Patent Application
20090029393
The present invention relates to a method for preparing an antibody selective for activating antibody Fc region receptors (FcRs) comprising an ITAM motif or motifs (immunoreceptor tyrosine-based activation motif), comprising the steps of obtaining monoclonal antibodies from a hybridoma, from a heterohybridoma, or from any animal, plant or human cell line, replacing each of the His 310 and His 435 residues (Kabat numbering) of the Fc region of said antibody with a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine, then selecting the antibodies for which the binding to inhibitory FcRs comprising ITIM motifs (immunoreceptor tyrosine-based inhibition motif) is reduced by at least 30%, preferably by at least 50%, 70%, 80% or also by at least 90% relative to the same antibody possessing a native Fc region. The present invention also relates to the use of an antibody originating from the method of the invention in obtaining a medicament intended in particular for the treatment of cancer and infectious pathologies.
Numerous antibody preparations for therapeutic use, of plasmatic or biotechnological origin, are currently on the market, or in the clinical development phase. Their properties are exploited in order to obtain therapeutic tools capable of binding specifically to their target, and effectively recruiting immune cells.
These last few years, research has been directed towards improving the effectiveness of antibodies, and more particularly towards the manipulation of their constant Fc region. It is the latter which is responsible for the “effector” properties of the antibodies, as it allows the mobilization of the effector immune cells and complement molecules. This ability is made possible by the presence, on certain immune cells, of glycoproteins, the Fc receptors or FcRs. These receptors are capable of binding to the constant region of the antibodies, in particular once the latter have bound, by their variable region, the target antigen. On contact with these cells, the antibodies trigger different cell mechanisms such as phagocytosis and ADCC (Antibody-Dependent Cell-mediated Cytotoxicity).
However, different human FcR classes exist. The latter are encoded by eight human genes, all located on chromosome 1. Certain of these genes exhibit an allelic polymorphism generating different receptor allotypes then having different IgG binding properties (Hulett M. D. & Hogarth P. M., Advances in Immunology, vol. 57, pp. 1-127, 1994) leading to differential effector properties (Carton et al. (2002) Blood, vol. 99, no. 3, 754-758). The main human FcR classes identified are FcγRI (CD64, possessing the A and B isoforms), the FcγRII receptor (CD32, possessing the A and B isoforms) and the FcγRIII receptor (CD16, possessing the A and B isoforms). The FcyRI and FcγRIII receptors, as well as the FcγRIIA receptor are receptors qualified as “activating”, as their activation, using the ITAM motifs comprised by their sequences or the sequences of associated chains, allows the triggering of effector functions such as the lysis of the target cells. Conversely, the FcγRIIB receptors are qualified as “inhibiting” receptors, as they inhibit the transduction pathways of the activating receptors via their ITIM motifs comprised by their sequences and negatively modulate the effector mechanisms such as those of the ADCC induced via the activating FcRs or other surface molecules such as the B-cell antigen receptor (BCR) or growth factor receptors such as c-kit.
Therefore, the diversity of the FcR receptors, in particular the existence of activating FcRs and inhibitory FcRs expressed on the same cells is capable of modulating the effectiveness of the therapeutic antibodies, the engagement of inhibitory FcRs counter-balancing the engagement of the activating FcRs.
It was therefore the intention of the Applicant to provide a therapeutic antibody the effectiveness of which, i.e. the ability to activate the effector immune cells, is not at all or only slightly modulated by the diversity of the FcR receptors, in particular by the inhibitory FcRs.
The Applicant, in the document WO 2005/040216, describes antibodies the primary sequence of which is modified, being particularly useful in IgG4 replacement therapy, or for preventing graft rejections, or also as anti-tetanus, anti-diphtheria agents or directed against certain pathogenic agents or derived toxins.
However, the Applicant surprisingly found that the antibody thus modified retained its ability to induce an effector function dependent on the activating receptors, whereas it no longer possesses the ability to recruit the inhibitory receptors.
