequence coding for the constant region of the a human antibody.
• a recombinant DNA comprising a promoter and a sequence coding for the variable region of the non-human antibody, and a sequence coding for the constant region of the a human antibody.
• humanized antibody is meant an antibody which contains CDRs regions derived from an antibody of non-human origin, the other parts of the antibody molecule being derived from one (or more) human antibodies.
• some of the skeletal segment residues (referred to as FR) can be modified to maintain binding affinity (Jones et al., 1986, Verhoeyen et al., 1988, Riechmann et al., 1988).
• the humanized antibodies according to the invention may be prepared by techniques known to those skilled in the art such as “CDR grafting", “resurfacing”, Superhumanization, "Human string content", “FR libraries” technologies. "Guided selection", “FR shuffling” and “Humaneering”, as summarized in the review of Almagro et al-2008.
• the inventors also generated humanized antibodies derived from two of the anti-CD303 antibodies originally obtained. For this purpose, for each of the two antibodies, several mutated regions of variable region of heavy chain (mutations to make the sequence closer to a human sequence) and several light chain variable region mutated sequences were generated and combined in pairs to attempt to obtain humanized antibodies with a capacity for binding to the human CD303 antigen (SEQ ID NO: 130) as strong as possible (advantageously at least 70% of the binding capacity of the murine or chimeric antibody, more preferably similar to the binding capacity of the murine or chimeric antibody, still more preferably greater than the binding capacity of the murine or chimeric antibody).
• SEQ ID NO: 130 human CD303 antigen
• the amino acid sequences of the heavy chain and light chain variable (humanized, ie mutated) regions that were tested are shown in Table 5 below:
• Humanized antibodies 122A2 are thus advantageously chosen from those whose heavy chains comprise a variable region having an amino acid sequence chosen from SEQ ID NO: 131 to 133, advantageously from those whose heavy chains comprise a variable region having a sequence of amino acid selected from SEQ ID NO: 131 and 132.
• Humanized antibodies 122A2 are also advantageously chosen from those whose light chains comprise a variable region having an amino acid sequence chosen from SEQ ID NO: 134 to 137, advantageously from those whose light chains comprise a variable region having a sequence of amino acid selected from SEQ ID NO: 134 and 135.
• the various humanized 122A2 antibodies generated by the inventors have heavy chains and light chains comprising variable regions having the amino acid sequences described in Table 6 below.
• the preferred humanized 122A2 antibodies (because having an antigen binding greater than or equal to that of the original chimeric antibody) are those whose heavy chains and light chains comprise variable regions having the amino acid sequences comprising those described for antibodies 122A2H5, 122A2H9, 122A2H6, 122A2H10, 122A2H1, 122A2H7, 122A2H1 1 and 122A2H1 in Table 6 below (shown in bold in Table 6 below).
• the most preferred humanized 122A2 antibodies are those with higher antigen binding than the original chimeric antibody: 122A2H5, 122A2H9, 122A2H6, 122A2H10, 122A2H14, 122A2H7, and particularly 122A2H5, 122A2H9, 122A2H7, and 122A2H10.
• Humanized 102E9 antibodies are thus advantageously chosen from those whose heavy chains comprise a variable region having an amino acid sequence chosen from SEQ ID NO: 138 to 140, advantageously from those whose heavy chains comprise a variable region having the following sequence: amino acids SEQ ID NO: 139.
• Humanized 102E9 antibodies are also advantageously chosen from those whose light chains comprise a variable region having an amino acid sequence chosen from SEQ ID NO: 141 to 143, advantageously from those whose light chains comprise a variable region having the following sequence: amino acids SEQ ID NO: 142.
• the various humanized 102E9 antibodies generated by the inventors have heavy chains and light chains comprising variable regions having the amino acid sequences described in Table 8 below.
• the preferred humanized antibodies 102E9 (because having a binding of the level of the binding of the chimeric antibody close to that of the chimeric antibody of origin: at least 70% of the binding of the chimeric antibody in the ELISA test) are those in which the heavy and light chains comprise variable regions having the amino acid sequences including those described for antibodies 102E9H6, 102E9H7, 102E9H9, and 102E9H10 in Table 8 below (shown in bold in Table 8 below). below), more preferably those whose heavy chains and light chains comprise variable regions having the amino acid sequences including those described for the antibody 102E9H10 in Table 8 below.
• an antibody, functional fragment or derivative thereof according to the invention which is chimeric with human constant regions, or else humanized, will advantageously comprise a human heavy chain constant region having as amino acid sequence SEQ ID NO: 53 or SEQ ID NO: 144 (which corresponds to the human heavy chain constant region sequence SEQ ID NO: 53 with an additional C-terminal lysine residue).
• an antibody, functional fragment or derivative thereof according to the invention which is chimeric with human constant regions, or else humanized, will advantageously comprise a human light chain constant region having a sequence of amino acids SEQ ID NO: 54.
