Patent Application
20240307538
The contents of the electronic sequence listing (SequenceListing.txt; Size: 34,364 bytes; and Date of Creation: Mar. 26, 2024) is herein incorporated by reference in its entirety.
The present invention relates to an immune cell comprising at least one Fc receptor on its surface, characterised in that a hybrid molecule is grafted at the level of the Fc receptor, said hybrid molecule comprising at least one antibody Fc fragment covalently bound to at least one fibrin-derived peptide comprising one or more citrullyl residue(s). The present invention also relates to the uses of such a grafted cell, as well as its method of production.
Rheumatoid arthritis is the most common type of inflammatory rheumatism or arthritis, but also the most common autoimmune disease. The disease is characterised by chronic inflammation of the synovial joints, resulting in irreversible joint destruction.
The presence of Immunoglobulin G (IgG) autoantibodies directed against citrullinated proteins, called anti-citrullinated protein autoantibodies (ACPA), is highly specific for rheumatoid arthritis. Serum from patients that contains ACPAs, the lymphocyte cells that express them on their membrane and the patients themselves are all said to be “ACPA-positive”. Several studies have demonstrated that these ACPAs are at the heart of the autoimmune reactions specific to rheumatoid disease and thus represent a therapeutic target of choice.
The antigenic targets of ACPAs have been characterised. They are directed, in particular, specifically against deiminated, or citrullinated, forms of the α and β polypeptide chains of fibrin, a protein abundant in inflamed synovial tissue. This citrullination corresponds to the enzymatic deimination of the arginyl residues of these polypeptide chains by the action of peptidyl-arginine deiminases (PAD).
More specifically, the immunodominant epitopes recognised by ACPAs on the α and β polypeptide chains of fibrin have been characterised and published, in particular in applications PCT/FR00/01857 or PCT/FR2007/000758. The five citrullinated peptides carrying immunodominant epitopes are more particularly the peptides named α36-50Cit (as shown in SEQ ID NO: 5), α171-185Cit (as shown in SEQ ID NO: 6), α501-515Cit (as shown in SEQ ID NO: 14), α621-635Cit (as shown in SEQ ID NO: 18) and β60-74Cit (as shown in SEQ ID NO: 19). Serum from ACPA-positive patients recognise one or more of these five peptides.
Secreted into the rheumatoid synovial tissue by local plasma cells, ACPAs are found there in high concentrations. They remain close to their main target, citrullinated fibrin, which is also abundant there in the form of interstitial deposits. The binding of ACPAs to these deposits and the resulting formation of immobilised immune complexes that in turn bind to rheumatoid factors, other autoantibodies associated with rheumatoid arthritis and also secreted by local plasma cells, triggers a cascade of pro-inflammatory events.
The stimulation of macrophage cells by these immune macro-complexes, essentially via their membrane Fc-gamma receptors, leads them to secrete pro-inflammatory cytokines and in particular TNF-alpha, which has been identified as the main cytokine responsible for rheumatoid inflammation.
To date, there is no curative treatment for rheumatoid arthritis. Treatments are aimed only at treating flare-ups and/or preventing their appearance.
An object of the present invention is thus to provide a treatment for rheumatoid arthritis.
ACPAs are oligoclonal and therefore secreted by only a few clonal plasma cells, themselves resulting from the differentiation of a few clonal B lymphocytes.
In the case of rheumatoid arthritis, clonal B lymphocytes express on their membranes immunoglobulins carrying ACPA specificity (these are ACPA-positive B lymphocytes), whereas plasma cells resulting from the differentiation of ACPA-positive clonal B lymphocytes (these are ACPA-positive plasma cells) secrete these same ACPAs in abundance into their microenvironment.
The present invention is based on the results of the inventors showing that it is possible to target clonal B lymphocytes expressing ACPAs on their surface and to eliminate them using an “armed” immune cell, i.e., a cell expressing an Fc receptor at its surface and onto which is grafted a hybrid molecule comprising (i) at least one fibrin-derived peptide having at least one citrullyl residue and (ii) an Fc fragment of human immunoglobulin. The hybrid molecule is bound to the Fc receptor through its Fc fragment. These hybrid molecules will specifically target ACPA-positive B lymphocytes (using the fibrin-derived peptide having at least one citrullyl residue, which is recognised by said ACPAs), which will then be eliminated, after the Fc fragment binds to the Fc receptors of immune cells expressing at least one Fc receptor. Such cells are, in particular, macrophages (ACPA-positive B lymphocytes are thus eliminated by phagocytosis) and/or NK cells (ACPA-positive B lymphocytes are thus eliminated by antibody-dependent cell-mediated cytotoxicity, or ADCC).
By targeting the clonal B lymphocytes expressing ACPAs, the grafted immune cells of the invention thus aim to make the ACPAs disappear from the body of the patients.
Immune Cell Grafted with a Hybrid Molecule According to the Invention
In a first aspect, the invention relates to an immune cell comprising at least one Fc receptor on its surface, characterised in that a hybrid molecule is grafted onto the Fc receptor, said hybrid molecule comprising at least one antibody Fc fragment covalently bound to at least one fibrin-derived peptide having at least one citrullyl residue, a spacer being optionally present between said Fc fragment and said peptide. The diagram of such a construction is presented in
According to the invention, an “immune cell” is a cell of the innate or adaptive immune system which expresses at least one Fc receptor on its surface. According to a preferred embodiment, the immune cell is chosen from among an NK cell or a macrophage. According to one embodiment, said cell is allogenic or autologous.
According to the invention, a “hybrid molecule” means a molecule having at least two components of different nature, in this case the antibody Fc fragment and said peptide.
According to the invention, an “Fc receptor” means the transmembrane protein that reacts with an Fc fragment, and which contributes to the protective functions of the immune system. Fc receptor refers to an Fc-gamma receptor, neonatal Fc-gamma receptor, Fc-alpha receptor and Fc-epsilon receptor. According to one embodiment, the Fc receptor is an Fc-gamma receptor, in particular FcγRIII (CD16).
