Patent Grant
10611843
The present invention is in to the field of therapeutic antibodies for treating or preventing hematopoietic tumors of the CD4+CD56+ phenotype or inflammatory diseases, notably autoimmune diseases, involving plasmacytoid dendritic cells (pDCs). It relates to five monoclonal antibodies directed against the CD303 antigen, and to functional fragments and derivatives thereof, to the nucleic acids encoding the heavy and/or light chain of the antibody, to expression vectors, host cells, transgenic non-human animals or transgenic plants expressing said antibodies, and to therapeutic uses of said antibodies.
Dendritic cells (called “DCs” throughout the present description) are antigen-presenting cells (APCs) of the immune system. Under certain conditions, DCs have cytoplasmic projections similar to the dendrites of neurons.
Dendritic cells have two main functions:
DCs are capable of differentiating into various subpopulations, according to the stimuli they receive. There are three major types of DCs: conventional DCs, plasmacytoid DCs (called “pDCs”) and inflammatory DCs.
Conventional DCs present self or non-self-antigens in lymphoid organs or in the periphery. Inflammatory DCs, probably derived from blood monocytes, appear only in the event of stimulation following inflammation or infection.
Plasmacytoid DCs (pDCs) are circulating, round and without dendrites in the basal state, but acquire dendrites after activation, generally by a viral antigen. After stimulation, they produce a large amount of type I interferons (IFNs), and are chiefly involved in the antiviral response or in autoimmune diseases. Phenotypically, they are notably characterized by the following markers: CD4+, CD11c−, Lin−, CD303+, CD304+.
pDCs can be the cause of hematopoietic tumors in which they acquire an additional marker (CD56). This is referred to as blastic plasmacytoid dendritic cell neoplasm (BPDCN). These tumors are also referred to as CD4+CD56+ hematopoietic tumors. These hematopoietic tumors are rare (1% of acute leukemias) and appear as cutaneous nodules associated with lymphadenopathy or swelling of the spleen and with frequent cytopenia. The cutaneous manifestations are very quickly followed by infiltration of the bone marrow. It is now accepted that the hematopoietic cells at the origin of these tumors are pDCs.
The current treatments for these blastic plasmacytoid dendritic cell neoplasms are based on chemotherapy. Although relatively effective at first, this treatment is characterized by frequent and early relapses (about 9 months), median overall survival being only about 13 months. Another treatment is based on allografting of hematopoietic cells, but it does not enable long-term survival either.
It has been proposed in WO2012/080642 to use anti-BDCA-2 (i.e., anti-CD303) antibodies to treat these neoplasms by tumor cell depletion.
pDCs are also involved in certain inflammatory diseases, and notably in certain autoimmune diseases, in particular via their secretion of type I IFN.
One treatment is based on the use of anti-IFNα antibody. However, this treatment leads to systemic neutralization of IFNα, thus potentially increasing the risk of opportunistic infections. It has thus also been proposed in WO01/365487 and WO2012/080642 to use anti-BDCA-2 (anti-CD303) antibodies (in particular antibody AC144) to treat autoimmune diseases, by removing the pDCs responsible for local inflammation.
However, in order for these treatments to be effective, it is necessary to have suitable monoclonal antibodies that allow, in humans, the most efficient as possible removal of pDCs. To that end, chimeric or humanized antibodies with high affinity for the CD303 antigen and effector capabilities (ADCC, CDC, phagocytosis, signaling via cross-linking of CD303 by Fc receptors, in particular Fcγ receptor III (FcγRIII, also called CD16) and/or apoptosis) enabling them to remove pDCs in physiological conditions are necessary. The anti-CD303 antibodies available to date being inadequate, there thus exists a need for new anti-CD303 antibodies having the desired properties.
WO2014/09339 describes humanized anti-CD303 antibodies, defined by the sequences of their heavy and light chains, and notably antibody BIIB059, which is the one having the best affinity for the CD303 antigen. Antibody BIIB059 was produced in CHO cells and characterized in terms of certain properties.
In the context of the present invention, the inventors generated five chimeric monoclonal antibodies having such properties. At least two of these chimeric antibodies (122A2 and 102E9) have a capacity to bind to the ectodomain of the human CD303 antigen that is greater than that of the antibody (BIIB059) described in WO2014/09339 (in particular 122A2). Moreover, humanized antibodies derived from these two chimeric antibodies were generated and characterized. These humanized antibodies can be produced with higher productivity than the original chimeric antibodies and, among those derived from chimeric antibody 122A2, certain have a capacity to bind to the ectodomain of the human CD303 antigen that is even higher than that of the original chimeric antibody and thus higher than that of the antibody (BIIB059) described in WO2014/09339. The chimeric and humanized antibodies produced in YB2/0 cells also have high affinity for FcγRIIIa (CD16a), and are capable of inducing strong ADCC responses, even at low antigen density. The antibodies also have CDC activity and are capable of inhibiting the secretion of IFN-α and of TNF-α.
In a first aspect, the present invention thus 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 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 present invention also relates to a nucleic acid encoding the heavy and/or 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 medicinal product.
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 or in the treatment or prevention of inflammatory diseases, notably autoimmune diseases.
Antibody, Functional Fragment or Derivative
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:
Advantageously, the heavy chains comprise three CDR-H (heavy-chain CDR according to IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences, and the light chains comprise three CDR-L (light-chain CDR according to IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences:
Table 1 below summarizes the amino acid sequences of the CDR-IMGT of the two families of antibodies according to the invention:
Advantageously, the heavy chains of an antibody, functional fragment or derivative thereof according to the invention comprise three CDR-H (heavy-chain CDR according to IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences, and the light chains comprise three CDR-L (light-chain CDR according to IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences:
Advantageously, the heavy chains of an antibody, functional fragment or derivative thereof according to the invention comprise a variable region having a sequence selected from SEQ ID NOs: 43 to 47 or a sequence having at least 80% identity with one of SEQ ID NOs: 43 to 47.
Additionally or alternatively, 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 NOs: 48 to 52 or a sequence having at least 80% identity with one of SEQ ID NOs: 48 to 52.
In a preferred embodiment, the antibody, functional fragment or derivative thereof according to the invention has heavy and light chains which variable regions have the following amino acid sequences or sequences having at least 80% identity with the following sequences:
Table 2 below summarizes the murine VH, JH and VL and JL gene segments used by the various antibodies according to the invention and the percent identity.
Table 3 below summarizes the amino acid sequences of the CDRs and the variable regions of the heavy and light chains of the anti-CD303 antibodies generated by the inventors:
The human CD303 antigen is C-type lectin domain family 4, member C (CLEC4), also called DLEC; HECL; BDCA2; CLECSF7; CLECSF11; or PRO34150 (see the Entrez Gene website for the CLEC4 gene). It is a type II transmembrane glycoprotein of 213 amino acids, comprising a short cytoplasmic domain with no evident signaling motif (amino acids 1-21), a transmembrane region (amino acids 22-41), a neck domain (amino acids 42-82), and a carbohydrate recognition domain (CRD; amino acids 83-213) (Dzionek et al.—2001). The mRNA sequence encoding this protein may be found in the 14 Feb. 2002 version of the GenBank database under accession number AF293615.1 (SEQ ID NO: 129), while the amino acid sequence is accessible in the 14 Feb. 2002 version of the GenBank database under accession number AAL37036.1 (SEQ ID NO: 130).
By “antibody” or “immunoglobulin” is meant a molecule comprising at least one binding domain for a given antigen and a constant domain comprising an Fc fragment capable of binding to Fc receptors (FcR). In most mammals, like humans and mice, an antibody consists of four polypeptide chains: two heavy chains and two light chains bound 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 consisting of a variable domain (VH) and three or four constant domains (CH1 to CH3 or CH1 to CH4) according to the isotype of the antibody. In a few rare mammals, such as camels and llamas, the antibodies consist of only two heavy chains, each heavy chain comprising a variable domain (VH) and a constant region.
The variable domains are involved in antigen recognition, while the constant domains are involved in the biological, pharmacokinetic and effector properties of the antibody.
The variable region differs from one antibody to another. Indeed, the genes encoding antibody heavy and light chains are respectively generated by recombination of 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 recombination and form the constant regions of the heavy and light chains, respectively. 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 three hypervariable zones or complementarity-determining regions (CDRs) called CDR1, CDR2 and CDR3, with CDR3 being the most variable since it is located in the recombination zone. These three CDRs, and particularly CDR3, are found in the portion of the antibody that will be in contact with the antigen and are thus very important for antigen recognition. Thus, antibodies retaining the three CDRs and each of the heavy and light chains of an antibody mostly retain the antigen specificity of the original antibody. In a certain number of cases, an antibody retaining only one of the CDRs, and notably CDR3, also retains the specificity of the original antibody. CDR1, CDR2 and CDR3 are each preceded by FR1, FR2 and FR3, respectively, corresponding to framework regions (FRs) that vary the least from one VH or VL segment to another. CDR3 is also followed by a framework region, FR4.
An antibody's CDRs are defined by the amino acid sequence of its heavy and light chains compared to criteria known to a person 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 according to the method chosen. The first determination method is that proposed by Kabat et al. (1991). In this method, CDRs are defined by looking for the amino acids responsible for antibody-antigen binding. A second method was proposed by the IMGT, based on determining hypervariable regions. In this method, a unique numbering has been defined to compare variable regions regardless of the antigen receptor, chain type or species (Lefranc et al. 2003). This numbering provides a standardized definition of framework regions ((FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and complementarity-determining regions (CDR1-IMGT: positions 27 to 38, CDR2-IMGT: positions 56 to 65 and CDR3-IMGT: positions 105 to 117). Finally, there is also a numbering called “common” in which the sequence of a particular CDR corresponds to the common sequence between the Kabat numbering and the IMGT numbering. Throughout the present description, the CDR sequences are indicated by the IMGT numbering. In particular, the CDRs have been determined by using the IMGT/V-QUEST program available at http://www.imgt.org/IMGT_vquest/share/textes/ and described in Brochet et al.—2008. Unlike the variable domains, whose sequence strongly varies from one antibody to another, the constant domains are characterized by an amino acid sequence that is very similar from one antibody to another, typical of the species and the isotype, with optionally a few somatic mutations. The Fc fragment naturally consists of the heavy chain constant region excluding the CH1 domain, i.e., the lower hinge region and the constant domains CH2 and CH3 or CH2 to CH4 (depending on the isotype). In human IgG1, the complete Fc fragment consists of the C-terminal portion of the heavy chain starting from the cysteine residue at position 226 (C226), the numbering of amino acid residues in the Fc fragment being throughout the present 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 may easily be identified by a person skilled in the art by sequence alignments.