The Applicant therefore sought to develop a method making it possible to provide therapeutic antibodies the effectiveness of which, i.e. the ability to activate the effector immune cells, is not at all or only slightly, modulated by the diversity of the FcR receptors, in particular by the inhibitory FcRs.
The invention relates firstly to a method for preparing an antibody possessing the ability to recruit the activating FcRs, but the ability of which to recruit inhibitory FcRs is reduced relative to the same antibody possessing a native Fc region (i.e. the Fc region of which selectively binds the activating antibody Fc region receptors (FcRs)), comprising the following steps:
For the purposes of the invention, by “antibody” is meant any antibody, whatever its specificity and its isotype, provided that it comprises an Fc region or a region possessing the same functions and the same characteristics as the Fc region. Thus, it can be a whole antibody or an antibody fragment, for example an Fc antibody fragment, or a fusion molecule comprising an Fc region at one of its ends. Moreover, the antibodies utilized in the method according to the invention can be IgGs, i.e. any IgG1, any IgG2, any IgG3 (G3m(s) or G3m(st) allotypes) and any IgG4, IgM, IgE, IgA or IgD, or also a mixture of one or more of these. Moreover, the antibodies utilized in the method according to the invention can be monoclonal or polyclonal. In the case where they are monoclonal antibodies, these antibodies can be chimeric, humanized, human or of animal origin.
Moreover, the word “antibody” also denotes an antibody composition, composed of molecules of antibodies possessing the same amino acid sequence, expressed in the same biological system, and comprising at least one excipient or pharmaceutically acceptable vehicle such that the composition can be formulated in order to allow pharmaceutical administration, for a prophylactic and/or therapeutic purpose.
At the end of step b), the sequences coding for the modified antibody of the invention are expressed in a suitable biological system (stage b′).
For the purposes of the invention, by “same antibody” is meant a non-modified antibody according to Step b) of the method of the invention, and produced in the same biological production system as the modified antibody of the invention. The “same antibody” possesses the same native primary sequence (apart from the His 310 and His 435 residues, which have been modified in the antibody of the invention) and has been subjected to the same post-translational modifications as the antibody obtained by the method of the invention, since it has been produced in the same biological system.
For the purposes of the invention, the antibodies of Step a) are obtained in the form of monoclonal antibody compositions. Each monoclonal antibody composition is composed of molecules of antibodies possessing the same amino acid sequence, and therefore the same specificity, expressed in the same biological system. These compositions can, if appropriate, contain at least one excipient or pharmaceutically acceptable vehicle such that these compositions can be formulated in order to allow pharmaceutical administration, for a prophylactic and/or therapeutic purpose.
The antibodies of Step a) comprise a native Fc region, i.e. they possess histidine residues at positions 310 and 435 (Kabat numbering) of their Fc region.
Preferably, the Fc region of such antibodies binds both to the activating receptors and to the inhibitory receptors. For the purposes of the invention, by “native Fc region of the antibody” is meant any Fc region not modified by any chemical or biotechnological methods on the His 310 and His 435 residues. More particularly, by “native Fc region of the antibody” is meant any Fc region the His 310 and His 435 residues of which have not been replaced, by chemical or biotechnological methods, with a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine.
By way of example, the antibody utilized in the invention can be chosen from anti-Ep-CAM, anti-KIR3DL2, anti-EGFR, anti-VEGFR, anti-HER1, anti-HER2, anti-GD, anti-GD2, anti-GD3, anti-CD20, anti-CD-23, anti-CD-25, anti-CD30, anti-CD33, anti-CD38, anti-CD44, anti-CD52, anti-CA125 and anti-Protein C, anti-HLA-DR, the anti-virals: HBV, HCV, HIV and RSV, and more particularly from the antibodies of Table 1 hereafter:
For the purposes of the invention, by “antibody selective for activating antibody Fc region receptors (FcRs)” or “antibody the Fc region of which selectively binds activating antibody Fc region receptors (FcRs)” is meant any antibody which possesses the ability to recruit activating FcRs but which possesses an ability to engage inhibitory FcRs which is reduced or even null, relative to the same antibody possessing a native Fc region.