According to the invention, a “hybrid molecule is grafted onto the Fc receptor” means that the immune cell expresses at least one Fc receptor on its surface and that a hybrid molecule as defined here is bound to the Fc receptor via an Fc fragment. Because of the binding of the Fc receptor with the Fc fragment, the hybrid molecule is bound to the immune cell. When an immune cell expresses several Fc receptors on its surface, several hybrid molecules can thus be grafted onto the immune cell, i.e., one hybrid molecule per receptor.
According to the invention, an “Fc fragment” of an antibody means the constant region of an immunoglobulin excluding the first immunoglobulin constant region domain (i.e. CH1-CL). The Fc fragment thus refers to a homodimer, each monomer comprising the last two constant domains of IgA, IgD, IgG (i.e. CH2 and CH3), or the last three constant domains of IgE and IgM (i.e. CH2, CH3 and CH4).
According to the invention, the expression “covalently bonded” means a covalent bond, that is, a chemical bond in which two atoms share two electrons. Said covalent bond can be polar or non-polar.
According to the invention, a “spacer” is a coupling agent that enables covalently linking an Fc fragment of an antibody to said fibrin-derived peptide having at least one citrullyl residue, while separating said Fc fragment from said peptide (thus reducing possible steric hindrance). It can be, for example, a molecule or a peptide. Preferably, the spacer does not modify the physical and chemical properties of the hybrid molecule.
The presence of at least one spacer is advantageous: it facilitates the independent accessibility of the two partners of the hybrid molecule (the Fc fragment is more easily accessible to bind to the Fc receptor, just as said peptide is more easily accessible to bind to ACPA-positive B lymphocytes), and/or to stabilise the hybrid molecule, and/or to increase the solubility of the hybrid molecule.
According to one embodiment, the hybrid molecule grafted onto the immune cell according to the invention may comprise one or more spacers. Preferably, the hybrid molecule comprises one or two spacers. According to one embodiment, when at least one spacer is present in said hybrid molecule of the invention, said Fc fragment is covalently bound to a spacer, said spacer being itself covalently bound to said peptide. According to another embodiment, when at least two spacers are present in said hybrid molecule of the invention, said Fc fragment is covalently bound to a first spacer, said first spacer being itself covalently bound to a second spacer and the second spacer being itself covalently bound to said peptide. The bond between the Fc fragment and the peptide can therefore be direct, or even indirect in the presence of spacers.
According to one embodiment, the hybrid molecule grafted onto the immune cell according to the invention may comprise at least one fibrin-derived peptide having at least one citrullyl residue. This means that the Fc fragment can be bound to one or two peptides. Indeed, the Fc fragment comprises two monomers, and the Fc fragment can thus be covalently bound to a peptide on only one of the two monomers, or the Fc fragment can be covalently bound to a peptide on each monomer. Preferably, when two peptides are bound to the Fc fragment, the two peptides are identical, for example two peptides of SEQ ID NO: 18 or SEQ ID NO: 19.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said spacer is a polymer containing one or more repeat units containing the ether group. According to a particular embodiment, said spacer is polyethylene glycol of formula PEGn, in which n represents an integer between 1 and 100, preferably between 1 and 10, and in particular 1, 2, 3, 4 or 8. According to the invention, said polyethylene glycol can be functionalised, for example with an amine group (PEGn-amine such as PEG-NH2). According to the invention “an integer between 1 and 100” represents all the integer values between 1 and 100, i.e.; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100.
According to the invention, the expression “fibrin-derived peptide having at least one citrullyl residue” means a molecule of fibrin or fibrinogen in which at least one arginyl residue has been substituted by a citrullyl residue. A “fibrin-derived peptide having at least one citrullyl residue” according to the invention means a peptide recognised by the ACPAs, and it can also be called a “citrullinated peptide”. Such peptides can be obtained from natural, recombinant or synthetic fibrin or fibrinogen fragments. Such peptides can also be obtained by direct synthesis. The amino acids constituting the peptide can be L or D amino acids, preferably L. Said substitution can be carried out for example by an enzymatic deimination step under the action of peptidyl-arginine deiminases (PAD). Such a peptide can also be obtained by directly incorporating one or more citrullyl residues into the synthesised peptide. A peptide according to the invention binds to ACPAs, and the binding between said fibrin-derived peptide having at least one citrullyl residue and an ACPA can be verified using an ELISA test or as described in the publication by Sebbag M, Moinard N, Auger I, Clavel C, Arnaud J, Nogueira L, Roudier J, Serre G, Epitopes of human fibrin recognised by the rheumatoid arthritis-specific autoantibodies to citrullinated proteins. Eur J Immunol 36:2250-2263, 2006.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide is derived from all or part of the sequence of the α or β chain of a vertebrate fibrin, by substitution of at least one arginyl residue with a citrullyl residue. Preferably, said peptide is derived from a sequence of at least 5 consecutive amino acids of the α chain (in particular represented by SEQ ID NO: 27) or β (in particular represented by SEQ ID NO: 28), of a vertebrate fibrin. Even more particularly, said vertebrate fibrin is a mammalian, preferably human, fibrin.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the peptide has a size of at least 2 consecutive amino acids, 3 consecutive amino acids, 4 consecutive amino acids, preferably 5 consecutive amino acids, and even more preferably between 5 and 25 amino acids. According to the invention, “between 5 and 25” means all the values: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. Preferably, said peptide has a size of between 10 and 20 amino acids, more particularly 15 amino acids.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the peptide is linear.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the peptide can be modified so as to improve its reactivity to ACPAs. By way of example, the peptides can be cyclised, the peptides can be retro peptides (L amino acids are bound according to a reverse sequence of the peptide to be reproduced), or retro-inverso peptides (the amino acids are type D instead of the natural L amino acids and are bound according to a reverse sequence of the peptide to be reproduced). According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal carboxyl function (COOH) of said peptide is replaced by a carboxamide function (CONH2). Preferably, in the peptide of SEQ ID NO: 12, the terminal carboxyl function (COOH) of said peptide is replaced by a carboxamide function (CONH2). By way of example, the β60-74Cit peptide (as represented in SEQ ID NO: 19) advantageously has such a carboxamide function.