The Fc fragment is glycosylated in the CH2 domain with the presence, on each of the two heavy chains, of an N-glycan bound to the asparagine residue at position 297 (Asn297).
The following binding domains, located in the Fc, are important for the biological properties of the antibody:
In the context of the invention, the Fc fragment of an antibody may be natural, as defined above, or else may be modified in various ways, provided that it comprises a functional FcR (FcγR for IgGs) binding domain, and preferably a functional FcRn binding domain. The modifications may include deletion of certain portions of the Fc fragment, provided that the latter contains a functional FcR (FcγR for IgGs) binding domain, and preferably a functional FcRn binding domain. The modifications may further include various amino acid substitutions able to affect the biological properties of the antibody, provided that the latter contains a functional FcR binding domain, and preferably a functional FcRn binding domain. In particular, when the antibody is an IgG, it may comprise mutations intended to increase FcγRIIIa (CD16a)-binding, as described in WO00/42072, Shields et al.—2001, Lazar et al.—2006, WO2004/029207, WO2004/063351, WO2004/074455. Mutations for increasing FcRn 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.—2006(a), WO00/42072, WO02/060919, WO2010/045193, or WO2010/106180. Other mutations, such as those for decreasing or increasing binding to complement proteins and thus the CDC response, may optionally be present (see WO99/51642, WO2004/074455, Idusogie et al.—2001, Dall'Acqua et al.—2006(b), and Moore et al.—2010).
In the context of the present invention, the preferred mutants comprising mutations for increasing FcRn binding and thus in vivo half-life are mutants comprising the following combinations of mutations in their Fc fragment, described in WO2010/106180:
Alternatively, just as fragments or derivatives without the Fc fragment of the antibody may be useful in the context of the invention, notably for the treatment of inflammatory, notably autoimmune, diseases involving pDCs, the antibody according to the invention may also have a mutated Fc fragment free of effector functions. Examples of mutations leading to a mutated Fc fragment free of effector functions are the single amino acid deletions at 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).
By “monoclonal antibody” or “monoclonal antibody composition” is meant a composition comprising antibody molecules having an identical and unique antigen specificity. The antibody molecules present in the composition are likely to vary in terms of their post-translational modifications, and notably in terms of their glycosylation structures or their isoelectric point, but have all been encoded by the same heavy and light chain sequences and thus have, before any post-translational modification, the same protein sequence. Certain differences in protein sequences, related to post-translational modifications (such as for example cleavage of the heavy chain C-terminal lysine, deamidation of asparagine residues and/or isomerization of aspartate residues), may nevertheless exist between the various antibody molecules present in the composition.
The percent identities referred to in the context of the disclosure of the present invention are determined on the basis of a global alignment of sequences to be compared, i.e., on an alignment of the sequences taken in their entirety over their entire length using any algorithm well-known to a person skilled in the art, such as the algorithm of Needleman and Wunsch (1970). This sequence comparison may be performed using any software well-known to a person skilled in the art, for example the Needle software by using the “Gap open” parameter equal to 10.0, the “Gap extend” parameter equal to 0.5 and a “Blosum 62” matrix. The Needle software is for example available on the website ebi.ac.uk under the name “Align”.
When the CDR or variable region of an antibody according to the invention has an amino acid sequence that is not 100% identical to one of those described above and in the sequence listing (reference sequences) but that 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 one such reference sequence, it may have insertions, deletions or substitutions with regard to the reference sequence. When it is a matter of substitutions, the substitution is preferably made with an “equivalent” amino acid, i.e., any amino acid whose structure is similar to that of the original amino acid and therefore unlikely to change the biological activity of the antibody. Examples of such substitutions are presented in Table 4 below:
The antibodies may be of several isotypes, according to the nature of their constant region: the γ, α, μ, ε and δ constant regions correspond to immunoglobulins IgG, IgA, IgM, IgE and IgD, respectively. Advantageously, the monoclonal antibody present in a composition used as a medicinal product in the context of the invention is of isotype IgG. Indeed, this isotype shows a capacity to generate antibody-dependent cell cytotoxicity (ADCC) activity in the greatest number of individuals (humans). The γ constant regions comprise several subtypes: γ1, γ2, γ3, these three types of constant regions having the feature of binding human complement, and γ4, thus creating the subtypes IgG1, IgG2, IgG3, and IgG4. Advantageously, the monoclonal antibody present in a composition used as a medicinal product in the context of the invention is of 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 heavy and light chain constant region is of human origin.
By “chimeric” antibody is meant an antibody that contains a natural variable region (light chain and heavy chain) derived from an antibody of a given species in combination with the light and heavy chain constant regions of an antibody of a species heterologous to said given species. Advantageously, if the monoclonal antibody composition for use as a medicinal product according to the invention comprises a chimeric monoclonal antibody, the latter comprises human constant regions. From a non-human antibody, a chimeric antibody can be prepared by using the genetic recombination techniques well-known to a person skilled in the art. For example, the chimeric antibody can be prepared by cloning the heavy and light chains of a recombinant DNA comprising a promoter and a sequence encoding the variable region of the non-human antibody, and a sequence encoding the constant region of a human antibody. For methods for preparing chimeric antibodies, reference may be made, for example, to the document by Verhoeyen et al.—1988.
By “humanized” antibody is meant an antibody that contains CDRs derived from an antibody of non-human origin, the other portions of the antibody molecule being derived from one (or from several) human antibodies. Moreover, certain residues of the framework regions (FR) may be modified to retain binding affinity (Jones et al.—1986; Verhoeyen et al. 1988; Riechmann et al.—1988). The humanized antibodies according to the invention can be prepared by techniques known to a person skilled in the art such as CDR grafting, resurfacing, superhumanization, human string content, FR libraries, guided selection, FR shuffling and humaneering technologies, as summarized in the review by Almagro et al.—2008.
In addition to the five chimeric antibodies comprising the CDR or variable region sequences described above, the inventors also generated humanized antibodies derived from two of the five anti-CD303 antibodies obtained initially. To that end, for each of the two antibodies, several mutated heavy chain variable region sequences (mutations directed at making the sequence more similar to a human sequence) and several mutated light chain variable region sequences were generated and combined with each other in pairs to try to obtain humanized antibodies having the strongest possible capacity to bind to the human CD303 antigen (SEQ ID NO: 130) (advantageously at least 70% of the binding capacity of the murine or chimeric antibody, more advantageously similar to the binding capacity of the murine or chimeric antibody, even more advantageously higher than the binding capacity of the murine or chimeric antibody).
For antibody 122A2, the amino acid sequences of the heavy and light chain variable regions (humanized, i.e., mutated) that were tested are presented in Table 5 below:
Humanized antibodies 122A2 are thus advantageously selected from those whose heavy chains comprise a variable region having an amino acid sequence selected from SEQ ID NOs: 131 to 133, advantageously from those whose heavy chains comprise a variable region having an amino acid sequence selected from SEQ ID NO: 131 and 132.
Humanized antibodies 122A2 are also advantageously selected from those whose light chains comprise a variable region having an amino acid sequence selected from SEQ ID NOs: 134 to 137, advantageously from those whose light chains comprise a variable region having an amino acid sequence selected from SEQ ID NO: 134 and 135.
The various humanized antibodies 122A2 generated by the inventors have heavy and light chains comprising variable regions having the amino acid sequences described in Table 6 below. The preferred humanized antibodies 122A2 (because they exhibit antigen-binding equal to or higher than that of the original chimeric antibody) are those whose heavy and light chains comprise variable regions having the amino acid sequences comprising those described for antibodies 122A2H5, 122A2H9, 122A2H6, 122A2H10, 122A2H14, 122A2H7, 122A2H11 and 122A2H15 in Table 6 below (shown in bold in Table 6 below). The most-preferred humanized antibodies 122A2 are those exhibiting higher antigen-binding than that of the original chimeric antibody: 122A2H5, 122A2H9, 122A2H6, 122A2H10, 122A2H14, 122A2H7, and in particular 122A2H5, 122A2H9, 122A2H7, and 122A2H10.
122A2H5
122A2-VHha
122A2-VKha
(SEQ ID NO: 131)
(SEQ ID NO: 134)
122A2H9
122A2-VHha
122A2-VKhb
(SEQ ID NO: 131)
(SEQ ID NO: 135)
122A2H6
122A2-VHhb
122A2-VKha
(SEQ ID NO: 132)
(SEQ ID NO: 134)
122A2H10
122A2-VHhb
122A2-VKhb
(SEQ ID NO: 132)
(SEQ ID NO: 135)
122A2H14
122A2-VHhb
122A2-VKhc
(SEQ ID NO: 132)
(SEQ ID NO: 136)
122A2H7
122A2-VHhc
122A2-VKha
(SEQ ID NO: 133)
(SEQ ID NO: 134)
122A2H11
122A2-VHhc
122A2-VKhb
(SEQ ID NO: 133)
(SEQ ID NO: 135)
122A2H15
122A2-VHhc
122A2-VKhc
(SEQ ID NO: 133)
(SEQ ID NO: 136)
For antibody 102E9, the amino acid sequences of the heavy and light chain variable regions (humanized, i.e., mutated) that were tested are presented in Table 7 below:
Humanized antibodies 102E9 are thus advantageously selected from those whose heavy chains comprise a variable region having an amino acid sequence selected from SEQ ID NOs: 138 to 140, advantageously from those whose heavy chains comprise a variable region having the amino acid sequence SEQ ID NO: 139.