• the preferred heavy (SEQ ID NO: 53 or SEQ ID NO: 144) or light (SEQ ID NO: 54) human chain constant sequence sequences, of IgG1 isotype, are shown in Table 9 below. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
• the human heavy chain constant region sequence SEQ ID NO: 144 corresponds to the human heavy chain constant region sequence SEQ ID NO: 53 with an additional C-terminal lysine residue.
• the heavy and light chains of the chimeric or humanized antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise the sequences described in Table 10 below (or consist essentially or consist of such sequences).
• the heavy chain and / or the light chain of the antibody, functional fragment or derivative thereof according to the invention advantageously furthermore comprises at least one heterologous signal peptide of sequence SEQ ID NO: 65 (MRWSWIFLLLLSITSANA, MB7 signal peptide) .
• this peptide has been shown to improve the expression and secretion of recombinant proteins in eukaryotic cell lines. higher (see WO201 1/1 14063).
• the heavy chains of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise an amino acid sequence chosen from the sequences SEQ ID NO: 66 to 70, consisting of the fusion of N- into C- terminal between the amino acid sequence of the signal peptide MB7 (SEQ ID NO: 65) and one of the amino acid sequences of the VH region of the antibodies according to the invention (SEQ ID NO: 43 to 47), or are essentially or consist of such sequences.
• the light chains of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise an amino acid sequence chosen from the sequences SEQ ID NO: 71 to 75, consisting of the fusion of N- in C-terminal between the amino acid sequence of the signal peptide MB7 (SEQ ID NO: 65) and one of the amino acid sequences of the VL region of the antibodies according to the invention (SEQ ID NO: 48 at 52), or consist essentially or consist of such sequences.
• SEQ ID NO: 65 amino acid sequence of the signal peptide MB7
• SEQ ID NO: 48 at 52 amino acid sequences of the VL region of the antibodies according to the invention
• preferred complete amino acid sequences of the antibodies according to the invention are obtained as described in Table 1 1 below.
• the constant region may further contain an additional C-terminal lysine residue.
• the heavy and light chains of the chimeric or humanized antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise the sequences described in Table 1 1 below (or consist essentially or
• the antibody, functional fragment or derivative thereof according to the invention can be produced from any host cell, any transgenic non-human animal or any transgenic plant described in the present description, and especially below in the part relating to the nucleic acids, vectors, host cells, transgenic non-human animals and transgenic plants according to the invention.
• functional fragment is meant an antibody fragment retaining the antigen-binding domain and therefore having the same antigenic specificity as the original antibody, such as Fv, ScFv, Fab, F (ab fragments). ') 2, Fab', scFv-Fc or di-antibodies ("diabodies").
• a functional fragment of antibody according to the invention is therefore advantageously chosen from Fv, ScFv, Fab, F (ab ') 2 , Fab', scFv-Fc fragments or diabodies.
• derivative of an antibody is meant a fusion protein formed of a carrier peptide and at least one of the CDRs of the original antibody to preserve its ability to recognize CD303.
• the antibody, functional fragment or derivative thereof according to the invention advantageously has a low fucose content of less than or equal to 65%.
• fucose content is meant the percentage of fucosylated forms within the N-glycans attached to the Asn297 residue of the Fc fragment of each heavy chain of each antibody.
• low fucose content is meant a fucose content less than or equal to 65%.
• the fucose content of an antibody composition plays a crucial role in the ability of this composition to induce a strong ADCC response via the Fc ⁇ RIII receptor.
• the fucose content is less than or equal to 65%, preferably less than or equal to 60%, 55% or 50%, or even less than or equal to 45%, 40%, 35% or 30%. , 25% or 20%.
• the fucose content can for example be between 5 and 65%, between 5 and 60%, between 5 and 55%, between 5 and 50%, between 5 and 45%, between 5 and 40%, between 5 and 35% between 5 and 30%, between 5 and 25%, between 5 and 20%, between 10 and 65%, between 10 and 60%, between 10 and 55%, between 10 and 50%, between 10 and 45%, between 10 and 40%, between 10 and 35%, between 10 and 30%, between 10 and 25%, between 10 and 20%, between 15 and 65%, between 15 and 60%, between 15 and 55%, between 15 and 50%, between 15 and 45%, between 15 and 40%, between 15 and 35%, between 15 and 30%, between 15 and 25%, between 15 and 20%, between 20 and 65%, between 20 and 60% between 20 and 55%, between 20 and 50%, between 20 and 45%, between 20 and 40%, between 20 and 35%, between 20 and 30%, between 20 and 25%.
• the antibody, functional fragment or derivative thereof according to the invention may moreover possess different types of glycosylation (oligomannose-type N-glycans or biantennane complexes, variable proportion of N-acetylglucosamine residues (GlcNAc)) or galactose residues. in the case of N-glycans of the biantennen complex type), provided that it has a low fucose content.