According to another embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the peptide can be modified so as to facilitate its synthesis and/or improve its stability, for example by alkylation. According to an even more particular embodiment, in said hybrid molecule according to the invention, the terminal amine function (NH2) of said peptide is acetylated. Preferably, in the peptide of SEQ ID NO: 1, the terminal amine function of said peptide is acetylated. By way of example, the α621-635Cit peptide (as shown in SEQ ID NO: 18) advantageously has such a terminal amine function (NH2).
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, the amine and carboxyl functions of the peptide can be in the form of the salt corresponding to the acid or the base.
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of:
According to a particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of:
According to an even more particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide comprises at least one citrullyl residue, and is chosen from the group consisting of:
According to an even more particular embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said peptide is chosen from the group consisting of: SEQ ID NO: 5 (α36-50cit38,42), SEQ ID NO: 6 (α171-185cit178,181), SEQ ID NO: 7 (α183-197cit186,190), SEQ ID NO: 8 (α246-260cit258), SEQ ID NO: 9 (α259-273cit263,271), SEQ ID NO: 10 (α366-380cit367), SEQ ID NO: 11 (α396-410cit404), SEQ ID NO: 13 (α411-425cit425), SEQ ID NO: 14 (α501-515cit510,512), SEQ ID NO: 15 (α546-560cit547), SEQ ID NO: 16 (α561-575cit573), SEQ ID NO: 17 (α588-602cit591), SEQ ID NO: 18 (α621-635cit621,627,630), SEQ ID NO: 19 (β60-74cit60,72,74), SEQ ID NO: 20 (β210-224cit224), SEQ ID NO: 21 (β281-295cit285,294), SEQ ID NO: 22 (β420-434cit421) and SEQ ID NO: 23 (β433-447cit436,445), more preferably chosen from the group consisting of: SEQ ID NO: 5 (α36-50cit38,42), SEQ ID NO: 6 (α171-185cit178,181), SEQ ID NO: 14 (α501-515cit510,512), SEQ ID NO: 18 (α621-635cit621,627,630) and SEQ ID NO: 19 (β60-74cit60,72,74), even more preferably chosen from SEQ ID NO: 18 (α621-635cit621,627,630) and SEQ ID NO: 19 (β60-74cit60,72,74).
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment is a human Fc fragment, in particular of IgG, more particularly of IgG1. The IgG1 can correspond to any allotypic variant, for example G1m3 or nG1m1. By way of example, the Fc fragment of IgG1 is represented by SEQ ID NO: 24, SEQ ID NO: 25 (Fc+Q-tag) or SEQ ID NO: 26 (Fc+Q-tag bis).
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment is wild-type or mutated. The mutation(s) may aim to increase or reduce the plasma half-life or modify the effector functions of the Fc fragment. According to an even more particular embodiment, said mutated Fc fragment comprises at least the following mutations:
According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment has a fucosylation rate of between 0% and 100% of the glycosylated forms. According to the invention “between 0% and 100%” represents all the integer values between 0 and 100, i.e.; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%. Weak fucosylation of the Fc fragment causes a strong ADCC response. This is why according to a particular embodiment said Fc fragment has a fucosylation rate of between 0% and 60% of the glycosylated forms, in particular 50%, 40%, 30%, 20%, 10% or 0%. According to the invention, the fucosylation rate is defined as the average proportion of fucose carried by the Fc fragment relative to the maximum quantity of fucose that an Fc fragment can carry.
The Fc fragment and the peptide each have N- and C-terminal ends. The Fc fragment can therefore be bound via its N- or C-terminal end to the N- or C-terminal end of the peptide. According to one embodiment, in said hybrid molecule grafted onto the immune cell according to the invention, said covalent bond is located between the C-terminal end of said Fc fragment and the N-terminal end of said peptide, or between the N-terminal end of said Fc fragment and the N-terminal end of said peptide. According to one embodiment, when a spacer is present, the spacer can be bound to the Fc fragment via its N- or C-terminal end. According to another embodiment, when two spacers are present, the first spacer can be bound to the Fc fragment via its N- or C-terminal end and the second spacer can be bound to the peptide via its N- or C-terminal end, in particular the N-terminal end. Alternatively, said covalent bond between said Fc fragment and said peptide (optionally in the presence of one or more spacers) can be created on all or part of the Fc fragment. According to the invention “all or part of the Fc fragment” means that different and/or several amino acids that constitute the Fc fragment can be involved in a covalent bond with said peptide.
According to a preferred embodiment, when at least one spacer is present in said hybrid molecule grafted onto the immune cell according to the invention, this makes it possible for the Fc fragment to bind to an azide or an alkyne that will itself be involved in the covalent bond with said peptide. According to one embodiment, when at least one spacer is present in said hybrid molecule grafted onto the immune cell according to the invention, this can also make it possible for the peptide to bind to an alkyne or an azide that will itself be involved in the covalent bond with the Fc fragment. According to a preferred embodiment, the hybrid molecule grafted onto the immune cell according to the invention comprises at least two spacers: a first spacer which makes it possible for the Fc fragment to bind to an azide or an alkyne and a second spacer which makes it possible for the peptide to bind to an azide (when the Fc fragment is bound to an alkyne) or an alkyne (when the Fc fragment is bound to an azide).