Thus, the antibodies of the invention possess the ability to recruit, i.e. to bind by their Fc region, the activating FcR receptors, but their ability to engage, i.e. to bind by their Fc region, the inhibitory FcR receptors, is reduced or eliminated relative to the same antibody possessing a native Fc region.
By activating FcR is meant FcγRIIIA, FcγRTIIB, FcyRIIA, FcγRIA and FcγRIB, FcγR, FcγRI and the human equivalent of FcγRIV described in mice. By inhibitory FcR, is meant the FcγRIIBs.
During Step c), it is possible to select the antibodies, i.e. different antibody compositions, for which the binding to the FcRs is reduced relative to the same antibody possessing a native Fc region. This reduction can for example be measured by quantifying the number or percentage of cells expressing the FcRs to which the antibodies of the invention are bound, and by comparing this number or this percentage to that of cells expressing the FcRs to which the antibodies possessing a native Fc region are bound.
In a preferred embodiment of the invention, in Step c) the antibodies, i.e. different antibody compositions, for which the binding to the inhibitory FcγRs is eliminated relative to the same antibody possessing a native Fc region, are selected according to a method similar to that described previously. Thus, the antibodies prepared according to the method of the invention possess an Fc region which binds to activating FcRs but does not bind to inhibitory receptors, whereas the same antibody produced by the same biological system but possessing an Fc region not modified according to the method of the invention binds, by its Fc region, to activating FcγRs and inhibitory FcγRs.
In a preferred embodiment of the invention, the His 310 and His 435 residues are replaced with a lysine residue.
In a preferred embodiment of the invention, the His 310 and His 435 residues are replaced by site-directed mutagenesis or by molecular evolution.
In another embodiment, the His 310 and His 435 residues are modified by means of a chemical treatment, for example by treatment with DEPC (diethylpyrocarbonate, which is a histidine modifying agent).
The Applicant surprisingly found that the mutation of the two particular histidine residues His 310 and His 435 situated at the CH2/CH3 interface of an antibody by a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine, and preferably by lysine, has a major impact on the binding of the antibody thus modified to inhibitory FcRs, and in particular to the FcγRIIBs, and has a moderate effect on its binding to activating FcRs, and in particular to the FcγRIIIAs. The Applicant has demonstrated that the antibody of the invention thus modified no longer, or virtually no longer, binds the human inhibitory FcγRIIB receptor in an in vitro binding test whereas its binding to the activating FcγRIIIA and FcγRIIA receptors is only very partially inhibited relative to the non-mutated antibody (see below). This mutated antibody is an antibody capable of engaging the activating receptors (FcγRIIA and FcγRIIIA) involved in the ADCC-type cytotoxicity by avoiding engaging the inhibitory FcyRIIB. Such an antibody therefore makes it possible to induce an ADCC against target cells (red blood cells, tumour cells, allo-reactive cells, cells infected with microbial pathogens) without this ADCC being negatively modulated following the engagement of the FcyRIIB. Such an antibody also makes it possible to optimize the antigen presentation by dendritic cells due to the fact that the immune complexes containing this mutated antibody are captured only by the activating FcγRs expressed on the dendritic cells, without activating the inhibitory FcγRIIBs, also present on these cells.
This “differential” behaviour on the activating and inhibitory receptors is an essential asset for using the antibodies of the invention therapeutically, in particular within the framework of cancerous or infectious pathologies. In fact, such an antibody is capable of inducing ADCC mechanisms via the activating FcγRs without the latter being negatively modulated by the inhibitory FcγRs. Moreover, the Applicant has found that such an antibody only recruits the activating FcγRs on the dendritic cells and does not recruit the inhibitory FcγRs at the surface of the same cells. It has recently been shown that the engagement of the inhibitory FcγRs at the surface of the dendritic cells has a negative effect on the maturation of these cells and on their ability to effectively present an antigen to the effector T lymphocytes, rendering these dendritic cells tolerogenic. If such an antibody complexed to an antigen recognizes only the activating FcγRs expressed at the surface of the dendritic cells and not the inhibitory FcγRs, the antigen presentation by the dendritic cells which results from this is optimal and the effect of activation of the optimized specific immune response is optimized.