Humanized antibodies 102E9 are also advantageously selected from those whose light chains comprise a variable region having an amino acid sequence selected from SEQ ID NOs: 141 to 143, advantageously from those whose light chains comprise a variable region having the amino acid sequence SEQ ID NO: 142.
The various humanized antibodies 102E9 generated by the inventors have heavy and light chains comprising variable regions having the amino acid sequences described in Table 8 below. The preferred humanized antibodies 102E9 (because they exhibit binding on the level of the chimeric antibody similar to that of the original chimeric antibody: at least 70% of the binding of the chimeric antibody in ELISA) are those whose heavy and light chains comprise variable regions having the amino acid sequences comprising those described for antibodies 102E9H6, 102E9H7, 102E9H9, and 102E9H10 in Table 8 below (shown in bold in Table 8 below), more advantageously those whose heavy and light chains comprise variable regions having the amino acid sequences comprising those described for antibody 102E9H10 in Table 8 below.
102E9H9
102E9-VHha
102E9-VKhb
(SEQ ID NO: 138)
(SEQ ID NO: 142)
102E9H6
102E9-VHhb
102E9-VKha
(SEQ ID NO: 139)
(SEQ ID NO: 141)
102E9H10
102E9-VHhb
102E9-VKhb
(SEQ ID NO: 139)
(SEQ ID NO: 142)
102E9H7
102E9-VHhc
102E9-VKha
(SEQ ID NO: 140)
(SEQ ID NO: 141)
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 the 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). Additionally or alternatively, an antibody, functional fragment or derivative thereof according to the invention, which is chimeric with human, or else humanized, constant regions will advantageously comprise a human light chain constant region having the amino acid sequence SEQ ID NO: 54. Preferred human heavy chain (SEQ ID NO: 53 or SEQ ID NO: 144) or light chain (SEQ ID NO: 54) constant region sequences, of isotype IgG1, are presented in Table 9 below.
Thus, 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 of or consist of such sequences).
The heavy and/or light chain of the antibody, functional fragment or derivative thereof according to the invention advantageously further comprises at least one heterologous signal peptide of sequence SEQ ID NO: 65 (MRWSWIFLLLLSITSANA, signal peptide MB7). Indeed, this peptide has been shown to improve the expression and secretion of recombinant proteins in higher eukaryotic cell lines (see WO2011/114063). Thus, the heavy chains of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise an amino acid sequence selected from SEQ ID NOs: 66 to 70, consisting of the N- to C-terminal fusion of the amino acid sequence of signal peptide MB7 (SEQ ID NO: 65) to one of the amino acid sequences of the VH region of the antibodies according to the invention (SEQ ID NOs: 43 to 47), or consist essentially of or consist of such sequences. Additionally or alternatively, the light chains of the antibodies, functional fragments or derivatives thereof according to the invention advantageously comprise an amino acid sequence selected from SEQ ID NOs: 71 to 75, consisting of the N- to C-terminal fusion of the amino acid sequence of signal peptide MB7 (SEQ ID NO: 65) to one of the amino acid sequences of the VL region of the antibodies according to the invention (SEQ ID NOs: 48 to 52), or consist essentially of or consist of such sequences.
By adding the preferred heavy and light chain constant regions, the preferred complete amino acid sequences of the antibodies according to the invention are obtained, as described in Table 11 below. For the heavy chain, the constant region may further contain an additional C-terminal lysine residue. Thus, 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 11 below (or consist essentially of or consist of such sequences).
The antibody, functional fragment or derivative thereof according to the invention may be produced from any host cell, any transgenic non-human animal or any transgenic plant described in the present description, and notably below in the section concerning the nucleic acids, vectors, host cells, transgenic non-human animals and transgenic plants according to the invention.
By “functional fragment” is meant an antibody fragment retaining the antigen-binding domain and thus having the same antigen specificity as the original antibody, such as the fragments Fv, ScFv, Fab, F(ab′)2, Fab′, scFv-Fc or diabodies. A functional antibody fragment according to the invention is thus advantageously selected from the fragments Fv, ScFv, Fab, F(ab′)2, Fab′, scFv-Fc or diabodies.
By “derivative” of an antibody is meant a fusion protein consisting of a carrier peptide and at least one CDR of the original antibody allowing it to retain 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%.
By “fucose content” is meant the percentage of fucosylated forms within N-glycans attached to the Asn297 residue of the Fc fragment of each heavy chain of each antibody. By “low fucose content” is meant a fucose content of less than or equal to 65%. Indeed, it is known today that the fucose content of an antibody composition plays a crucial role in the capacity of said composition to induce a strong ADCC response via FcγRIII. Advantageously, the fucose content is less than or equal to 65%, preferably less than or equal to 60%, 55% or 50%, even less than or equal to 45%, 40%, 35%, 30%, 25% or 20%. However, it is not necessary that the fucose content is nil, and it may for example be greater than or equal to 5%, 10%, 15% or 20%. The fucose content may 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 have different types of glycosylation (N-glycans of the oligomannose or biantennary complex type, with a variable proportion of bisecting N-acetylglucosamine (GlcNAc) residues, or of galactose residues in the case of N-glycans of the biantennary complex type), provided that they have a low fucose content. Thus, oligomannose-type N-glycans may be obtained by culturing in the presence of various glycosylation inhibitors, such as α1,2-mannosidase I inhibitors (such as deoxymannojirimycin (DMM)) or α-glucosidase inhibitors (such as castanospermine (Cs)); or by producing the antibody in the CHO cell line Lec1. Production in the milk of transgenic goats also leads to an antibody whose majority N-glycan is of the oligomannose type, with as minority forms fucosylated biantennary complex forms with one or two galactoses, without bisecting GlcNAc and without sialylation (G1F or G2F) (see WO2007/048077). N-glycans of the biantennary complex type may be obtained in most mammalian cells, but also in bacteria, yeasts or plants whose glycosylation machinery has been modified. To limit the fucose content, cell lines naturally having low activity of the enzyme 1,6-fucosyltransferase (FUT8) responsible for the addition of fucose on the GlcNAc bound to the Fc fragment; such as the YB2/0 cell line, duck embryonic cell line EB66®, rat hepatoma cell lines H4-II-E (DSM ACC3129) and H4-II-Es (DSM ACC3130) or the lines NM-H9D8-E6 (DSM ACC 2807) and NM H9D8-E6Q12 (DSM ACC 2856) may be used. Lines mutant for other genes and whose expression or overexpression leads to a low fucose content may also be used, such as the CHO cell line Lec13, a mutant of the CHO line having decreased synthesis of GDP-fucose. It is also possible to select a line of interest and to decrease or abolish (notably by using interfering RNAs or by mutation or deletion of the gene expressing the protein of interest) the expression of a protein involved in the N-glycan fucosylation pathway (notably FUT8, see Yamane-Ohnuki et al.—2004; but also GMD, a gene involved in GDP-fucose transport, see Kanda et al.—2007). Another alternative consists in selecting a cell line of interest and in overexpressing a protein that interferes in some way with the fucosylation of N-glycans, such as the GnTIII (β(1,4)-N-acetylglucosaminetransferase III) protein. In particular, antibodies having low fucosylated N-glycans were notably obtained by:
Oligomannose-type N-glycans have a reduced in vivo half-life compared to biantennary complex-type N-glycans. Consequently, advantageously, the antibodies according to the invention have, on their Fc-fragment N-glycosylation sites, biantennary complex-type glycan structures with a low fucose content as defined above.
In particular, the monoclonal antibody according to the invention may have a content of G0+G1+G0F+G1F forms greater than 60% and a low fucose content as defined above. It may also have a content of G0+G1+G0F+G1F forms greater than 65% and a low fucose content as defined above. It may also have a content of G0+G1+G0F+G1F forms greater than 70% and a low fucose content as defined above. It may also have a content of G0+G1+G0F+G1F forms greater than 75% and a low fucose content as defined above. It may also have a content of G0+G1+G0F+G1F forms greater than 80% and a low fucose content as defined above. It may also have a content of G0+G1+G0F+G1F forms greater than 60%, 65%, 70%, 75% or 80% and a content of G0F+G1F forms of less than 50%. The forms G0, G1, G0F and G1F are as defined below:
Such antibody compositions may notably be obtained by production in YB2/0 cells, in CHO Lec13 cells, in wild-type CHO cell lines cultured in the presence of small interfering RNAs directed against FUT8 or GMD, or in CHO cell lines in which both alleles of the FUT8 gene encoding 1,6-fucosyltransferase or both alleles of the GMD gene encoding the GDP-fucose transporter in the Golgi apparatus have been deleted.
However, in another embodiment, the antibody, functional fragment or derivative thereof according to the invention has a high oligomannose-type N-glycans content.
By “oligomannose-type N-glycans” is meant N-glycans whose pentasaccharide core, consisting of two N-acetylglucosamine (GlcNAc) residues (one of them being bound to the Asn297 residue of the Fc fragment of the antibody) and three mannose residues, is supplemented by one to six additional mannoses bound to the terminal mannose residues of the pentasaccharide core. The oligomannose-type N-glycans are not fucosylated.
By “oligomannose-type N-glycans content” is meant the percentage of oligomannose forms within N-glycans attached to the Asn297 residue of the Fc fragment of each heavy chain of each antibody. By “high oligomannose-type N-glycans content” is meant an oligomannose-type N-glycans content of 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 even greater than or equal to 95%.
In addition or alternatively to a low fucose content, the antibody, functional fragment or derivative thereof according to the invention has a high galactose content.