According to one embodiment according to the invention, in said hybrid molecule grafted onto the immune cell according to the invention, said Fc fragment:
According to one embodiment according to the invention, in said hybrid molecule grafted onto the immune cell according to the invention,
According to one embodiment according to the invention, in said hybrid molecule grafted onto the immune cell according to the invention:
According to one embodiment, in the molecules described above that are grafted onto the immune cell, more particularly from page 9, line 13 to page 9, line 36, the spacer used is preferably a PEGn spacer, n representing more particularly an integer between 1 and 10. When the hybrid molecule according to the invention comprises at least two PEGn spacers, the n numbers of the two spacers can be identical or different. For example, the first spacer can be a PEG2 and the second spacer can be a PEG3 or PEG4.
According to a particularly preferred embodiment, in the molecules described above that are grafted onto the immune cell, more particularly from page 9, line 13 to page 9, line 36, the peptide is derived from human fibrin or fibrinogen and the Fc fragment is a human Fc, preferably human IgG1.
According to the invention, the formation of the covalent bond between the azide and the alkyne corresponds to what is known as a “click-chemistry” step, the N3 part of the azide reacting with an alkyne. Azide means salts of hydrazoic acid HN3, or organic azides in which one of the nitrogen atoms is covalently bound with a carbon atom of an organic compound (for example methyl azide CH3N3). Preferably, the azide is represented by the formula N3. Alkyne means molecules having the general formula CnH2n-2, which are characterised by the presence of at least one triple bond. The alkyne is preferably a cyclooctyne, even more preferably dibenzocyclooctyne (DBCO).
The Fc fragment, said peptide and optionally said spacer, are coupled to the alkyne or to the azide by any conventionally used molecular coupling technique (such as conjugation). Any technique may also be used to covalently bind the Fc fragment to the spacer and/or the peptide to the spacer.
More particularly, a conjugation technique means an enzymatic conjugation or a chemical conjugation. An enzymatic conjugation means, for example, a conjugation using a transglutaminase that catalyses the formation of covalent bonds between free amine groups and glutamine or lysine residues or even using a transpeptidase such as sortase. For more information on enzymatic conjugation, see, for example, patent applications US20160361434 or US20170313787, or the publication by Ohtsuka et al., Bioscience, Biotechnology, and Biochemistry Volume 64, 2000-Issue 12, Comparison of Substrate Specificities of Transglutaminases Using Synthetic Peptides as Acyl donors. The transglutaminase substrate is, for example, a peptide comprising a glutamyl residue (a Q-tag), as represented by SEQ ID NO: 29 (LLQG). A chemical conjugation means, for example, a covalent bond between a cysteine isolated or participating in a disulphide bridge after reduction of the latter and, for example, a maleimide. An example of such a conjugation is shown in
According to the invention, the term “coupled” or “molecular coupling” means the establishment of a covalent bond, thus the Fc fragment and/or the peptide and/or the spacer are covalently bound to a alkyne or an azide. The term “bound” also means a covalent bond. Thus, by way of example, the expression “the Fc fragment is coupled to an azide and said peptide is bound to a spacer, itself coupled to an alkyne, such as a cyclooctyne, and in particular DBCO” can also read “the Fc fragment is covalently bound to an azide and said peptide is covalently bound to a spacer, said spacer being itself covalently bound to an alkyne, such as a cyclooctyne, and in particular DBCO”.
According to the invention, the hybrid molecule is grafted onto the immune cell by virtue of the bond between the Fc receptor and the Fc fragment.
According to one embodiment according to the invention, said hybrid molecule grafted onto the immune cell according to the invention is represented by:—a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74) or SEQ ID NO: 18 (α621-635cit621,627,630),
According to one embodiment according to the invention, said hybrid molecule is represented by:
According to an even more particular embodiment according to the invention, said hybrid molecule is represented by:
In a second aspect, the present invention also relates to an immune cell onto which is grafted a hybrid molecule as defined above for use as a medicinal product.
More particularly, according to the invention, said immune cells onto which the hybrid molecules are grafted are intended to target inside the patient's body and lyse, by ADCC and/or phagocytosis and/or complement activation, all the “ACPA-positive” B cells (i.e., B lymphocytes that express anti-citrullinated protein autoantibodies [ACPA] on their surface). Indeed, the hybrid molecule according to the invention binds to “ACPA-positive” B lymphocytes thanks to the fibrin-derived peptide that has at least one citrullyl residue: this is the target epitope of said ACPA. The hybrid molecule according to the invention also binds to the cells, making it possible to destroy the “ACPA-positive” B lymphocytes, the immune cells expressing an Fc receptor, due to their Fc fragment, a natural Fc receptor ligand (for example, Fc-gamma receptor [FcγR] if the Fc fragment is derived from an IgG), present in particular on the surface of macrophages but also of NK cells (“Natural killer” cells).
According to a particular embodiment, the invention relates to an immune cell onto which is grafted a hybrid molecule, as defined above, for its use in the treatment of autoimmune diseases associated with the production of autoantibodies against citrullinated proteins, in particular Gougerot-Sjögren syndrome and rheumatoid arthritis. In this embodiment, the severe forms of said autoimmune diseases associated with the production of anti-citrullinated protein autoantibodies are targeted, as well as various so-called “borderline” forms with other chronic arthritis, such as psoriatic arthritis or systemic lupus erythematosus.
According to one embodiment, the invention also relates to a pharmaceutical composition comprising an immune cell onto which is grafted a hybrid molecule, as defined previously, in combination with a pharmaceutically acceptable vehicle.
According to the invention, “a pharmaceutically acceptable vehicle” means any formulation that makes the composition suitable for administration to a patient, in any galenic form.
According to one embodiment, the invention also relates to a set of elements (or a kit) comprising: (a) a cell of the innate or adaptive immune system that expresses on its surface at least one Fc receptor, in particular an NK cell or a macrophage, and (b) a hybrid molecule as previously defined.