Advantageously, the Fc region of the antibody originating from the method of the invention binds to activating Fc receptors whereas it does not bind to inhibitory Fc receptors.
Advantageously, the antibody of the method of the invention does not recruit inhibitory Fc receptors, in particular the inhibitory Fc receptors expressed by the B lymphocytes.
Particularly advantageously, the antibody of the method of the invention does not recruit inhibitory Fc receptors but binds a molecule at the surface of the B lymphocytes.
Advantageously, the antibody of the invention does not recruit inhibitory Fc receptors but binds a molecule at the surface of tumour cells, in particular at the surface of tumorous B lymphocytes.
Advantageously, the antibody of the invention does not recruit inhibitory Fc receptors but binds a molecule at the surface of B-CLL tumorous lymphocytes. Advantageously, the antibody of the invention does not recruit inhibitory Fc receptors but binds the CD20 molecule at the surface of B-CLL tumorous lymphocytes.
Particularly advantageously, the antibody of the method of the invention binds to the FcyRIII receptor (A and B isoforms) and/or to the FcγRIIA receptor and/or to the FcγRI receptor (A and B isoforms), whereas it does not bind to the FcγRIIB receptors (B1 and B2 isoforms). In a particular embodiment, the FcγRIIB receptor is the FcyRIIB1 receptor.
Preferably, the receptors involved in the method of the invention are human receptors.
In a particular embodiment of the invention, the receptors of the Fc region of the antibodies are located on the monocytes, macrophages, dendritic cells, NK cells, B lymphocytes, monocytes, macrophages and B lymphocytes.
In another particular embodiment of the invention, the inhibitory receptors of the Fc region of the antibodies are located on tumour cells, such as malignant melanoma cells, tumorous B lymphocytes such as lymphomas, B-CLL (B chronic lymphoid leukaemia) cells and myeloma cells.
Advantageously, the antibody of the method of the invention binds, by its variable region, the CD20 molecule at the surface of B-CLL tumorous lymphocytes. Such an antibody of the invention does not recruit inhibitory Fc receptors, in particular at the surface of the B-CLL tumorous lymphocytes.
Advantageously, the antibodies utilized in the m of the invention are monoclonal antibodies. These monoclonal antibodies can be produced by any appropriate biological system, in the form of monoclonal antibody compositions which can contain at least one excipient or pharmaceutically acceptable vehicle such that these compositions can be formulated in order to allow pharmaceutical administration, for a prophylactic and/or therapeutic purpose. By “biological system” is meant animal or plant cell lines transfected using one or more vectors in order to express said antibody, plants or non-human transgenic animals, as well as any hybridoma, heterohybridoma. Among the cells, it is possible to choose cells originating from cell lines, transfected using a vector comprising the gene coding for said antibody, for example eukaryotic or prokaryotic cells, in particular cells of mammals, insects, plants, bacteria or yeast. Preferentially, rat myeloma cells such as YB2/0 (ATCC No. CRL 1662) are used.
It is also possible to use CHO cells, in particular CHO-K, CHO-Lec1O, CHO-Lec1O CHO Pro-5, CHO dhfr- or other cell lines from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293-HEK, K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8.653, PERC6 or BHK.
Another object of the invention is the use of an antibody for which each of the His 310 and His 435 residues (Kabat numbering) of its Fc region has been replaced by a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine, or an antibody produced by the method of the invention, for obtaining a medicament intended for anti-infection and anti-tumour vaccination, the antibody of the invention being engaged in an immune complex which can be bound only by the activating FcRγs of the antigen-presenting cells (monocytes, macrophages, dendritic cells, epidermal Langerhans' cells, B lymphocytes).
Preferably, for the preparation of such a medicament an antibody for which each of the His 310 and His 435 residues (Kabat numbering) of its Fc region has been replaced with a lysine residue is used.