By “galactose content” or “galactosylation level” of the antibody is meant a percentage calculated from an analytical chromatogram of the N-glycans released from the antibody, according to the following formula:
wherein:
By “high galactose content” is meant a galactose content of 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 affinity of the Fc fragment of an antibody for FcγRIIIa (CD16a) may be measured by several methods, including surface plasmon resonance (SPR, notably using a BIAcore 2000 device—Pharmacia Biosensor, Upsala, Sweden) and Scatchard analysis. Eight genes encoding FcγR have been identified in humans, but only five encode expressed receptors (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb). All are effector cell-activating receptors, except for FcγRIIb, which is an immune cell activation-inhibiting receptor. FcγRIa is characterized by high affinity for immunoglobulins (Kd of 5·10−7 to 10−10 M) while FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb are characteristic of low-affinity receptors (Kd below 10−7 M). The antibodies according to the invention have improved affinity for FcγRIIIa (CD16a) compared to antibodies directed against the ectodomain of the human CD303 antigen produced in CHO cells. In the context of this comparison, reference is preferably made to a wild-type CHO cell line producing in normal conditions antibodies in which the Fc fragment is highly fucosylated (at least 80%, preferably at least 90% of the oligosaccharides attached to the Fc fragment are fucosylated), such as one of the following lines: CHO-K-1 (ATCC® CCL-61™), CHO Pro-5 (ATCC® CRL-1781™), CHO dhfr-(ATCC® CRL-9096™), CHO-DP12 (ATCC® CRL-12444™ or ATCC® CRL-12445™), CHO DUKX-B11 (ATCC CRL-9010), and CHO DG-44 (Urlaub et al., Cell 33[2], 405-412, 1983). The antibodies according to the invention which have improved affinity for FcγRIIIa (CD16a) compared to antibodies directed against the ectodomain of the human CD303 antigen produced in CHO cells (as defined above) have the advantage that this improved affinity enables them not to be displaced, or to be less displaced, from FcγRIIIa (CD16a) by polyclonal IgG antibodies, notably the IgG present in serum. Such antibodies advantageously have high affinity for FcγRIIIa (CD16a), i.e., affinity at least equal to 2·106 M−1, at least equal to 2·107M−1, 2·108M−1 or 2·109M−1, as determined by Scatchard analysis or BIAcore (label-free surface plasmon resonance-based) technology.
High affinity for FcγRIIIa (CD16a), significantly improved compared to antibodies produced in CHO cells, may be obtained in the various ways described above for the antibody, functional fragment or derivative thereof according to the invention defined by its capacity to compete for binding to the CD303 ectodomain with the specific antibodies developed by the inventors and by the fact that the constant regions of its light and heavy chains are constant regions from a non-murine species. In particular, high affinity for FcγRIIIa (CD16a), significantly improved compared to antibodies produced in CHO cells, may be obtained by:
Such an antibody according to the invention may in addition have any characteristic or combination of characteristics described above for the antibody, functional fragment or derivative thereof according to the invention defined by its capacity to compete for binding to the CD303 ectodomain with the specific antibodies developed by the inventors and by the fact that the constant regions of its light and heavy chains are constant regions from a non-murine species.
Nucleic Acids, Vectors, Host Cells, Transgenic Non-Human Animals and Transgenic Plants
The present invention also relates to a nucleic acid (also called nucleic or nucleotide sequence) encoding the heavy and/or light chain of an antibody, functional fragment or derivative thereof according to the invention as described above.
All the different nucleic sequences, because of degeneration of the genetic code, encoding a particular amino acid sequence are within the scope of the invention.
In particular, the sequence of a nucleic acid according to the invention may be optimized to promote the expression thereof in a host cell, a transgenic non-human animal or a transgenic plant of interest. Indeed, there are in general several three-nucleotide combinations encoding the same amino acid (except for methionine and tryptophan), called synonymous codons. However, some of these combinations are in general used preferentially by a cell or a given organism (this is referred to as genetic code usage bias). This preference depends notably on the producing organism from which the cell is derived. Consequently, when a protein derived from one or more organisms is produced in a heterologous organism or a cell of such a heterologous organism, it may be useful to modify the nucleic sequence encoding the protein to use mainly the preferred codons of the heterologous organism. Data are available in the literature concerning the use of codons preferred by different species and a person skilled in the art knows how to optimize the expression of a given protein in a heterologous organism or a cell of a heterologous organism.
A nucleic acid according to the invention advantageously comprises at least one of SEQ ID NOs: 86 to 95 (chimeric antibodies), as described in Table 12 below, which encode the amino acid sequences of the VH and VL regions of the antibodies according to the invention and have been optimized for expression in Rattus norvegicus cells.
A nucleic acid according to the invention may also advantageously comprise at least one of SEQ ID NOs: 181 to 187 (humanized antibodies derived from antibody 122A2), as described in Table 13 below, which encode the amino acid sequences of the VH and VL regions of the humanized antibodies according to the invention and have been optimized for expression in Rattus norvegicus cells.
A nucleic acid according to the invention may also advantageously comprise at least one of SEQ ID NOs: 188 to 193 (humanized antibodies derived from antibody 102E9), as described in Table 14 below, which encode the amino acid sequences of the VH and VL regions of the humanized antibodies according to the invention and have been optimized for expression in Rattus norvegicus cells.
The nucleic sequences encoding the preferred heavy or light chain constant regions were also optimized for expression in Rattus norvegicus cells and are preferably those described in Table 15 below.
Thus, a nucleic acid encoding the heavy and/or light chain of an antibody according to the invention preferably comprises (or consists essentially of, or consists of) at least one nucleic sequence described in Table 16 below, consisting of the 5′ to 3′ fusion:
A nucleic acid encoding the heavy and/or light chain of the antibody, functional fragment or derivative thereof according to the invention advantageously comprises a nucleic sequence encoding heterologous signal peptide MB7 (MRWSWIFLLLLSITSANA, SEQ ID NO: 65), and notably the nucleic sequence SEQ ID NO: 108 (ATGAGGTGGTCCTGGATCTTCCTGCTGCTGCTGAGCATCACCAGCGCCAACGCC). Indeed, this peptide has been shown to improve the expression and secretion of recombinant proteins in higher eukaryotic cell lines (see WO2011/114063).
Thus, 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 selected from SEQ ID NOs: 109 to 113 (chimeric antibodies), SEQ ID NOs: 207 to 209 (humanized antibodies derived from chimeric antibody 122A2) and SEQ ID NOs: 214 to 216 (humanized antibodies derived from chimeric antibody 102E9), consisting of the 5′ to 3′ fusion of the nucleic sequence encoding signal peptide MB7 (SEQ ID NO: 108) to one of the nucleic sequences encoding the VH region of the antibodies according to the invention (SEQ ID NOs: 86 to 90 for the chimeric antibodies, SEQ ID NOs: 181 to 183 for the humanized antibodies derived from chimeric antibody 122A2, and SEQ ID NOs: 188 to 190 for the humanized antibodies derived from chimeric antibody 102E9).
Additionally or alternatively, 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 selected from SEQ ID NOs: 114 to 118, (chimeric antibodies), SEQ ID NOs: 210 to 213 (humanized antibodies derived from chimeric antibody 122A2) and SEQ ID NOs: 217 to 219 (humanized antibodies derived from chimeric antibody 102E9), consisting of the 5′ to 3′ fusion of the nucleic sequence encoding signal peptide MB7 (SEQ ID NO: 108) to one of the amino acid sequences of the VL region of the antibodies according to the invention (SEQ ID NOs: 91 to 95 for the chimeric antibodies, SEQ ID NOs: 184 to 187 for the humanized antibodies derived from chimeric antibody 122A2, and SEQ ID NOs: 191 to 193 for the humanized antibodies derived from chimeric antibody 102E9).
By adding the preferred heavy and light chain constant regions, the preferred complete amino acid sequences of the antibodies according to the invention are obtained, as described in Table 17 below.
Thus, a nucleic acid encoding the heavy and/or light chain of a chimeric or humanized antibody, a functional fragment or a derivative thereof according to the invention advantageously comprises at least one sequence described in Table 17 below (or consists essentially of, or consists of, such sequences).
The present invention also relates to a vector comprising a nucleic acid according to the invention. Such a vector comprises the elements necessary for the expression of said nucleic sequence, and notably a promoter, a transcription initiation codon, termination sequences, and suitable transcription regulatory sequences. These elements vary according to the host used for the expression and are easily selected by persons skilled in the art based on their general knowledge. In particular, for a vector designed for expression in eukaryotic cells, the vector advantageously comprises a Kozak consensus sequence, i.e., a conserved sequence found at the translation start site of eukaryotic messenger RNA, around the AUG start codon (generally GCCGCC(A/G)CCATGG, the translation initiation codon being underlined). The vector can notably be a plasmid or viral vector. It is used to clone or express the nucleic acids according to the invention. Examples of preferred vectors able to be used in the context of the invention include:
Advantageously, the transcription unit comprised in the vector comprises the hCMVie virus enhancer, the CDK9 promoter region, the eIF4GI gene 5′ UTR and the EF1a gene intron, and has the nucleotide sequence SEQ ID NO: 231 (“HKgenEFss” vector).
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 in particular be selected from bacterial, insect, plant, yeast or mammalian cells. The antibody, functional fragment or derivative according to the invention may then be produced by culturing the host cell under suitable conditions. A host cell according to the invention can notably be obtained by transforming a cell line by the expression vector(s) for the heavy and light chains of an antibody, functional fragment or derivative thereof according to the invention, and separating the various cell clones obtained. The transformed cell line is preferably of eukaryotic origin, and may in particular be selected from insect, plant, yeast or mammalian cells. Suitable cell lines for antibody production notably include lines selected from: SP2/0; YB2/0; IR983F; human myeloma Namalwa; PERC6; CHO cell lines, notably CHO-K-1, CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr-, CHO-DP12, CHO DUKX-B11, CHO DG-44, or the CHO cell line deleted for both alleles encoding the FUT8 gene and/or the GMD gene; Wil-2; Jurkat; Vero; Molt-4; COS-7; 293-HEK; BHK; K6H6; NSO; SP2/0-Ag 14, P3X63Ag8.653, duck embryonic cell line EB66® (Valneva); rat hepatoma cell lines H4-II-E (DSM ACC3129) and H4-II-Es (DSM ACC3130) (see WO2012/041768), NM-H9D8 (DSM ACC2806), NM-H9D8-E6 (DSM ACC 2807) and NM H9D8-E6Q12 (DSM ACC 2856) (see WO2008/028686).