As indicated above, the present invention aims to destroy “ACPA-positive” B lymphocytes. However, “ACPA-positive” B lymphocytes that have differentiated into ACPA-secreting plasma cells are no longer targeted by the hybrid molecules previously described. According to a particular embodiment, the present invention thus relates to an immune cell as previously described for use as a medicinal product in combination with a hybrid molecule (this second hybrid molecule not being grafted onto the immune cell). Said second hybrid molecule comprises at least one fibrin-derived peptide having at least one citrullyl residue, said peptide being covalently bound to at least one antibody or to at least one antibody fragment, said antibody or fragment being capable of binding to CD38 and/or CD138, one or more spacers being optionally present between said peptide and said antibody or said fragment. Such a combination therapy makes it possible to specifically target and destroy ACPA-positive clonal plasma cells and B lymphocytes, and thus eliminate ACPAs from the patient's body. The antibody used in this second hybrid molecule is preferably a monoclonal antibody.
According to a particular embodiment, in this second hybrid molecule the fragment is understood as a portion of antibody that carries the antigen-binding site. Such antibody fragments are, for example, Fab, F(ab′)2, Fv, dsFv, scFv, single-chain fragments such as isolated VH or VL fragments, camelid VHHs, cartilaginous fish VNARs, or even multispecific antibodies consisting of different fragments, such as bi-specific antibodies, or “nanobodies”, “diabodies”, “triabodies”, “tetrabodies”, or scFvs in tandem, etc. These fragments are well known to those skilled in the art. Further information concerning these fragments and constructions is described, for example, in international application WO 2017137579, in Bird et al., 1988 Science 242:423-426; and in Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883, or in Nelson, mAbs 2:1, 77-83; January/February 2010. The fragment is preferably a Fab or F(ab′)2. According to a particular embodiment, in this second hybrid molecule, said antibody or F(ab′)2 fragment is a bispecific antibody or F(ab′)2 fragment directed against CD38 and another plasma cell target. Alternatively, in this second hybrid molecule, said antibody or F(ab′)2 fragment is a bispecific antibody or F(ab′)2 fragment directed against CD138 and another plasma cell target. According to the invention, “another plasma cell target” means any marker expressed on the surface of the plasma cells.
According to another particular embodiment, in this second hybrid molecule, said antibody or F(ab′)2 fragment is a bispecific antibody or F(ab′)2 fragment directed against CD38 and CD138.
According to this particular embodiment of the invention, the grafted immune cell and the second hybrid molecule are administered simultaneously, separately or gradually over time.
According to another embodiment, the hybrid molecule according to the invention grafted onto the immune cell as previously described can be coupled to at least one radioisotope or to at least one fluorochrome, such as A488 or A647. Such molecules can advantageously be used as molecular tracer tools.
According to another embodiment, the invention thus relates to the in vitro or ex vivo use as a molecular tool of an immune cell comprising at least one Fc receptor on its surface onto which is grafted a hybrid molecule, said hybrid molecule comprising at least one antibody Fc fragment covalently bound to at least one fibrin-derived peptide having at least one citrullyl residue, at least one spacer being optionally present between said Fc fragment and said peptide. Such constructions can in particular be used to analyse the binding of hybrid molecules to ACPAs and to the Fc receptors of macrophages and NK cells, as well as to analyse the reactivity of macrophages and NK cells to the binding of hybrids followed by the bridging of the latter by the ACPAs . . . . The radioisotopes and/or fluorochromes are preferably coupled to the Fc fragment, even more particularly at the level of the Q-tag (if present) or at the level of the lysines.
In another aspect, the invention also relates to a method that enables obtaining an immune cell grafted with a hybrid molecule as defined previously.
According to one embodiment, the invention thus relates to a method for producing an immune cell grafted with a hybrid molecule as defined above, comprising the following steps:
According to one embodiment, the invention thus relates to a method for producing an immune cell grafted with a hybrid molecule as defined above, comprising the following steps:
According to another embodiment, the invention thus relates to a method for producing an immune cell grafted with a hybrid molecule as defined above, comprising the following steps:
The sequences of the invention are shown in Table 1 below.
Other characteristics, details and advantages of the invention will appear on reading the appended figures and the examples that illustrate the invention and in no way intend to limit it.
In the hybrid molecules exemplified, it is the N-terminal end of the Fc fragment that is bound to a PEGn spacer (except in the case indicated with Cter-PEG [see
In the examples below, an “armed or pre-armed” cell means a cell to which a hybrid molecule according to the invention is attached. Thus, the hybrid molecule is bound to the Fc receptor of the cell due its Fc fragment.
A spacer (which is optional) is shown between the antibody Fc fragment and the fibrin-derived peptide having a citrullyl residue (referred to as “citrullinated peptide”).
The NK cell will thus be able to destroy the B lymphocyte by ADCC. More specifically, this figure shows a B cell (or B lymphocyte) that expresses on its surface an ACPA-type BCR and that interacts specifically with the citrullinated peptide carried by the hybrid molecule. The binding of the Fc fragment carried by the same hybrid molecule to the FcγRIIIa (CD16a) expressed on the surface of an NK cell will activate the ADCC and induce the specific destruction of the ACPA-positive B lymphocyte. (ACPA, Anti-Citrullinated Protein Autoantibodies; ADCC: Antibody-Dependent Cell Cytotoxicity; BCR, B-Cell Receptor; FcγR, Fc-gamma Receptor; NK cell, Natural Killer cell).
The macrophage will thus be able to destroy the B lymphocyte by phagocytosis. More specifically, this Figure shows B cells (or B lymphocytes) that express an ACPA-type BCR on their surface and that interact specifically with citrullinated peptides carried by the hybrid molecules. The binding of the Fc fragments carried by these same hybrid molecules to different FcγRs expressed on the surface of a macrophage will activate ADP, i.e. phagocytosis, and induce the specific destruction of ACPA-positive B lymphocytes. (ACPA, Anti-Citrullinated Protein Autoantibodies; ADP, Antibody-Dependent Phagocytosis; BCR, B-Cell Receptor; FcγR, Fc-gamma Receptor).