• glycosylation oligomannose-type N-glycans or biantennane complexes, variable proportion of N-acetylglucosamine residues (GlcNAc)
• galactose residues in the case of N-glycans of the biantennen complex type
• oligomannose N-glycans can be obtained by culturing in the presence of different glycosylation inhibitors, such as ⁇ 1,2-mannosidase I inhibitors (such as Deoxymannojirimycin or "DMM”) or ⁇ -glucosidase (such as castanospermine or "Cs"); or by production of the antibody in the CHO Lec 1 line.
• ⁇ 1,2-mannosidase I inhibitors such as Deoxymannojirimycin or "DMM”
• ⁇ -glucosidase such as castanospermine or "Cs”
• lines naturally possess a low activity of the enzyme FUT8 (1, 6-fucosyltransferase) responsible for the addition of fucose to the GlcNAc linked to the Fc fragment; such as the YB2 / 0 line, the EB66® duck embryonic cell line, the H4-II-E rat rat hepatoma lines (DSM ACC3129), the H4-ll-Es (DSM ACC3130), or the NM lines -H9D8-E6 (DSM ACC 2807), and NM H9D8-E6Q12 (DSM ACC 2856) can be used.
• FUT8 (1, 6-fucosyltransferase
• Mutant lines for other genes whose subexpression or overexpression leads to a low fucose content may also be used, such as the CHO Lec13 line, a mutant of the CHO line having decreased GDP-fucose synthesis. It is also possible to select a line of interest and to reduce or abolish (in particular by using interfering RNA or by mutation or deletion of the gene expressing the protein of interest) the expression of a protein involved in the pathway. fucosylation of N-glycans (notably FUT8, see Yamane-Ohnuki et al-2004, but also GMD, a gene involved in the transport of GDP-fucose, see Kanda et al-2007).
• N-glycans such as the GnTIII protein (6 (1,4) -N-acetylglucosaminetransferase III ).
• GnTIII protein (6 (1,4) -N-acetylglucosaminetransferase III ).
• antibodies possessing weakly fucosylated N-glycan have been obtained in particular by:
• NM-H9D8 (DSM ACC2806), NM-H9D8-E6 (DSM ACC 2807), and NM H9D8-E6Q12 (DSM ACC 2856) (see WO2008 / 028686).
• Oligomannose N-glycans have a reduced half-life in vivo compared to biantennane-type N-glycans. Therefore, advantageously, the antibodies according to the invention have, on their N-glycosylation sites of the Fc fragment, glycan structures of the bantennate complex type, with a low fucose content, as defined above.
• the monoclonal antibody according to the invention may have a content of G0 + G1 + G0F + G1 F forms greater than 60% and a low fucose content, as defined above. It may also have a G0 + G1 + G0F + G1 F content content greater than 65% and a low fucose content, as defined above. It may also have a G0 + G1 + G0F + G1 F content content greater than 70% and a low fucose content, as defined above. It may also have a G0 + G1 + G0F + G1 F content content greater than 75% and a low fucose content, as defined above.
• G0 + G1 + G0F + G1 F content content greater than 80% and a low fucose content, as defined above. It may also have a G0 + G1 + G0F + G1 F content content greater than 60%, 65%, 70%, 75% or 80% and a G0F + G1 F content content of less than 50%.
• the forms G0, G1, G0F and G1 F are as defined below:
• Such antibody compositions can in particular be obtained by production in YB2 / 0, in CHO Lec13, in wild-type CHO lines cultured in the presence of small interfering RNAs directed against FUT8 or GMD, in CHO lines whose two alleles of the FUT8 gene coding for 1,6-fucosyltransferase or the two alleles of the GMD gene coding for the GDP-fucose transporter in the Golgi have been deleted.
• the antibody, functional fragment or derivative thereof according to the invention has a high content of N-glycans of the oligomannose type.
• oligomannose N-glycans N-glycans whose pentasaccharide core consists of two N-acetylglucosamine (GlcNAc) residues (one of which is bound to the Asn297 residue of the Fc fragment of the antibody). ) and three mannose residues, is supplemented with one to six additional mannoses attached to terminal mannose residues of the pentasaccharide core. Oligomannose N-glycans are not fucosylated.
• oligomannose-type N-glycans content is meant the percentage of oligomannose forms within the N-glycans attached to the Asn297 residue of the Fc fragment of each heavy chain of each antibody.
• high content of oligomannose N-glycans means an N-glycans content of oligomannose type greater than or equal to 30%, advantageously greater than or equal to 35%, greater than or equal to 40%, greater than or equal to 45%, greater than or equal to 50%, greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65%, greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80 %, greater than or equal to 85%, greater than or equal to 90%, or greater than or equal to 95%.
• the antibody, functional fragment or derivative thereof according to the invention has a high galactose content.
• ⁇ Y 1 (number of A) x (% relative area)
• N represents the number of N-glycans analyzed on a chromatogram, for example a normal phase high performance liquid chromatography spectrum (NP HPLC), "Gai number” represents the number of galactoses on the glycan antenna corresponding to the peak,
• a number represents the number of N-acetyl-glucosamine antennas of the glycan form corresponding to the peak
• % relative area is the percentage of the area under the corresponding peak.