A transgenic non-human animal according to the invention may be obtained by directly injecting the gene(s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) into a fertilized egg (Gordon et al.—1980). A transgenic non-human animal may 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 the animal by a chimera aggregation method or a chimera 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 may also be obtained by a cloning technique in which a nucleus, into which the gene(s) of interest (here, the rearranged genes encoding the heavy and light chains of the antibody) has/have 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 methods above. The antibody may then be accumulated in the transgenic animal and harvested, notably from the animal's milk or eggs. For producing antibodies in the milk of transgenic non-human animals, preparation methods are notably described 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). The transgenic non-human animals of interest notably include mice, rabbits, rats, goats, bovines (notably cows), and poultry (notably chicken). A transgenic plant according to the invention may be selected from any plant allowing antibody production. Numerous antibodies have already been produced in transgenic plants and the technologies required for obtaining a transgenic plant expressing an antibody of interest and for recovering the antibody are well-known to a person skilled in the art (see Stoger et al.—2002, Fisher et al.—2003, My et al.—2003, Schillberg et al.—2005). It is also possible to influence the glycosylation obtained in the plants in order to obtain glycosylation similar to that of natural human antibodies (without xylose), but with, in addition, slight fucosylation, for example by means of small interfering RNAs (Forthal et al.—2010).
Therapeutic Uses of the Antibodies
The present invention also relates to an antibody, functional fragment or derivative thereof according to the invention, for use as a medicinal product.
In a first embodiment, 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. It is notably the case of blastic plasmacytoid dendritic cell neoplasms (BPDCN), of phenotype CD4+, CD11c−, Lin−, CD303+, CD304+, CD56+.
In a second embodiment, the antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of inflammatory diseases, notably autoimmune diseases. The antibody, functional fragment or derivative thereof according to the invention is advantageously used in the treatment or prevention of diseases involving pDCs, and more particularly diseases involving IFN-α secretion by pDCs. In particular, 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 for pDCs has been established (see Wollenberg et al.—2002 and Cao-2014): atopic dermatitis, contact dermatitis, psoriasis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, type 1b diabetes, autoimmune thrombocytopenia (or thrombopenia) (notably idiopathic thrombocytopenic purpura, or ITP), systemic scleroderma (also called progressive systemic scleroderma or systemic sclerosis), rheumatoid arthritis.
The present invention also concerns:
The present invention also concerns:
The present invention also concerns:
By “treatment” is meant an improvement, observed at the clinical or biochemical level, of the patient's disease.
By “prevention” is meant the fact of preventing or delaying the onset of, or of decreasing the intensity of, the clinical or biochemical manifestations associated with the disease. Persons skilled in the art know, on the basis of their general knowledge, how to determine which clinical or biochemical manifestations are associated with a given disease and which are likely to be improved (treatment) or prevented, delayed or decreased in intensity (prevention). In the context of hematopoietic tumors expressing the CD303 antigen, and notably blastic plasmacytoid dendritic cell neoplasms (BPDCN), a biological parameter of interest may be the number of blasts. In the context of inflammatory diseases, a biological parameter of interest may be the number of pDCs or the number of local or systemic molecules contributing to the inflammation (inflammatory cytokines and notably INFα).
The following examples aim at illustrating the present invention.
Five chimeric monoclonal antibodies with murine variable regions and human constant regions of IgG1 type were generated and their structures characterized. In addition, humanized antibody B11B059, the sequences of which are described in the application WO2014/09339, was reproduced for comparison with the chimeric antibodies according to the invention.
Materials and Methods
Sequencing of the Heavy and Light Chains from Murine Hybridomas
Total RNA from each hybridoma was extracted using the NucleoSpin RNA II Kit (column purification) from Macherey-Nagel.
The mRNA was 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 by the M13 vector and clones positive for the M13 sequences were sequenced.
Determination of the Heavy Chain VH, DH, JH Segments and the Light Chain VL and JL Segments
The variable portion, the V and J segments used by the heavy and light chains and the sequences of the heavy and light chain CDRs were determined by using IMGT's Domain Gap Align tool (see Ehrenmann et al.—2010 and Ehrenmann et al.—2011) available at the following address: http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi.
Construction of Expression Vectors for the Chimeric Antibodies
The sequences of the variable regions, VH and VL, of the five murine antibodies were optimized for preferential use of codons from Rattus norvegicus. A sequence encoding heterologous signal peptide MB7 was in addition introduced at the 5′ end of the sequence encoding the variable region, VH or VL, of each antibody.
The sequences of the human constant portions were extracted from the expression vector CHK622-21 for a human anti-Rhesus D antibody (T125) by ApaI/AscI digestion for the H chain (IgG1m1.17) and DraIII/XbaI for the Kappa chain.
Lastly, the variable and constant portions of the same chain were introduced simultaneously into the generic HKgenEFss vector by ligation with KAPA T4 ligase, generating the vectors HKBDCA-2-122A2, HKBDCA-2-102E9, HKBDCA-2-104C12, HKBDCA-2-114D11, and HKBDCA-2-104E10 (see
Production of Chimeric Antibodies
Vectors HKBDCA-2-122A2, HKBDCA-2-102E9, HKBDCA-2-104C12, HKBDCA-2-114D11, and HKBDCA-2-104E10 (see
Production of an Antibody Corresponding to the Antibody (B11B059) Described in WO2014/09339
Based on the sequences described in said patent application, the sequences of the heavy (H) chain and of the light (K) chain were synthesized by adding a Kozak sequence and restriction sites on each side of the two H and K sequences, to allow cloning by digestion/ligation.
The expression vector used to produce humanized antibody B11B059 is a bicistronic vector, HKgenEFss, of 10,835 bp, which makes it possible to optimally produce the antibody in CHO cells (
Humanized antibody B11B059 is produced by transient transfection from the CHO cell line, in order to meet the conditions of the application WO2014/09339. This line is cultured in ProCHO4 medium, supplemented with 4 mM glutamine. In the laboratory, the line is maintained at 2·105 to 3·105 cells/mL, every 2 days. The solutions used for the transfection are buffer (OptiPRO) and transfection agent (Freestyle Max Reagent).
Once produced, antibody BIIB059 was purified by affinity chromatography.
Results
The data concerning the heavy chain VH, DH, JH segments and the light chain VL and JL segments of the five antibodies are presented in Table 2 above. It is noted that:
IGHV1S137*01
IGKV10-96*01
IGKJ1*01
IGHV1S137*01
IGKV10-96*02
IGKJ1*02
IGHV9-2-1*01
IGHJ4*01
IGKV4-57*01
IGKJ1*02
IGHV9-2-1*01
IGHJ4*01
IGKV4-57*01
IGKJ1*02
IGHV9-2-1*01
IGHJ4*01
IGKV4-57*01
IGKJ1*02
Furthermore, the data concerning the amino acid sequences of the CDRs and of the variable regions of the five antibodies are presented in Table 3 above. It is noted that:
GYTFTDYS
ISTYYGDS
ARNGNFYVMDY
QDISNY
YTS
QQGNTLPWT
GYTFTDYS
ISPYYGDT
ARNDDYYRFAY
QDINNY
YTS
QQGKTLPWT
GYTFTDYS
INTETGEP
SSVIY
STS
QQRRSYPFT
GYTFTDSS
INTETGGP
SSVFY
STS
QQRRSYPYT
GYTFTDYS
INTETGEP
SSVIY
STS
QQRRSYPYT
The data concerning the amino acid sequences of the constant regions of the five antibodies are presented in Table 9 above.
Moreover, the nucleic acid sequences of the variable regions (VH and VL) and of the constant regions of each antibody are presented in Tables 12 and 15 above. Lastly, the maps of the expression vectors for the five antibodies are presented in
Conclusions
Five chimeric monoclonal antibodies, with murine variable regions and human constant regions of IgG1 type, and directed against the CD303 antigen, were generated and their structures characterized. It turns out that two antibodies (122A2 and 104C12) have similar structures and form an antibody subfamily (family 1), and that the other three antibodies (102E9, 114D11 and 104E10) are also very similar structurally and form another antibody subfamily (family 2).
A humanized antibody having the sequences of the antibody (B11B059) described in the application WO2014/09339 was also produced in CHO cells (as in the application WO2014/09339).
These antibodies were then characterized in terms of their biological properties (see Example 2).
The five chimeric monoclonal antibodies, with murine variable regions and human constant regions of IgG1 type, and directed against the CD303 antigen, generated in Example 1 were tested for various biological properties. In certain cases, these properties were compared to those of an antibody corresponding to the antibody (B11B059) described in the application WO2014/09339 (same sequences and same production line).
Materials and Methods
Tested Antibodies
Tested antibodies are those prepared as described in Example 1.
Antigen-Binding
Binding to CD303+ cells (Fcγ chain-CD303 Jurkat, CAL-1, and CAL-1 overexpressing CD303)
Various types of cells expressing CD303 at various antigen densities were used:
The CD303-expressing cells and the antibodies are prepared in diluent (PBS+1% FCS). 1·105 cells are incubated at 4° C. for 30 minutes with 100 μL of antibody (anti-CD303 or negative control) at various concentrations (0-40 μg/mL, final concentration). After washing with the diluent, the antibodies are visualized by addition of a phycoerythrin (PE)-coupled goat anti-mouse IgG F(ab′)2 fragment (100 μL diluted to 1:100 in the diluent) for 45 minutes at 4° C. The cells are then washed and analyzed by flow cytometry (FC500, Beckman Coulter).
FcγRIIIa (CD16a)-Binding
NK cells were isolated from peripheral blood mononuclear cells (PBMCs), then incubated with varying concentrations of the antibodies tested (0 to 100 μg/mL) simultaneously incubated with the phycoerythrin-coupled murine antibody (3G8-PE, Beckman Coulter) at 10 μL/test.
After washing, the binding of 3G8-PE to CD16 expressed by the NK cells was evaluated by flow cytometry. Mean fluorescence intensity (MFI) values are expressed as a percentage, 100% being the value obtained with the 3G8-PE antibody alone, and 0% the value obtained in the absence of 3G8-PE.