β60-74 represents the peptide of SEQ ID NO: 19. Fc-WT-β60-74 represents a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74). Fc-LALA-β60-74 represents an Fc fragment comprising the L234A, L235A and P329G mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19. Fc-SDH-β60-74 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a peptide represented by SEQ ID NO: 19. Fc-WT-PEG-β60-74 represents a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 (β60-74ct60,72,74). Fc-SDH-PEG-60-74 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a PEGn spacer, itself bound to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74). Fc-SDH(Cter)-PEG-β60-74 corresponds to Fc-SDH-PEG-β60-74, except that in the hybrid Fc-SDH(Cter)-PEG-β60-74, the first PEGn is attached at the C-terminal end of the Fc fragment, unlike the other examples in which the first PEGn is attached at the N-terminal end. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
α621-635 represents the peptide of SEQ ID NO: 18. Fc-WT-PEG-α621-635 represents a wild-type (WT) Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18. Fc-SDH-PEG-α621-635 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
FcWT-PEG-α621-635 represents a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18. FcSDH-PEG-α621-635 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations, which is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18. n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The results of the 6 experiments presented in Example 6 are indicated (N=6). The P value is as follows: *<0.05 **<0.01 ***<0.001.
Nalm6 represents the case where only Nalm6 cells were incubated; Nalm6+NK the case where the Nalm6 cells were incubated with the NK cells; WTαCIT the case where the WTαCIT hybrid was incubated with Nalm6 cells and NK cells; WTαArg the case where the WTαArg hybrid was incubated with Nalm6 cells and NK cells; SDHαCIT the case where the SDHαCIT hybrid was incubated with the Nalm6 cells and the NK cells; and SDHαArg the case where the SDHαArg hybrid was incubated with the Nalm6 cells and the NK cells.
The transduced lymphoblastic malignant human B cell line expressing a recombinant monoclonal ACPA on its membrane corresponds to the Nalm6 line as described in Example 11 and in
A—Analysis by flow cytometry of the transduction efficiency of the Nalm6 B line for expression on its membrane of the monoclonal 2H06 ACPA, using double labelling with an IgG anti-Fab antibody and a α621-635cit peptide tetramer (SEQ ID NO: 18). B—Analysis by flow cytometry of the enrichment in 2H06 Nalm6 transduced cells following cell sorting carried out using an anti-Fab antibody. C-Flow cytometry analysis of the interaction between the membrane 2H06 ACPA expressed by the transduced Nalm6 line and hybrids Fc-WT-PEG-α621-635cit (cit) or Fc-WT-PEG-α621-635arg (arg) coupled to fluorochrome A647. NT Nalm6: non-transduced control Nalm6 line. Fc-WT-PEG-α621-635 represents a wild-type (WT) Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18. Fc-WT-PEG-α621-635arg represents a wild-type (WT) Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 18 in that the X residues are not citrullinated (i.e., they are arginyl residues).
The Nalm6 cells expressing the monoclonal 2H06 ACPA correspond to the transduced Nalm6 line mentioned in the examples.
A—The figure shows the results of a flow cytometry experiment in which the cell populations are represented in the form of scatter plots. TO represents the result at time 0, after bringing into contact the Nalm6 target cells and the macrophages. T2 represents the result at time 2h, after 2 hours of bringing Nalm6 target cells and macrophages into contact with one another in the absence of hybrid molecules (spontaneous phagocytosis). All of the other cytometry diagrams represent the results obtained after 2h of bringing Nalm6 target cells into contact with macrophages armed with Fc-PEG-α621-635cit (citrullinated) hybrids or Fc-PEG-α621-635arg (non-citrullinated) control hybrids. The hybrids are identical to those described in
These figures show that the double-positive cells observed in cytometry in the preceding experiments correspond well to the phagocytosis of the Nalm6 target cells by the macrophages. The four panels on the left show flow cytometry results obtained after bringing together 2H06 Nalm6 cells labelled with pHRodo SE and macrophages armed with Fc-WT-PEG-α621-635 hybrids in the presence or absence of cytochalasin D, a phagocytosis inhibitor. The two panels on the right show the cell populations at the end of the experiment, observed by optical microscopy after staining with MGG. The hybrids are identical to those described in
The figure shows the results of an experiment analysed by flow cytometry: the point clouds correspond to cell populations. TO represents the result at time 0, just after bringing into contact the Nalm6 target cells and the macrophages. T2 represents the result at 2 hours after bringing into contact the Nalm6 target cells and the macrophages in the absence of hybrid molecules (spontaneous phagocytosis). The remaining cytometry diagrams show the results obtained after 2h of bringing Nalm6 target cells and macrophages into contact, either pre-armed with the FcWT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, citrullinated or not, or incubated with the WT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, citrullinated or not, solubilised in the incubation medium. The hybrids are identical to those described in
Cells showing double fluorescence (PKH-67 green/PKH-26 red) correspond to macrophages that have phagocytosed Nalm6 target B cells (% double positives). The cells exhibiting a single PKH-67 fluorescence correspond to the residual Nalm6 target B cells, i.e. not phagocytosed after 2h in the presence of macrophages. In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population.