• High galactose content means a galactose content greater than or equal to 30%, advantageously greater than or equal to 50%, advantageously greater than or equal to 55%, greater than or equal to 60%, greater than or equal to 65% , greater than or equal to 70%, greater than or equal to 75%, greater than or equal to 80%, greater than or equal to 85%, greater than or equal to 90%, greater than or equal to 95%, or even equal to 100%.
• the present invention also relates to a monoclonal antibody directed against the ectodomain of the human CD303 antigen (SEQ ID NO: 130), or a functional fragment or a derivative thereof, characterized in that:
• the constant regions of the light and heavy chains are constant regions from a non-murine species.
• the affinity of the Fc fragment of an antibody for the Fc ⁇ RIIIa receptor can be measured by several methods, including surface plasmon resonance (RPS, in particular using a BIAcore 2000-type device - Pharmacia Biosensor, Upsala , Sweden) and Scatchard analysis.
• RPS surface plasmon resonance
• Eight genes encoding Fc ⁇ R have been identified in humans, but only five code for expressed receptors (Fc ⁇ RIa, Fc ⁇ RIla, Fc ⁇ RIIb, Fc ⁇ RIIIa, Fc ⁇ RIIIb). All are effector cell activating receptors, except for Fc ⁇ RIIb, which is an inhibitory receptor for the activation of immune cells.
• the FcyRIa receptors are characterized by a high affinity for immunoglobulins (Kd of 5.10 "7 to 10" 10 M) whereas the Fc? RIIa receptor, Fc? RIIb, Fc? RIIIa, FcyRIIIb are characteristic of low affinity receptors (Kd of less than 10 "7 M).
• the antibodies according to the invention have an affinity for the Fc ⁇ RIIIa receptor (CD16 ⁇ ) improved with respect to antibodies directed against the ectodomain of the human CD303 antigen produced in the CHO cell line.
• a wild-type CHO line producing in normal conditions antibodies whose Fc fragment is very strongly fucosylated (At least 80%, preferably at least 90% of oligosaccharides attached to the Fc fragment are fucosylated), such as one of the CHO-K-1 (ATCC® CCL-61 TM), CHO Pro-5 (ATCC) lines.
• the antibodies according to the invention which have an affinity for the Fc ⁇ RIIIa receptor (CD16a) improved with respect to antibodies directed against the ectodomain of the human CD303 antigen produced in the CHO cell line (as defined above) have advantage that this improved affinity allows them to not be displaced or to be less displaced from the Fc ⁇ RIIIa receptor (CD16a) by polyclonal IgG antibodies, especially IgG present in the blood serum.
• Such antibodies advantageously have a high affinity for the Fc ⁇ RIIIa receptor (CD16a), that is to say an affinity at least equal to 2 ⁇ 10 6 M -1 , at least equal to 2 ⁇ 10 7 M -1 , 2 ⁇ 10 8 M -1 or 2x10 9 M "1, as determined by Scatchard analysis or BIAcore technology (Label-free surface plasmon resonance based technology).
• a high affinity for the Fc ⁇ RIIIa receptor (CD16a), significantly improved over antibodies produced in the CHO cell line, can be obtained in various ways described above for the antibody, functional fragment or derivative thereof according to US Pat. invention defined by its ability to compete for ectodomain binding of CD303 with the particular antibodies developed by the inventors and the fact that the constant regions of its light and heavy chains are constant regions derived from a non-murine species.
• a high affinity for the Fc ⁇ RIIIa receptor (CD16a) significantly improved compared to antibodies produced in the CHO cell line, can be obtained by:
• Such an antibody according to the invention may furthermore possess any characteristic or combination of characteristics described above for the antibody, functional fragment or derivative thereof according to the invention defined by its ability to compete for binding to the ectodomain of CD303 with the particular antibodies developed by the inventors and in that the constant regions of its light and heavy chains are constant regions from a non-murine species.
• the present invention also relates to a nucleic acid (also called a nucleic acid or nucleotide sequence) coding for the heavy chain and / or the light chain of an antibody, functional fragment or derivative thereof according to the invention as described above. .
• a nucleic acid also called a nucleic acid or nucleotide sequence
• sequence of a nucleic acid according to the invention may have been optimized to promote its expression in a host cell, a transgenic non-human animal or a transgenic plant of interest.
• a host cell a transgenic non-human animal or a transgenic plant of interest.
• synonymous codons there are in general several combinations of three nucleotides encoding the same amino acid (except for methionine and tryptophan), called synonymous codons.
• some of these combinations are generally used preferentially by a given cell or organism (this is called genetic code usage bias). This preference depends in particular on the organism producing or from which the cell comes.
• a protein derived from one or more organisms is produced in a heterologous organism or in a cell of such a heterologous organism, it may be useful to modify the nucleic acid sequence coding for the protein so as to use primarily the preferred codons of the heterologous organism.
• Data are available in the literature regarding the preferred codon usage by different species, and one skilled in the art knows how to optimize the expression of a given protein in an organism or cell of a heterologous organism.