IC50 values (concentration of anti-CD303 antibody necessary to induce 50% inhibition of 3G8-binding) are calculated using the PRISM software.
ADCC
Fcγ chain-CD303 Jurkat cells (35,000 cells/well) are incubated in a 96-well flat-bottom plate with NK cells and increasing concentrations of anti-CD303 antibody for 4 hours at 37° C. After incubation, the supernatant is collected. Lysis of the target cells induced by the anti-CD303 antibodies is measured chromogenically by quantifying the intracellular lactate dehydrogenase (LDH) enzyme released into the supernatant by the lysed target cells (Cytotoxicity Detection Kit (LDH), Roche Diagnostics).
The percentage of lysis is calculated according to the following formula:
% lysis=[(ER−SR)/(100−SR)]−[(NC−SR)/(100−SR)]
Where ER and SR represent the experimental release (ER) and the spontaneous release (SR) of LDH, respectively, and NC represents the natural cytotoxicity of the NK cells.
The results (% lysis) are expressed as a function of antibody dilution factor. For each antibody, the “50% activity” value corresponds to the antibody dilution factor necessary to induce 50% of the plateau value obtained for this antibody. This value was calculated with the PRISM software.
Inhibition of IFN-α Secretion
Preparation of the Cells
Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood of healthy donors by Ficoll density gradient. The pDCs are purified by negative depletion (Miltenyi Biotec—Plasmacytoid Dendritic Cell Isolation Kit, human). The cells are counted and resuspended at 2·105 cells/mL in diluent (RPMI 1640+10% fetal calf serum (FCS)).
IFN-α Secretion by Purified pDCs
This suspension (205 μL/well) is then transferred to a 24-well flat-bottom culture plate. CpG ODN (10 μM, 50 μL), IL-3 (100 ng/mL, 50 μL), antibody (100 μg/mL, 50 μL) and diluent (100 μL) are added. The plate is then incubated overnight at 37° C., 7% CO2.
IFN-α Assay
The culture supernatants of each well are collected and assayed by flow cytometry by using the FlowCytomix Human IFN-α Kit (Bender MedSystems BMS216FF+Basic kit BMS8420FF).
The percentage of inhibition of each sample is evaluated in relation to the negative control (CPG-activated pDCs without anti-CD303 monoclonal antibody).
The samples leading to less than 20% inhibition are considered non-inhibitors, those leading to between 20% and 75% inhibition are considered weak inhibitors, those leading to greater than 75% inhibition are considered strong inhibitors.
Inhibition of TNF-α Secretion
Preparation of the Cells
Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood of healthy donors by Ficoll density gradient. The pDCs are purified by negative depletion (Miltenyi Biotec—Plasmacytoid Dendritic Cell Isolation Kit, human). The cells are counted and resuspended at 2·105 cells/mL in diluent (RPMI 1640+10% fetal calf serum (FCS)).
TNF-α Secretion by Purified pDCs
This suspension (205 μL/well) is then transferred to a 24-well flat-bottom culture plate. CpG ODN (10 μM, 50 μL), IL-3 (100 ng/mL, 50 μL), antibody (100 μg/mL, 50 μL) and diluent (100 μL) are added. The plate is then incubated overnight at 37° C., 7% CO2.
TNF-α Assay
The culture supernatants of each well are collected and assayed by flow cytometry by using the FlowCytomix Human IFN-α Kit (Bender MedSystems BMS216FF+Basic kit BMS8420FF).
The percentage of inhibition of each sample is evaluated in relation to the negative control (CPG-activated pDCs without anti-CD303 monoclonal antibody).
The samples leading to less than 20% inhibition are considered non-inhibitors, those leading to between 20% and 75% inhibition are considered weak inhibitors, those leading to greater than 75% inhibition are considered strong inhibitors.
Measurement of Complement-Dependent Cytotoxicity (CDC) Activity
Fcγ chain-CD303 Jurkat cells are incubated with increasing concentrations of anti-CD303 antibody (0 to 5,000 ng/mL) and in the presence of baby rabbit serum as source of (1:10 dilution).
After 2 hours of incubation at 37° C., the amount of intracellular lactate dehydrogenase (LDH) enzyme released into the supernatant by the lysed target cells is measured with the Cytotoxicity Detection Kit (LDH) (Roche Diagnostics, product no. 11644793001).
Results
Antigen-Binding
Binding to Fcγ Chain-CD303 Jurkat Cells
The results of the tests for binding of the antibodies according to the invention to their CD303 antigen on Fcγ chain-CD303 Jurkat cells are presented in
These relative Kd results and the Bmax values calculated after dose-response modeling make it possible to classify the antibodies into two groups: A first group that contains the antibodies 104C12 (Bmax: MFI=27.7; Kd=0.17 μg/mL) and 122A2 (Bmax: MFI=26.2 Kd=0.13 μg/mL) which are comparable and exhibit higher relative affinity than the antibodies of the second group: 114D11 (Bmax: MFI=24.7; Kd=3.9 μg/mL), 104E10 (Bmax: MFI=23.7; Kd=6 μg/mL) and 102E9 (Bmax: MFI=24.9; Kd=3.8 μg/mL).
These results show that all the chimeric antibodies generated efficiently bind the CD303 antigen expressed on the surface of Jurkat cells, for which they are specific.
Binding to CAL-1 Cells
The results of the tests for binding of the chimeric antibodies according to the invention to their CD303 antigen on CAL-1 cells are presented in
These relative Kd results and the Bmax values calculated after dose-response modeling make it possible to classify the antibodies into two groups: A first group that contains the antibodies 104C12 (Bmax: MFI=29.02 Kd=0.34 μg/mL) and 122A2 (Bmax: MFI=25.2 Kd=0.20 μg/mL) which are comparable and exhibit higher relative affinity than the antibodies of the second group: 114D11 (Bmax: MFI=30.1; Kd=1.7 μg/mL), 104E10 (Bmax: MFI=29.2; Kd=1.93 μg/mL) and 102E9 (Bmax: MFI=30.47; Kd=1.81 μg/mL).
These results notably correlate with the results for binding to Jurkat-CD303 cells.
In another experiment, the CAL-1 cell-binding of chimeric antibodies 122A2 and 102E9 and of humanized antibody B11B059 was tested. The results are presented in
These data show that the two chimeric antibodies according to the invention, 122A2 and 102E9 (and in particular chimeric antibody 122A2), bind more strongly than humanized antibody 6116059 to the CD303 antigen on the surface of CAL-1 cells.
Binding to CAL-1 Cells Overexpressing the CD303 Antigen
The results of the tests for binding of the chimeric antibodies 122A2 and 102E9 according to the invention and of humanized antibody 6116059 to their CD303 antigen on CAL-1 cells transfected with a CD303 expression vector and expressing about 40,000-50,000 CD303 molecules/cell (or about 10 times more than wild-type CAL-1 cells) are presented in
These data again show that the two chimeric antibodies according to the invention, 122A2 and 102E9 (and in particular chimeric antibody 122A2), bind more strongly than humanized antibody 6116059 to the CD303 antigen on the surface of CAL-1 cells transfected with a CD303 expression vector and expressing about 40,000-50,000 CD303 molecules/cell.
FcγRIIIa (CD16a)-Binding
The results of the tests for binding to FcγRIIIa (CD16a) of the five chimeric antibodies according to the invention are presented in
The results of the tests for FcγRIIIa (CD16a)-binding of the two chimeric antibodies according to the invention, 122A2 and 102E9, and of humanized antibody 8116059 are presented in
ADCC
The results of the tests for ADCC of the five chimeric antibodies according to the invention on Fcγ chain-CD303 Jurkat target cells are presented in
These results show that the five anti-CD303 chimeric antibodies induce lysis of Jurkat-CD303 cells (Emax about 40%). The EC50 values for 104C12 (EC50: 0.21 ng/mL), 122A2 (EC50: 0.16 ng/mL), 114D11 (EC50: 3.6 ng/mL), 102E9 (EC50: 3.4 ng/mL) and 104E10 (EC50: 8.3 ng/mL) suggest that the antibodies having high affinity are more effective with regard to ADCC than the antibodies having lower affinity.
The results of the tests for ADCC of an irrelevant antibody (anti-factor VIII inhibitor antibodies, Anti Id FVIII), of the two chimeric antibodies according to the invention, 122A2 and 102E9, and of humanized antibody B1113059 on CAL-1 target cells are presented in
These results show that, unlike humanized antibody 6116059, the two chimeric antibodies according to the invention, 122A2 and 102E9, are capable of inducing lysis of CAL-1 cells very weakly expressing the CD303 antigen. Moreover, the stronger ADCC response of the chimeric antibody according to the invention 122A2 compared to the chimeric antibody according to the invention 102E9 also suggests than the antibodies having high affinity are more effective with regard to ADCC than the antibodies having lower affinity.
Inhibition of IFN-α Secretion
The results of the tests for inhibition of IFN-α secretion by activated pDCs are presented in
These results show that the five anti-CD303 chimeric antibodies induce inhibition of IFN-α secreted by CpG-activated pDCs and this at both concentrations tested (10 and 0.1 μg/mL). At the concentration of 0.1 μg/mL, the IFN-α assays show an advantage for antibodies 104C12 and 122A2 compared to the other three antibodies (114D11, 102E9, 104E10).
Inhibition of TNF-α Secretion
The results of the tests for inhibition of TNF-α secretion by activated pDCs are presented in
These results show that the five anti-CD303 chimeric antibodies at 10 μg/mL induce inhibition of TNF-α secreted by CpG-activated pDCs.
Complement-Dependent Cytotoxicity (CDC) Activity
The results of the CDC tests are presented in
Materials and Methods
Definition of Humanized Sequences
A work of humanization in silico, making use of the IMGT.org database and modeling, was undertaken to define various sequences of the variable portions of the heavy and light chains containing “human amino acids” instead of their murine homologues.