Another subject of the invention relates to the use of an antibody, each of the His 310 and His 435 residues (Kabat numbering) of which is replaced by a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine, or an antibody produced by the method of the invention, for obtaining a medicament intended for the treatment of cancers, and more particularly for the treatment of lymphomas, leukaemias, myelomas, sarcomas, solid tumours such as mammary carcinomas, colorectal tumours, pancreatic tumours, prostate tumours, stomach tumours, pulmonary tumours, ovarian tumours, cervical tumours, ocular tumours, thyroid tumours, tumours of the ENT sphere, malignant melanoma, nerve tumours such as glioblastomas, and neuroblastomas.
Preferably, for the preparation of such a medicament, an antibody for which each of the His 310 and His 435 residues (Kabat numbering) of its Fc region has been replaced with a lysine residue is used.
Another subject of the invention is the use of an antibody, each of the His 310 and His 435 residues (Kabat numbering) of which is replaced by a residue chosen from lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine or threonine, or an antibody produced by the method of the invention, for obtaining a medicament intended for the treatment of infectious diseases such as infection with HIV, HBV, HCV, RSV (respiratory syncytial virus), SARS virus, rotavirus, influenza virus, variola, bacterial infections such as infections caused by Koch's bacillus, meningococci, Criptoccocus neoformans, Clostridium, the bacteria responsible for botulism, anthrax, tetanus, tuberculosis, and enterococcal infections.
Preferably, for the preparation of such a medicament an antibody, for which each of the His 310 and His 435 residues (Kabat numbering) of its Fc region has been replaced with a lysine residue, is used.
Finally, a last aspect of the invention relates to an antibody composition for which the binding to inhibitory FcRs is reduced by at least 30%, preferably at least 50%, 70%, 80% or also at least 90% or 100% relative to the same antibody possessing a native Fc region.
Preferably, the composition of the invention is a composition the Fc region of the antibodies of which possesses an ability to bind to the inhibitory receptors which have been eliminated relative to the same antibody possessing a native Fc region.
Particularly advantageously, the composition of the invention is an anti-CD20 antibody composition.
Other aspects and advantages of the invention are described in the examples which follow, which must be considered as illustrative and do not limit the scope of the invention.
The line YB2/0 (rat myeloma, line ATCC No. CRL 1662) transfected and producing the EMAB5 antibody (described in the document WO 2005/040216), which is a human IgG1 (γ) directed against the Rh(D) antigen, was adapted for culture in medium without serum.
EMAB5 was purified by affinity chromatography on Sepharose-protein A.
By HPCE-LIF, it was shown that the majority glycanic structure is a biantenna-type oligosaccharide, containing approximately 25% fucose.
The purified EMAB5 antibody is dialyzed overnight against 50 mM Tris buffer, pH 8.0. The antibody solution, adjusted to 50 mM CaC12 and 10 mM cysteine, is incubated for 30 minutes at 37° C. before adding the trypsine solution (1 mg/ml) in an enzyme/substrate ratio of 1/25.
After incubation for 5 hours at 37° C., the reaction is stopped by the addition of diisopropyl fluorophosphate (1 mM final). The hydrolysate is dialyzed overnight against 50 mM Imidazole buffer, pH 7.8.
For the purification of the Fc fragment, the dialyzed hydrolysate is brought into contact with protein L-affarose at a rate of 1 ml of gel per 3.6 mg of antibodies. After incubation for 4 hours at ambient temperature under stirring, the gel is loaded in a column and washed with 50 mM Imidazole buffer, pH 7.8. The effluent and the washing buffer which contain the Fc fragments are combined and concentrated by centrifugation on a Vivaspin 20 using the conditions described by the manufacturer.