• a nucleic acid according to the invention advantageously comprises at least one of the sequences SEQ ID NO: 86 to 95 (chimeric antibodies), as described in Table 12 below, which encode the amino acid sequences of the VH and VL antibodies according to the invention and were optimized for expression in cells of the species Rattus norvegicus.
• VH VL Antibody chimeric antibodies
• nucleic acid according to the invention may also advantageously comprise at least one of the sequences SEQ ID NO: 181 to 187 (humanized antibodies derived from the 122A2 antibody), as described in Table 13 below, which encode the sequences of amino acids of the VH and VL regions of humanized antibodies according to the invention and have been optimized for expression in cells of the species Rattus norvegicus.
• a nucleic acid according to the invention may also advantageously comprise at least one of the sequences SEQ ID NO: 188 to 193 (humanized antibodies derived from the antibody 102E9), as described in Table 14 below, which encode the sequences of amino acids of the VH and VL regions of humanized antibodies according to the invention and have been optimized for expression in cells of the species Rattus norvegicus.
• Table 14 Preferred nucleotide sequences for humanized antibodies derived from the 102E9 antibody, optimized for expression in cells of the Rattus norvegicus species encoding the VH and VL regions of the antibodies, functional fragments or derivatives thereof according to US Pat. 'invention.
• Nucleic sequences encoding the preferred heavy or light chain constant regions have also been optimized for expression in cells of the Rattus norvegicus species and are preferably those described in Table 15 below.
• Table 1 Preferred nucleotide sequences coding for the preferred heavy or light human chain constant regions.
• a nucleic acid encoding the heavy chain and / or the light chain of an antibody according to the invention preferably comprises (or consists essentially of) at least one of the nucleic sequences described in Table 16. below, consisting of the 5 'to 3' fusion:
• sequences SEQ ID NO: 86 to 90 coding for the VH region of the preferred chimeric antibodies according to the invention and of the sequence SEQ ID NO: 96 coding for the preferred constant region of human heavy chain (see SEQ ID No. NO: 98 to 102);
• sequences SEQ ID NO: 91 to 95 coding for the VL region of the preferred chimeric antibodies according to the invention and of the sequence SEQ ID NO: 97 coding for the preferred constant region of human light chain see SEQ ID No. NO: 103 to 107
• sequence SEQ ID NO: 181 to 183 coding for the VH region of the humanized antibodies derived from the 122A2 chimeric antibody of the invention and of the sequence SEQ ID NO: 96 coding for the preferred constant chain region heavy human see SEQ ID NO: 194-196
• sequences SEQ ID NO: 191 to 193 coding for the VL region of the humanized antibodies derived from the chimeric antibody 102E9 which are preferred according to the invention and of the sequence SEQ ID NO: 97 coding for the preferred constant chain region light human (SEQ ID NO: 204-206).
• a nucleic acid encoding the heavy chain and / or the light chain of the antibody, functional fragment or derivative thereof according to the invention advantageously comprises a nucleic sequence encoding the heterologous signal peptide MB7 (MRWSWIFLLLLSITSANA, SEQ ID NO: 65), and in particular the nucleic acid sequence SEQ ID NO: 108 (ATG AG GTG GTCCTG G ATCTTCCTG CTG CTG CTG AG ATC ACC AG CG CCAACG CC).
• this peptide has been shown to improve the expression and secretion of recombinant proteins in higher eukaryotic cell lines (see WO2011 / 114063).
• a nucleic acid encoding the heavy chain of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprises (or consists essentially of) or consists of a nucleic sequence chosen from the sequences SEQ ID NO: 109 to 113 (chimeric antibodies), SEQ ID NO: 207 to 209 (humanized antibodies derived from the chimeric antibody 122A2) and SEQ ID NO: 214 to 216 (humanized antibodies derived from the chimeric antibody 102E9), constituted by the fusion 5 'to 3' between the nucleic sequence coding for the MB7 signal peptide (SEQ ID NO: 108) and one of the nucleic sequences coding for the VH region of the antibodies according to the invention (SEQ ID NO: 86 to 90 for the antibodies chimeric, SEQ ID NO: 181-183 for humanized antibodies derived from chimeric antibody 122A2, and SEQ ID NO: 188-190 for humanized antibodies derived from chimeric antibody 102E9).
• a nucleic acid encoding the light chain of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprises (or consists essentially of) or consists of a nucleic sequence chosen from sequences SEQ ID NO: 114 to 118, (chimeric antibodies), SEQ ID NO: 210 to 213 (humanized antibodies derived from the chimeric antibody 122A2) and SEQ ID NO: 217 to 219 (humanized antibodies derived from the chimeric antibody 102E9 ), constituted by the 5 'to 3' fusion between the nucleic acid sequence coding for the MB7 signal peptide (SEQ ID NO: 108) and one of the amino acid sequences of the VL region of the antibodies according to the invention (SEQ ID No.
• preferred complete amino acid sequences of the antibodies according to the invention are obtained, as described in Table 17 below.