For each VH or VL domain, the human gene encoding a VH or VL domain having the amino acid sequence closest to that of the murine sequence of the original chimeric antibody was determined. The closest human VH and VL genes determined for chimeric antibodies 122A2 and 102E9 are mentioned in Table 28 below:
For each VH and VL domain, based on an alignment between the murine amino acid sequence of the original chimeric antibody and the amino acid sequence of the closest human gene, the different mutation positions between the two amino acid sequences (murine/human) were determined, and several murine sequences incorporating a variable number of mutations replacing a murine amino acid with a human amino acid were generated.
Thus, for each of antibodies 122A2 and 102E9, three versions of each heavy chain variable portion (Hha, Hhb and Hhc) and light chain variable portion (Kha, Khb and Khc) were determined. A fourth version of the light chain variable portion (Khd) is added, concerning antibody 122A2. These various sequences comprise variable numbers of mutations relative to the VH and VL regions of the original chimeric antibody, as described in Table 29 below:
S49G/A76V/T85S/A92S/E97D/S99T/I101V/S123L
P9S/D11E/T18S/K43R/K48Q/D69Q/A77V/E81D/S82T/
S86E/S88T/R93S/E95Q/A99F
Preparation of the Expression Vectors
The various sequences were synthesized as linear double-stranded DNA, called “String”, with codon optimization for Rattus norvegicus.
The construction of the expression vectors encoding the humanized anti-CD303 antibodies is carried out by homologous recombination, by means of the In-Fusion® HD Cloning Kit (Clontech® Laboratories). The expression vector selected for each Ig heavy and light chain is the commercial vector pCEP4 (Invitrogen). It has the advantage of being highly productive when used in combination with the human cell line FreeStyle HEK-293, a line used to produce sufficient amounts of antibody quickly in order to carry out certain tests. pCEP4 is monocistronic, 10,186 bp in size and has all the elements necessary for expressing the genes of interest, i.e., an ampicillin-resistance gene, an origin of replication (pUC origin), a CMV promoter and an SV40 poly A (see
Once all the gene elements are prepared, they can be assembled by means of the In-Fusion® kit. Thus, the mixture, i.e., the linearized vector (0.5 μL), the “string” (1 μL) at an initial concentration of 50 ng/μL, and the heavy or light chain constant portion (1 μL) brought up to 10 μL with sterile water, is added into the kit's tube containing the lyophilized product notably consisting of the high-fidelity PCR enzyme CloneAmp™ HiFi. The mixture is then incubated for 15 minutes at 37° C., then 15 minutes at 50° C. In parallel, ligation with only the linearized vector, without the variable and constant portions, is carried out and will be used as the negative control for the experiment.
The vectors were then cloned into highly competent E. coli NEB 5-alpha bacteria, and the sequences of the various clones of each vector were confirmed by sequencing. When the clone is declared to match in sequence, a bacterial culture (300 mL) of the selected clone is prepared in order to purify the vector of interest contained therein, using the NucleoBond Xtra EF kit (Macherey-Nagel). The plasmids obtained are sterile and in sufficient amounts to carry out cotransfections in the production line.
Production of the Antibodies in FreeStyle HEK-293 Cells
The antibodies are produced by transient cotransfection from the FreeStyle human embryonic kidney (HEK)-293 cell line. This last is an immortalized cell line that has the capacity to proliferate in suspension and in the absence of serum. This HEK line, cultured in Freestyle F17 Expression Medium (Life Technologies), supplemented with 8 mM glutamine, also allows greater and faster productivity of recombinant proteins.
During the cotransfections, the controls used to validate the experiments are the growth control, which corresponds to an HEK cell culture not having undergone transfection, and a line that has been transfected with a vector, pMAX, encoding a fluorescent protein, GFP, which is used after 24 hours to determine the transfection rate. The buffer used to dilute the DNA, the transfection agent and the cells is OptiMEM® (Life Technologies). Polyethylenimine (PEI) is the transfection agent (TA), which will make it possible to introduce the vectors into the cells. A 1:2 ratio of DNA to TA and a 1:3 ratio of vector comprising the H chain to vector comprising the K chain are observed. After contacting the DNA/TA complex with the cells, the latter are then cultured with shaking at 37° C., with 8% CO2.
At the conclusion of 7 days of production post-cotransfection, the cell cultures are centrifuged for 15 minutes at 3,000 g. The clarified supernatant, containing the antibodies produced, is collected and assayed using a commercial kit, FastELISA (RD-Biotech), in order to precisely estimate the amount of antibody of each supernatant for carrying out thereafter affinity studies of the humanized antibodies with respect to the CD303 protein.
Production of Certain Humanized Derivatives of Chimeric Antibody 122A2 by YB2/0 Clones Stably Transfected with an Expression Vector
YB2/0 clones were stably transfected with an expression vector comprising nucleic sequences encoding humanized antibodies 122A2H5, 122A2H7, 122A2H9 and 122A2H10 derived from chimeric antibody 122A2. The antibodies were then produced and their amount in the supernatant was assayed.
From the monocistronic vectors used for transient transfection in the HEK line, the light chains (122A2H5: pCEP4_Kha_122 A2; 122A2H9: pCEP4_Khb_122 A2; 122A2H10: pCEP4_Khb_122 A2; 122A2H7: pCEP4_Kha_122 A2) were introduced by SpeI-XbaI digestion into the HKgenEFss vector to obtain an intermediate vector. Then, on these intermediate vectors, NheI-AscI digestion was carried out to introduce the heavy chain (122A2H5: pCEP4_Hha_122 A2; 122A2H9: pCEP4_Hha_122 A2; 122A2H10: pCEP4_Hhb_122 A2; 122A2H7: pCEP4_Hhc_122 A2).
Characterization of the Antigen-Binding of the Antibodies Produced, by ELISA
The study of the binding capacity of the various antibodies produced in FreeStyle HEK-293 cells is carried out by enzyme-linked immunosorbent assay (ELISA).
This technique, which uses a colorimetric approach, makes it possible to detect and visualize antigen-antibody interactions. The principle of ELISA is, first, to coat each well of a MaxiSorp plate with 10 ng of the antigen, in this case the CD303 ectodomain. The latter was purchased from the supplier Mybiosource. Second, the various antibodies produced, contained in the supernatant, are all diluted to the same concentration, and contacted with the antigen to allow them to bind. A third step consists in adding the secondary antibodies, HRP-conjugated anti-human antibody, necessary for amplified detection of the antigen-binding of the anti-CD303 antibodies. Lastly, detection is carried out using a solution containing the substrate for the enzyme (3,3′,5,5′-tetramethylbenzidine, or TMB). The appearance of coloring of the substrate indicates the formation of the antigen-antibody complex. The intensity of this coloring is proportional to the amount of enzyme present and thus to the amount of antibody tested bound to the antigen. The reaction is stopped by adding H2SO4 (acid). These colors are read using a spectrophotometer at a wavelength of 450 nm.
Characterization of the Antigen-Binding of the Antibodies Produced, by Flow Cytometry
With the aim of confirming the results obtained by ELISA, the various antibodies produced in FreeStyle HEK-293 cells are evaluated by flow cytometry. Flow cytometry makes it possible to confirm that, in a cellular context, the recombinant antibody is capable of binding to its antigen, and thus to confirm the results obtained by ELISA.
The line used to carry out this technique, CAL-1, is a leukemic cell line of plasmacytoid dendritic cells obtained from a BPDCN patient. The antibodies produced were tested in supernatant, but also in forms purified by affinity chromatography with protein A-sepharose (not elaborated upon herein).
For cell staining, the cells and the antibodies are prediluted with PBS/1% FCS. In a 100 μL reaction volume, 1·105 cells are incubated at 4° C. for 30 minutes with the anti-CD303 antibodies to be tested, at two concentrations: 0.1 μg/mL or 1 μg/mL. This incubation is followed with several washings. The cell/antibody complexes thus formed are then contacted for 30 minutes at 4° C. with the secondary antibody diluted 1:50, an anti-human IgG Fc F(ab′)2 coupled to a fluorochrome, phycoerythrin. The cells are finally washed and studied by flow cytometry (FC500, Beckman Coulter), by measuring notably the mean fluorescence intensity (MFI). Various negative controls are used to eliminate possible nonspecific binding, such as: the cells alone or directly in contact with the secondary antibody, or replacing the antibody tested with an irrelevant chimeric antibody (factor VIII anti-idiotype). Finally, other controls, such as the use of the supernatant (mock), make it possible to show that the latter does not influence the affinity of the antibodies to the membrane antigen.
Inhibition of IFN-α Secretion
Preparation of the Cells
Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood of healthy donors by Ficoll density gradient. 107 cells/well (200 μL) are transferred to a 24-well flat-bottom culture plate, in the presence of 1 μM CpG ODN 2216-1 (Invivogen) and 10 ng/mL IL-3 (product no.: 130-093-909, Miltenyi Biotec). The anti-CD303 or irrelevant antibodies were added at various concentrations (0 to 1 μg/mL) in RPMI 10% FCS medium. The plate was then incubated overnight at 37° C. with 7% CO2.
IFN-α Assay
The culture supernatants of each well are collected and assayed by ELISA by using the Human IFN-α Module Set kit (product no.: BMS216MST, eBioscience).
Induction of IL-2 Secretion by Jurkat-CD16 Cells
This test evaluates the capacity of the anti-CD303 antibodies to bind to CD16 (Fcγ receptor III) expressed on CD16 Jurkat cells and to induce IL-2 secretion.
This test consists in contacting, in a 96-well plate: anti-CD303 antibodies, target cells expressing CD303, CD16 Jurkat cells and phorbol myristate acetate PMA).
After incubating at 37° C. overnight, the plates are centrifuged and the amount of IL-2 secreted is assayed in the supernatant.
Results
Cotransfection of the Vectors Produced in HEK-293F Cells
From vectors expressing the humanized heavy and light chain sequences, various heavy (H) chain/light (L or K) chain combinations were produced by transient cotransfection in HEK-293F cells, in order to produce various representative versions of the two candidate antibodies. In this manner, the following were produced (see Table 30 below):
During the various transient cotransfections carried out to produce the set of 36 molecules, transfection efficiency was determined on day 1 using the vector pMAX.