The expression vector containing the cDNA encoding the amino acid sequence of the heavy chain of the anti-Rh(D) antibody EMAB5, served as a matrix for carrying out a double site-directed mutagenesis by PCR (“PCR-based site-directed mutagenesis”). The following four nucleotide substitutions were introduced:
The heavy chain of the mutated antibody has, for a nucleotide sequence, the sequence SEQ ID NO: 1 and, for a peptide sequence SEQ ID NO: 2 (the mutated amino acids appear in the sequence SEQ ID NO: 2 at position 338 and 463 respectively for the amino acids Lys310 and Lys435). The numbering takes account of the leader sequences (338 and 463) or it does not (in the latter case, it is the so-called Kabat numbering which was used: 310 and 435).
The YB2/0 cells, co-transfected by electroporation with the mutated vector EMAB5-H-K338-K463-1 and the vector EMAB5-dhfr-K-SpeI coding for the light chain of the EMAB5 antibody, are cultured in RPMI medium supplemented with 5% dialyzed FCS, 0.5% G418 and 25 nM of Methotrexate (MTX). The clones secreting the highest level of human IgGs are cultured on 24-well plates in medium without MTX. The supernatants, collected after culture for 7 days, are used to carry out the tests described below.
In order to study the impact of a modification of the histidines of a human IgG1 on the human IgG1/FcγR interactions, the anti-RhD monoclonal antibody, T125(YB2/0) was treated with diethylpyrocarbonate (DEPC). The DEPC modifies the histidine residues by the creation of a covalent bond between a nitrogen atom of the histidine ring and a carbon atom of the DEPC molecule. The monoclonal antibodies, treated or not treated with DEPC, are fractionated on a column of protein A-Sepharose. Histidine 435 being important for binding IgG to the protein A, the fraction of monoclonal antibodies treated with DEPC and not retained on protein A corresponds to IgG1s at least the His435 residues of which have been modified. The monoclonal antibodies T125(YB2/0) not treated or treated with DEPC and not retained on protein A were compared for their binding to different types of human FcRγs (
In conclusion, the blocking of the histidines present in T125(YB2/0) by the DEPC reduces its binding to the FcγRIIIAs (
The preceding results indicate that the modification of the His residues of a monoclonal IgG1 affect its interactions with the human FcγRs. However, the treatment with DEPC does not make it possible to determine which His's have been modified.
Structural studies have shown the importance of the His435 and His310 residues situated on either side of the hinge region of the IgG1s in the IgG1/FcγR interactions. We therefore studied the effect of the mutation of the His435 and His310 residues of T125(YB2/0) to lysine on the binding of the monoclonal antibody to the human FcRγs (
We analyzed the effect of the mutation of the His310 and His435 residues of T125(YB2/0) on one of the activating effector functions of this antibody.
The abilities of the double mutant and of the unmutated monoclonal antibody to induce a production of IL-2 by the Jurkat-huFcγRIIIA FcγRIIIA+ cells were compared by ELISA (
These results indicate that the modification of the His310 and His435 residues at the CH2/CH3 interface of a monoclonal IgG1 partially reduces the binding of this mAb to the human FcγRIIIAs as well as its ability to induce a production of cytokine dependent on these activating FcRγs. Nevertheless, the double mutant monoclonal antibody T125(YB20) His310Lys/His435Lys is also capable of recruiting activating FcγRIIIAs and of inducing activating effector functions dependent on these receptors.
The preceding experiments show that the double mutant monoclonal antibody T125(YB2O) His310Lys/His435Lys is an antibody capable of engaging the activating receptors (FcγRIIA and FcγRIIIA), but the ability of which to bind the inhibitory FcγRIIBs is eliminated. In order to better characterize the behaviour of this monoclonal antibody, we compared its binding to human FcγRIIIAs and FcγRIIBs with that of T125 (CHO) for which the structural properties and the profile of engagement with the different types of FcγR have been precisely defined (
(B) and (D), the abilities of the monoclonal antibodies T125(YB2/0), T125(YB2/0) His310Lys/His435Lys and T125 (CHO) to inhibit the binding of the 3G8-PE antibodies (anti-human FcγRIIIA/IIIB) (B) or AT10-FITC (anti-human FcγRIIA/IIB) (D) were compared. The indicator cells Jurkat-huFcγRIIIA (B) or IIA. 1.6-huFcγRIIB1 (D) are incubated with different concentrations of T125(YB2/0), or T125(YB2/0) His310Lys/His435Lys or T125 (CHO), then with 40 ng/ml of 3G8-PE (B) or 40 ng/ml of AT10-FITC (D), respectively. The percentage of inhibition of the binding of 3G8-PE or AT10-FITC as a function of the concentration of the competing monoclonal antibodies is calculated: T125 (CHO), which is more fucosylated than T125(YB2/0), is capable of inducing inhibiting functions dependent on the FcγRIIB1s. On the other hand, it only weakly binds the human FcγRIIIAs and is a poor inductor of the activating functions dependent on the FcγRIIIAs.