• nucleic acid encoding the heavy chain and / or the light chain of a chimeric or humanized antibody, a functional fragment or a derivative thereof according to the invention advantageously comprises at least one of the sequences described in Table 17 below (or essentially consists, or consists of such sequences).
• the present invention also relates to a vector comprising a nucleic acid according to the invention.
• a vector comprises the elements necessary for the expression of said nucleic sequence, and in particular a promoter, a transcription initiation codon, termination sequences, and appropriate transcriptional regulatory sequences. These elements vary according to the host serving for the expression and are readily selected by those skilled in the art in view of his general knowledge.
• the vector advantageously comprises a "Kozak sequence", i.e. a conserved sequence found on eukaryotic messenger RNAs at the site start of translation, around the AUG start codon (usually GCCGCC (A / G) CCATGG, the initiation codon of the translation being underlined).
• the vector may especially be plasmidic or viral. It is used to clone or express the nucleic acids according to the invention.
• a vector as described in WO2013 / 061010 comprising at least one transcription unit comprising the following regulatory elements: the enhancer of the virus hCMVie said enhancer having the nucleotide sequence SEQ ID NO: 220, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 220 and having essentially transcriptional activation properties, and the promoter region of kinase-dependent cyclin 9 (CDK9), said region promoter having the nucleotide sequence SEQ ID NO: 221, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 221 and having essentially a promoter activity.
• the enhancer of the virus hCMVie said enhancer having the nucleotide sequence SEQ ID NO: 220, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 220 and having essentially transcriptional activation properties
• CDK9 kinase-dependent cyclin 9
• the transcription unit included in the vector further comprises at least one of the following two additional elements:
• a 5 'untranslated region (5' UTR) located downstream of the promoter region in particular chosen from:
• LTR long terminal repeat
• NF- ⁇ repressing gene Factor having the nucleotide sequence SEQ ID NO: 223, or a nucleotide acid having at least 70%> sequence identity with SEQ ID NO: 223,
• eukaryotic initiation Factor 4GI eukaryotic initiation Factor 4GI having the nucleotide sequence SEQ ID NO: 224, or a nucleotide acid having at least 70%> sequence identity with SEQ ID NO: 224,
• nucleotide acids having at least 70% sequence identity with the aforesaid SEQ ID NO: 222, 223, 224 sequences having essentially mRNA stabilizing and translation facilitating properties
• an intron located downstream of the promoter region and upstream of the translation initiation site in particular chosen from:
• Murine ROSA intron having the nucleotide sequence SEQ ID NO: 226, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 226,
• ⁇ intron 5 'LTR of the HTLV-1 virus having the nucleotide sequence SEQ ID NO: 227 or a nucleotide acid having at least 70%> sequence identity with SEQ ID NO: 227,
• ⁇ intron of the ubiquitin gene having the nucleotide sequence SEQ ID NO: 229, or a nucleotide acid having at least 70%> sequence identity with SEQ ID NO: 229,
• nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 230 or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 230.
• the transcription unit included in the vector comprises the enhancer of the hCMVie virus, the promoter region of CDK9, the 5 'UTR region of the elF4GI gene and the EFla gene intron, and has the nucleotide sequence SEQ ID NO: 231 (vector "HKgenEFss").
• the enhancer of the hCMVie virus said enhancer having the nucleotide sequence SEQ ID NO: 232, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 232 and having essentially activation properties transcription, o the promoter region of ⁇ -actin, said promoter region having the nucleotide sequence SEQ ID NO: 233, or a nucleotide acid having at least 70% sequence identity with the sequence SEQ ID NO: 233 and having essentially a promoter activity, and
• nucleotide acid located downstream of said promoter region and upstream of the translation initiation site, said nucleotide acid comprising at least one of the 5 'untranslated regions (5' UTR) chosen from the following:
• NF- ⁇ repressing gene Factor having the nucleotide sequence SEQ ID NO: 235, or a nucleotide acid having at least 70%> sequence identity with SEQ ID NO: 235
• elF4GI gene eukaryotic initiation Factor 4GI
• nucleotide acids having at least 70% sequence identity with one of the sequences represented by SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236 having essentially mRNA stabilizing properties and facilitator of the translation.
• the transcription unit included in the vector may furthermore comprise an intron, in particular chosen from the following:
• o intron of the Elongation Factor la (EFla) gene having the nucleotide sequence SEQ ID NO: 237, or a nucleotide acid having at least 70% sequence identity with SEQ ID NO: 237, o murine ROSA intron having the nucleotide sequence SEQ ID NO:
• nucleotide acid having at least 70% sequence identity with SEQ ID NO: 238,
• said intron being located:
• the transcription unit included in the vector comprises the enhancer of the hCMVie virus, the ⁇ -actin promoter region, the HTLV-1 virus 5 'UTR region (U1) and the 5' UTR region of the elF4GI gene. (U3), and has the nucleotide sequence SEQ ID NO: 240.
• the present invention also relates to a host cell, a transgenic non-human animal or a transgenic plant comprising at least one nucleic acid according to the invention or a vector according to the invention.