At the conclusion of production on day 7, the supernatants, containing the proteins produced, are collected, centrifuged, filtered and finally assayed to measure the titer in IgG produced (see Tables 31 and 32 below). The concentrations thus obtained vary as a function of the type of H chain and K chain, i.e., chimeric or humanized, possessed by the various versions of the antibodies produced.
It can be noted that the antibody concentration is dependent on the combinations produced but also on the type of H and/or K chain (humanized or chimeric). In this context, the productivity of chimeric antibodies 122A2 and 102E9, as well as that of most of the hybrids (combination of a chimeric H or K chain and a humanized H or K chain, as indicated above), is much lower than the rest of the molecules. This observation is related to expression of the chimeric H and K chains, which is unfavorable to satisfactory cellular expression.
What differentiates the chimeric antibodies from the humanized antibodies are the amino acids that have been mutated; the variations in productivity are uniquely dependent on a few amino acids.
Thus, for the antibodies derived from 102E9, the hybrid antibodies having the chimeric heavy or light chain and the humanized antibodies with the Hhc heavy chain (102E9H7, 102E9H11 and 102E9H15) all have lower productivity.
In the case of the antibodies derived from 122A2, it is the chimeric K and humanized H Hha chains that lead to lower productivity. Chimeric antibody 122A2 is found to have the lowest productivity compared to certain of its hybrid and humanized derivatives. Fortuitously, humanization made it possible to increase the volume titer by up to a factor of 100, in the case of 122A2H10 (Hhb, Khb).
Lastly, it is noted that humanized antibodies 122A2 are produced better overall than humanized antibodies 102E9. That confirms that expression of antibodies of the same isotype is highly dependent on their primary sequence and chiefly on their variable portions.
Characterization of the Antigen-Binding of the Antibodies Produced, by ELISA
ELISAs were carried out from the culture supernatants collected after production. First, they are all diluted to the same concentration, so that all the samples are under the same conditions. Furthermore, the initial dilutions are made using centrifuged, filtered supernatant from the cell cultures used as growth controls during the transfections. These cultures do not express an antibody, and are thus referred to as “mock” cultures. The samples are then diluted to half, on MaxiSorp 96-well plates, using buffer containing PBS, 4% BSA and 0.05% Tween.
All the antibodies produced, whether derived from 122A2 or 102E9, give a color signal whose intensity is quantifiable when read by spectrophotometry. This may be interpreted as being the capacity of the various antibodies to bind specifically to the CD303 ectodomain. There are however differences in affinity for the antigen, which are expressed as differences in OD450.
Thus, it was possible, for each antibody produced, to graphically represent OD450 as a function of antibody concentration. The result is a biphasic curve with an exponential phase leading to a plateau. This graphical representation made it possible to classify the antibodies tested and the chimeric antibody from which they are derived as a function of their affinity for the CD303 antigen.
In the case of the antibodies derived from 102E9, the antibodies deposited in each well range from 1.25 ng/mL to 20 ng/mL. Saturation of the OD450 signal appears as of a concentration of about 10 ng/mL antibody.
None of the humanized antibodies binds to the CD303 antigen as well as the chimeric antibody. Nevertheless, all the humanized antibodies preserve a reasonable capacity to bind to the CD303 antigen. Humanized antibody 102E9H10 has the best CD303 antigen-binding capacity among all the humanized antibodies. It represents a loss of binding of only about 20% compared to the original chimeric antibody, 102E9 (
In the case of the antibodies derived from 122A2, the antibody concentrations tested range from 1.75 to 10 ng/mL. The controls used are the same ones as before: the supernatant alone (mock) and the chimeric antibody, here 122A2.
All the antibodies tested are capable of binding to the CD303 ectodomain. The “supernatant” negative control sample has an OD450 near zero. There is thus no nonspecific binding due to the supernatant. Moreover, a dose-response effect is also observed for all the samples tested. Most of the curves representing the various humanized antibodies are similar in appearance to that of chimeric antibody 122A2.
Lastly, several humanized antibodies of 122A2 (122A2H5, 122A2H6, 122A2H7, 122A2H9, 122A2H10, 122A2H11, 122A2H14 and 122A2H15) appear to have a CD303 ectodomain-binding capacity at least equal to (122A2H11 and 122A2H15), and sometimes significantly higher than (122A2H5, 122A2H6, 122A2H7, 122A2H9, 122A2H10, and 122A2H14), that of the chimeric antibody.
Characterization by Flow Cytometry of the Antigen-Binding of the Antibodies Produced
Antibody-binding was also studied by flow cytometry to confirm the results obtained by ELISA.
For antibody 102E9, humanized antibodies 102E9H10, 102E9H6, 102E9H7, 102E9H9 and chimeric antibody 102E9 were selected to be characterized by this technique.
For antibody 122A2, humanized antibodies 122A2H5, 122A2H6, 122A2H9, 122A2H7, 122A2H10, 122A2H14, 122A2H17, 122A2H15, hybrid antibodies 122A2H8, 122A2H3 and chimeric antibody 122A2 in supernatant were selected for the characterization. 122A2H8 represents the best hybrid antibody with affinity higher than that of the chimeric antibody, while 122A2H3 has much lower affinity than that of the parent antibody. The humanized antibodies selected count among those that have better affinity for the target protein compared to the original chimeric antibody.
In the case of antibody 102E9, there are few differences between the humanized and the chimeric antibodies (not illustrated here).
In the case of antibody 122A2, the results are presented in
The same antibodies were evaluated in purified form and they show the same results.
Inhibition of IFN-α Secretion
CpG motifs induce IFN-α secretion from pDCs contained in PBMCs. In this experiment (
The IC50 values (Table 33 below) were:
These results show that the four humanized monoclonal antibodies 122A2 tested are capable of inhibiting IFN-α secretion, with an IC50 lower than or equal to that of the chimeric antibody.
Production by YB2/0 Clones Stably Transfected with an Expression Vector for Humanized Antibodies Derived from Chimeric Antibody 122A2
Humanized antibodies 122A2H5, 122A2H7, 122A2H9 and 122A2H10, derived from chimeric antibody 122A2, appeared particularly advantageous in terms of productivity and antigen-binding. They were thus selected to produce YB2/0 clones stably transfected with an expression vector for these antibodies.
The maps of the HKgenEFss vectors expressing humanized antibodies 122A2H5, 122A2H7, 122A2H9 and 122A2H10 are presented in
The supernatant assay data for the four clones thus obtained are presented in Table 34 below, and show that these four humanized antibodies can be produced with satisfactory productivity, with antibody 122A2H9 having the best productivity.
Induction of IL-2 Secretion by Jurkat-CD16 Cells
The capacity of humanized antibody 122A2H9, derived from chimeric antibody 122A2 and produced by stable YB2/0 clones, to bind CD16 and to induce IL-2 secretion by Jurkat-CD16 cells was tested and compared to that of chimeric antibody 122A2.
An exemplary result obtained is presented in
These results show that, despite an Fc fragment sequence identical to that of the chimeric antibody, humanized antibody 122A2H9 induces slightly higher IL-2 secretion by Jurkat-CD16 cells than that of the chimeric antibody.
Conclusions
In order to optimize the immunogenic tolerance of the antibodies, humanization was carried out on two of the five chimeric antibodies generated by the inventors, with the objective of preserving similar or not significantly different affinity for the CD303 antigen. To that end, several humanized heavy and light chains were tested in combination for each of the original antibodies, and transfected into HEK-293F cells.
Following the assay of the samples, it was noted that the antibody concentration is dependent on the original amino acid sequence (humanized antibodies derived from 122A2 are produced better than humanized antibodies derived from 102E9), on the combinations produced, and also on the type of H and/or K chain (humanized or chimeric). These data confirm that, overall, expression of antibodies of the same isotype is highly dependent on their primary sequence and chiefly on their variable portions. Moreover, numerous humanized antibodies, derived from one or the other of the original two chimeric antibodies, are produced better than the original chimeric antibodies.
The antibodies thus produced were characterized by ELISA. Insofar as the antibodies tested were similar to one another, it was difficult to identify the humanized antibodies having the same antigen-binding characteristics as the original chimeric antibodies. Where the antibodies were tested at several concentrations, the analyses were focused on the concentrations where a real difference in OD450 between the various antibodies is observed. Thus, a concentration of 5 ng/mL made it possible to show that the best humanized antibody from 102E9, 102E9H10, has a CD303 ectodomain-binding capacity that is 80% of that of the chimeric antibody. In this manner, four humanized antibodies (102E9H10, 102E9H6, 102E9H7 and 102E9H9) were identified and selected to be confirmed by flow cytometry, as humanized antibodies of interest.
In the case of the humanized antibodies derived from 122A2, several antibodies are clearly identified as of the first ELISA as having a CD303 ectodomain-binding capacity at least equal to (122A2H11 and 122A2H15), indeed higher than (122A2H5, 122A2H6, 122A2H7, 122A2H9, 122A2H10, and 122A2H14), that of the chimeric antibody. These results obtained from the supernatants were supplemented by an orthogonal method using flow cytometry.
On the whole, the results presented above show that, for each chimeric antibody 122A2 and 102E9, it was possible to generate humanized antibodies (thus with reduced immunogenicity in humans), which can be produced with better productivity than the original chimeric antibody, and which have a CD303 antigen-binding capacity that is:
Moreover, humanized antibodies derived from 122A2 are capable of inhibiting IFN-α secretion as well as chimeric antibody 122A2.
For certain humanized antibodies derived from 122A2, stably transfected YB2/0 clones were able to be obtained, which produce the antibody with satisfactory productivity. These antibodies have a strong capacity to bind to CD16 and to induce IL-2 secretion by Jurkat-CD16 cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15 52757 | Mar 2015 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/056995 | 3/31/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/156450 | 10/6/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20160319025 | Nakao | Nov 2016 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20180086834 A1 | Mar 2018 | US |