Indirect immunofluorescence experiments show that, although the binding of the double mutant monoclonal antibody T125(YB20) His310Lys/His435Lys to the FcRγIIIAs are slightly reduced relative to that of T125(YB2/0), its binding remains much greater than that of T125 (CHO) (
The indirect immunofluorescence experiments show that the binding of the double mutant T125(YB20) His310Lys/His435Lys to the HA.1.6-huFcγRIIB1 (FcγRIIB1+) indicator cells is completely eliminated, whereas that of T125 (CHO) is maintained despite a reduction in the ability of this last mAb to bind to the FcRγIIB1s relative to T125(YB2/0) (
Therefore, these immunofluorescence experiments show that the double mutant T125(YB2O) His310Lys/His435Lys has a behaviour different from that of T125 (CHO), in terms of interactions with the FcγRs: T125(YB20) His310Lys/His435Lys effectively recruits the activating FcγRIIIAs. On the other hand, this monoclonal antibody is incapable of engaging the FcγRIIBs.
The ability to induce an ADCC lysis of the Rhesus positive erythrocytes induced by different anti-Ds in the presence of mononucleated cells (source of effector cells) and of Tegeline (2500 μg/ml) was compared for different anti-D antibodies. The AD1 antibody is an anti-D antibody triggering no ADCC response and therefore serves as a negative control. The T125 antibody was expressed in two different cell types: YB2/0 (producing the T125 antibody (YB2/0) and CHO (producing the T125 antibody (CHO)). Moreover, the T125 antibody was mutated to in order to replace each of its His 310 and His 435 residues with lysine residues (T125(YB2/0) His310Lys/His435Lys).
The results appear in
Comparison of the crystallographic structures of the Fc fragments of T125(YB2/0) and T125H310K-H435K(YB2/0) (cf Example 6) shows that the mutations of histidines 310 and 435 to lysines have not modified the general conformation of the Fc fragment. In the document WO 2005/040216 (which is incorporated here by way of reference), the Applicant shows that the non-mutated antibodies, therefore carrying the His 310 and His 435 residues, possess the ability to bind Zinc cations (Zn2+). The Fc fragment carrying the lysines has lost its ability to bind the Zn2+ cations; however, the position of the side chain of these residues as well as their dimensions are superimposed with those of the histidine residues of the Fc fragment of T125 (YB2/0). This fact is important for use of this type of antibody in human therapeutics since this suggests that the binding of the T125 antibody H310K-H435K(YB2/0) to the FcnR, and therefore its catabolism, will not be modified. By contrast, the mutation of the histidine 310 and 435 residues to lysines has an impact on the orientation of the CH2 domains of the Fc fragment. In fact, comparison of the crystallographic structures of the Fc fragments of T125(YB2/0) (in the presence of zinc) and the double mutant T125H310K-H435K(YB2/0) show that the latter has a more closed conformation in which the two CH2 domains are closer to each other relative to the CH2 domains of the Fc fragment of T125 (YB2/0). This difference in conformation could be at the origin of the impact of the H310K and H435K mutations on the binding of the antibody to the human FcγRs (cf Examples 3, 4 and 5).
| Number | Date | Country | Kind |
|---|---|---|---|
| 0512812 | Dec 2005 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2006/002748 | 12/15/2006 | WO | 00 | 10/9/2008 |