• the host cell may be of prokaryotic or eukaryotic origin, and may especially be chosen from bacterial cells, insect cells, plants, yeast or mammals.
• the antibody, functional fragment or derivative according to the invention can then be produced by culturing the host cell under appropriate conditions.
• a host cell according to the invention may in particular be obtained by transformation of a cell line by the heavy and light chain expression vector (s) of an antibody, a functional fragment or derivative thereof according to US Pat. invention, and separation of the different cell clones obtained.
• the transformed cell line is preferably of eukaryotic origin, and may in particular be chosen from insect, plant, yeast or mammalian cells.
• Cell lines suitable for the production of antibodies include the lines selected from: SP2 / 0; YB2 / 0; IR983F; human myeloma Namalwa; PERC6; CHO lines, in particular CHO-K-1, CHO-Lec10, CHO-Led, CHO-Lec13, CHO Pro-5, CHO dhfr-, CHO-DP12, CHO DUKX-B11, CHO DG-44, or CHO deleted line.
• a transgenic non-human animal according to the invention can be obtained by direct injection of the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) in a fertilized egg (Gordon and al-1980).
• a transgenic non-human animal can also be obtained by introducing the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) into an embryonic stem cell and preparing it. animal by a chimera aggregation method or a chimeric injection method (see Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)).
• a transgenic non-human animal can also be obtained by a cloning technique in which a nucleus, in which the gene (s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) has been introduced, is transplanted into an enucleated egg (Ryan et al 1997, Cibelli et al., 1998, WO00 / 26357).
• a transgenic non-human animal producing an antibody of interest can be prepared by the above methods. The antibody can then be accumulated in the transgenic animal and harvested, in particular from the milk or eggs of the animal.
• transgenic methods of preparation are described in particular in WO90 / 04036, WO95 / 17085, WO01 / 26455, WO2004 / 050847, WO2005 / 033281, WO2007 / 048077.
• Methods for purifying proteins of interest from milk are also known (see WO01 / 26455, WO2007 / 106078).
• Non-human transgenic animals of interest include mice, rabbits, rats, goats, cattle (including cows), and poultry (especially chicken).
• a transgenic plant according to the invention may be chosen from any plant that allows the production of antibodies. Many antibodies have already been produced in transgenic plants and the technologies necessary to obtain a transgenic plant expressing an antibody of interest and to the recovery of the antibody are well known to those skilled in the art (see Stoger and al-2002, Fisher et al-2003, Ma et al. 2003, Schillberg et al. 2005). It is also possible to influence the glycosylation obtained in plants to obtain glycosylation close to that of natural human antibodies (without xylose), but with, in addition, low fucosylation, for example with the aid of small interfering RNAs (Forthal et al. al, 2010).
• the present invention also relates to an antibody, functional fragment or derivative thereof according to the invention, for use as a medicament.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of hematopoietic tumors expressing the CD303 antigen.
• CD303 antigen include neoplasms of blast plasmacytoid dendritic cells (BPDCN), CD4 + phenotype, CD11 c-, Lin-, CD303 +, CD304 +, CD56 +.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of inflammatory diseases, in particular autoimmune diseases.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of diseases involving pDC, and more particularly diseases involving secretion of IFN- ⁇ by pDC.
• the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of the following diseases, for which a role of pDC has been established (see Wollenberg et al.
• atopic dermatitis atopic dermatitis, contact dermatitis, psoriasis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, type 1b diabetes, autoimmune thrombocytopenia (or thrombocytopenia) (especially idiopathic thrombocytopenic purpura or PTI), systemic scleroderma (still called progressive systemic sclerosis or systemic sclerosis), rheumatoid arthritis.
• the present invention also relates to:
• an antibody, functional fragment or derivative thereof according to the invention for the preparation of a medicament for the treatment or prevention of hematopoietic tumors expressing the CD303 antigen, and in particular cell neoplasms Blastic plasmacytoid dendritic (BPDCN), CD4 + phenotype, CD1 1 c-, Lin-, CD303 +, CD304 +, CD56 +.
• BPDCN Blastic plasmacytoid dendritic
• CD4 + phenotype CD1 1 c-, Lin-, CD303 +, CD304 +, CD56 +.
• an antibody, functional fragment or derivative thereof according to the invention for the preparation of a medicament for the treatment or prevention of inflammatory diseases, in particular autoimmune diseases, and in particular the diseases mentioned above.
• the present invention also relates to:
• an antibody, functional fragment or derivative thereof in the treatment or prevention of hematopoietic tumors expressing the CD303 antigen, and in particular blast plasmacytoid dendritic cell neoplasms (BPDCN), CD4 + phenotype, CD1 1 c-, Lin-, CD303 +, CD304 +, CD56 +.
• BPDCN blast plasmacytoid dendritic cell neoplasms
• the present invention also relates to:
• BPDCN blast plasmacytoid dendritic cells
• a method of treating or preventing a patient from an inflammatory disease, particularly an autoimmune disease, and in particular one of the diseases mentioned above, comprising administering to said patient an effective amount of an antibody, functional fragment or derivative thereof according to the invention.