The Figure shows the results of two independent identical experiments, (n=2) of phagocytosis of the 2H06 Nalm6 ACPA+ transduced line by pre-armed macrophages with Fc-PEG-peptide α621-635 hybrids, in the presence or absence of human IgG at physiological serum concentration. The figure shows, for each experimental condition, the average residual percentage of the population of target cells, 2h after phagocytosis by the pre-armed macrophages of the citrullinated (cit) or non-citrullinated (arg) Fc-PEG-α621-635 hybrids, in the presence (grey histograms) or absence (black histograms) of competing human IgGs. The hybrids are identical to those described in
The figure shows the results of an experiment analysed by flow cytometry where the cell populations are represented by scatter plots. TO corresponds to the result obtained immediately after bringing into contact the 2H06 Nalm6 target cells and the macrophages. T2 shows the result 2h after bringing the target cells and the macrophages into contact in the absence of hybrid molecules (spontaneous phagocytosis). The remaining diagrams represent the results obtained 2h after bringing the target cells and macrophages into contact, incubated with the FcWT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, citrullinated (cit) or not (arg), and this in the presence or absence of human IgG at the physiological serum concentration or human serum. CFSE+ CD11b+ double-positive cells correspond to macrophages that have phagocytosed target cells. The CFSE+ single-positive cells correspond to the residual Nalm6 target cells, not phagocytosed after 2h. In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in
The figure shows the results of an experiment of phagocytosis of cells of the 2H06 Nalm6 ACPA+ transduced line by macrophages pre-armed with the Fc-peptide α621-635 hybrids, in the presence of cells of the same wild-type, non-transduced Nalm6 line. The results of the flow cytometry analysis appear in the form of point clouds representing cell populations. TO shows the situation at time 0, just after bringing into contact the 2H06 Nalm6 target cells, the non-transduced NT Nalm6 cells and the macrophages. T2 represents the result after 2h in the absence of hybrid molecules (spontaneous basal phagocytosis). The remaining cytometry diagrams represent the results obtained after 2h in the presence of macrophages armed with the FcWT-PEG-α621-635 or FcGASDIE-PEG-α621-635 hybrids, citrullinated (cit) or non-citrullinated (arg). The CFSE+ CD19+ double-positive cells correspond to the residual 2H06 Nalm6 target cells, i.e. not phagocytosed after 2h in the presence of macrophages. CFSE− CD19− double-negative cells are macrophages. The CFSE− CD19+ single-positive cells correspond to residual non-transduced (NT) Nalm6 cells, i.e. not phagocytosed after 2h in the presence of macrophages. The CFSE+ CD19− single-positive cells correspond to macrophages that have phagocytosed 2H06 Nalm6 target cells. In each quadrant of the cytometry diagrams, the different cell populations are expressed as a percentage of the total cell population. The hybrids are identical to those described in
The figure shows the destruction by human NK cells, in the peritoneal cavity of SCID/BEIGE immunodeficient mice, of ACPA+ target cells (transduced 2H06 Nalm6 line) in the presence of citrullinated Fc-peptide α621-635 hybrids. A—The figure shows the analysis by flow cytometry of the populations of non-transduced 2H06 Nalm6 and NT Nalm6 cells. The peaks observed correspond to the target cells (2H06 Nalm6 CTVhigh) or to the control cells (NT Nalm6 CTVlow). B—The figure shows all the results obtained for four groups of 7 SCID/BEIGE immunodeficient mice. These results are expressed as a ratio of CTVhigh/CTVlow fluorescences, corresponding respectively to 2H06 Nalm6 cells (CTVhigh) and NT Nalm6 cells (CTVlow). A decrease in the ratio reflects the disappearance of the 2H06 Nalm6 target cells. The p value is as follows: * p<0.05; ** p<0.01; *** p<0.001. The hybrids are identical to those described in
The figure shows the destruction by human macrophages, in the peritoneal cavity of SCID/BEIGE immunodeficient mice, of ACPA+ target cells (2H06 Nalm6 transduced line) in the presence of citrullinated Fc-peptide α621-635 hybrids. The figure shows the results obtained for 4 groups of 5 SCID/BEIGE immunodeficient mice. The number of residual 2H06 Nalm6 CTVhigh target cells for each group at the end of the experiment is presented, indicating the efficiency of the phagocytosis. The p value is as follows: * p<0.05. The hybrids are identical to those described in
The hybrid molecule described here comprises the following construction: an Fc fragment covalently bound to a PEGn, itself coupled to an azide, said azide being covalently bound to an alkyne that is itself bound to a spacer coupled to a citrullinated peptide. Such a molecule can be read: Fc-PEGn-N3-DBCO-PEGn-citrullinated peptide. The two PEGn can be identical or different (for example the first PEGn can be PEG3 and the second PEG2).
Similarly, an Fc-PEGn-DBCO and an N3-PEGn-citrullinated peptide can be obtained, then coupled to obtain Fc-PEGn-DBCO-N3-PEGn-citrullinated peptide.
Similarly an Fc-PEGn-DBCO and an N3-citrullinated peptide can be obtained, or an Fc-PEGn-N3 and a citrullinated DBCO-peptide, then coupled to obtain respectively Fc-PEGn-DBCO-N3-citrullinated peptide or Fc-PEGn-N3-DBCO-citrullinated peptide.
The ACPAs (here ACPA, pH3 fraction) used are ACPAs obtained from the serum of patients suffering from ACPA-positive rheumatoid arthritis. Such ACPAs were obtained by a standard column affinity chromatography method known to those skilled in the art, using as bound antigens one or more of the five immunodominant peptides of the invention (see SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 18 or SEQ ID NO: 19).
a. Examples with Hybrid Molecules Containing the β60-74Cit Peptide
Microtiter ELISA plate wells were coated by passive adsorption using 100 μL of a solution containing either the peptide 60-74cit (SEQ ID NO: 19, also called β60-74), or a hybrid molecule Fc-WT-β60-74cit, Fc-LALA-β60-74cit, Fc-SDH-β60-74cit, Fc-WT-PEG-β60-74cit, Fc-SDH-PEG-β60-74cit or Fc-SDH(Cter)-PEG-β60-74cit. These solutions were each used at a concentration of 5 μg/ml in PBS (Phosphate Buffered Saline) buffer and incubated overnight at 4° C. The non-citrullinated peptide and the hybrid molecules constructed with the non-citrullinated peptide (β60-74arg, corresponding to a peptide of SEQ ID NO: 19 in which the citrullyl residues were replaced by arginyl residues) were used at the same concentration as negative controls. The wells were then saturated with 2% BSA (Bovine Serum Albumin) for 1h at 4° C. After washing, the purified ACPAs were incubated at 1, 0.5 or 0.25 μg/ml, diluted in PBS 2% BSA 2M NaCl buffer for 1h at 4° C. After washing, ACPA reactivity was detected using a human IgG anti-Fab secondary antibody diluted to 1/2500 in PBS 2% BSA buffer. The results are expressed in ΔOD (change in Optical Density), corresponding to the OD obtained with the β60-74cit peptide or with the hybrid molecules constructed with the 60-74cit peptide, subtracted respectively from the OD obtained with the β60-74arg peptide or with the corresponding hybrid molecules constructed with the β60-74arg peptide.