• treatment is meant a clinical or biochemical improvement of the patient's pathology.
• prevention is meant to prevent or delay the occurrence or decrease the intensity of the clinical or biochemical manifestations associated with the disease.
• a biological parameter of interest may be the number of blasts.
• a biological parameter of interest may be the number of pDCs or the amount present locally or systemically of molecules contributing to inflammation (inflammatory cytokines and in particular INFa).
• RNAs of each hybridoma were extracted using the Macherey nagel kit; Nucleospin RNAII (column purification).
• the mRNAs were converted to cDNA and the heavy and light chains of the antibody were amplified using the Generacer kit (invitrogen) and cloned into an M13 vector. Bacteria were then transformed with the M13 vector and clones positive for the M13 sequences were sequenced.
• VH, DH, JH segments of heavy chains and light chain VL and JL segments The variable part, the V and J segments used by the heavy and light chains and the CDR sequences of the heavy and light chains were determined by using the IMGT Domain Gapalign tool (see Ehrenmann et al-2010 and Ehrenmann et al-201 1) available at the following address: http: //www.imgt.org/3Dstructure- DB / cgi / DomainGapAlign.cgi . Construction of expression vectors for chimeric antibodies
• VH and VL variable region sequences of the murine antibodies were optimized for preferential use of codons of the species Rattus norvegicus.
• a sequence encoding the heterologous signal peptide MB7 was further introduced 5 'to the sequence coding for the variable region VH or VL of each antibody.
• the sequences of the human constant parts were extracted from the CHK622-21 expression vector of a human anti-Rhesus D antibody (T125) by Apal / Ascl digestion for the H (IgG1 m1 .17) and Dralll / XbaI chain for the Kappa chain.
• variable and constant parts of the same chain were introduced simultaneously into the generic vector HKgenEFss by ligation with KAPA T4 ligase, generating the vectors HKBDCA-2-122A2, HKBDCA-2-102E9, HKBDCA-2-104C12 , HKBDCA-2- 1 14D1 1, and HKBDCA-2-104E10 (see FIGS. 1A-1E).
• the vectors HKBDCA-2-122A2, HKBDCA-2-102E9, HKBDCA-2-104C12, HKBDCA-2-1 14D1 1, and HKBDCA-2-104E10 were transfected into cells of the YB2 / 0 line, and a clone producing each antibody was selected for the production of chimeric antibodies.
• sequences of the heavy chain (H) and of the light chain (K) were synthesized by adding a Kozak sequence and restriction sites on both sides of the H and K sequences, to allow cloning by digestion / ligation.
• the expression vector used, to produce the humanized antibody BIIB059 is a bicistronic vector, HKgenEFss, of 10835 bp, which makes it possible to produce the antibody optimally in the CHO line (FIG. 1F).
• Cloning of heavy and light chains is done in 2 sequential steps. The first step is to insert by digestion / ligation the H chain between cloning sites Nhel / Ascl to result in an intermediate vector containing only this chain.
• the K-chain is inserted by the same technique between the Spel / XbaI cloning sites.
• the ligation enzyme is KAPA T4 DNA Ligase (Kapa Biosystems).
• a dephosphorylation step prior to insertion of the K chain is necessary to prevent the linearized plasmid from closing on itself, due to the Spel / XbaI restriction sites that generate blunt ends.
• the production of the humanized antibody BIIB059 is made by transient transfection from the CHO line, in order to meet the conditions of the application WO2014 / 09339.
• This line is cultured in ProCH04 medium, supplemented with 4 mM Glutamine.
• the line is maintained at 2-3x10 5 cells / ml, every 2 days.
• the solutions used for transfection are OptiPRO buffer and Freestyle Max Reagent transfection agent.
• the BIIB059 antibody was purified by affinity chromatography.
• the 2 antibodies of family 1 (122A2 and 104C12) share the use of the same VH segment (IGHV1 S137 * 01), as well as the use of VL segments (IGKV10- 96 * 01 / IGKV10-96 * 02) and JL (IGKJ1 * 01 / IGKJ1 * 02) of the same family, as shown in Table 18 below. These two antibodies therefore have a close structure.
• the two antibodies of family 1 (122A2 and 104C12) have the same CDR1 -H and CDR2-L sequences, and moreover have extremely close CDR2-H, CDR1 -L and CDR3-L sequences (only 1 or 2 amino acids of difference). Even the CDR3-H sequences show strong homology (5/1 common amino acids), as shown in Table 19 below. This confirms that these two antibodies have a very close structure.
• the 3 antibodies of family 2 (102E9, 1 14D1 1 and 104E10) have the same CDR2-L sequences, and moreover have extremely close CDR1 -H, CDR2-H, CDR1-L and CDR3-L sequences. (Only 1 amino acid difference). Even CDR3-H sequences show strong homology (12/14 common amino acids), as shown in Table 19 below. This confirms that these three antibodies have a very close structure.

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