The results are shown in
b. Examples with Hybrid Molecules Containing the α621-635Cit Peptide
ELISA plate wells were coated with 100 μL of a solution containing the peptide α621-635cit (SEQ ID NO: 18, also called α621-635) or a hybrid molecule Fc-WT-PEG-α621-635cit or even Fc-SDH-PEG-α621-635cit, at a concentration of 5 μg/ml in PBS buffer (Phosphate Buffered Saline), incubated overnight at 4° C. The non-citrullinated peptide α621-635arg and the corresponding hybrid molecules constructed with the non-citrullinated peptide (α621-635arg, corresponding to a peptide of SEQ ID NO: 18 in which the citrullyl residues were replaced by arginyl residues) were used at the same concentration as negative controls. The wells were then saturated with 2% BSA (Bovine Serum Albumin) in PBS buffer for 1h at 4° C. After washing, the purified ACPAs were incubated at 4, 2 and 1 μg/ml diluted in PBS 2% BSA 2M NaCl buffer for 1h at 4° C. After washing, ACPA reactivity was detected using a human IgG anti-Fab secondary antibody diluted to 1/2500 in PBS 2% BSA buffer. The results are expressed in ΔOD (change in Optical Density), corresponding to the OD obtained with the α621-635cit peptide or with the hybrid molecules constructed with the α621-635cit peptide, subtracted respectively from the OD obtained with the α621-635arg peptide or with the corresponding hybrid molecules constructed with the α621-635arg peptide.
The results are shown in
The ACPA used here is a recombinant monoclonal human ACPA (clone 022014CCP14CFCT2H06, known as 2H06). Such an ACPA can be obtained as described in Titcombe P. J., Wigerblad G., Sippl N., Zhang N., Shmagel A. K., Sahlstrom P., Zhang Y., Barsness L. O., Ghodke-Puranik Y., Baharpoor A., et al. Pathogenic Citrulline-Multispecific B Cell Receptor Clades in Rheumatoid Arthritis. Arthritis & Rheumatology 2018, 70 (12), 1933-1945. https://doi.org/10.1002/art.40590. The 2H06 ACPA VH has accession number MH629710.1 under Genbank, and VL has accession number MH629700.1.
ELISA plate wells were coated with 100 μL of a solution containing either the hybrid molecule Fc-WT-PEG-α621-635cit or Fc-SDH-PEG-α621-635cit, used at a concentration of 5 μg/ml in PBS buffer (Phosphate Buffered Saline) incubated overnight at 4° C. Hybrid molecules constructed with a non-citrullinated peptide (α621-635arg) were used at the same concentration as negative controls. The wells were then saturated in PBS 2% BSA (Bovine Serum Albumin) buffer for 1h at 4° C. After washing, the WT and SDH recombinant monoclonal 2H06 ACPAs were incubated at 40, 20 and 10 μg/ml, diluted in PBS 2% BSA 2M NaCl buffer for 1h at 4° C. After washing, their reactivities were detected using a human IgG anti-Fab secondary antibody diluted to 1/2500 in PBS 2% BSA buffer. The results are expressed in ΔOD (change in Optical Density), corresponding to the OD obtained with the hybrid molecules constructed with the peptide α621-635cit, subtracted from the OD obtained with the hybrid molecules constructed with the α621-635arg negative control peptide.
The Fc-WT-PEG-α621-635cit and Fc-SDH-PEG-α621-635cit hybrid molecules are identical to those used in Example 2.
The results are shown in
Human macrophages, differentiated in vitro in the presence of M-CSF (100 ng/ml) from CD14+ monocytes isolated from the peripheral blood of a healthy subject, were incubated at 500,000 cells/well in the presence of hybrid molecules at 5 μg/ml, FcWT-N3-DBCO-β60-74Cit (a wild-type Fc fragment that is bound to a PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19, referred to as WT in Tables 2 and 3), FcSDHFSTIE-N3-DBCO-β60-74Cit (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to a PEGn spacer that is itself coupled to an azide covalently bound to a DBCO that is coupled to a peptide represented by SEQ ID NO: 19, referred to as SDH in Tables 2 and 3) or FcLALAPG-N3-DBCO-β60-74Cit (an Fc fragment comprising the L234A, L235A and P329G mutations that is bound to a PEGn spacer that is itself coupled to an azide bound covalently to a DBCO cyclooctyne that is coupled to a peptide represented by SEQ ID NO: 19, called LALAPG in Tables 2 and 3), for 2h or 20h at 37° C. The attachment of the hybrid molecules to the surface of the macrophages is demonstrated and quantified by cytofluorimetry (FACS Canto II) after incubation of the macrophages with an anti-Fc antibody coupled to FITC, used at 1/1000. In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The results are presented in Tables 2 and 3 below. “NM” and “ANTI FC” respectively represent the wells containing the macrophages incubated without antibodies or with the anti-Fc antibody alone.
The results presented in Tables 2 and 3 show that hybrid molecules FcWT-N3-DBCO-β60-74Cit and FcSDHFSTIE-N3-DBCO-β60-74Cit at the physiological temperature of 37° C. bind from the 2nd hour to the macrophage membrane and are still present there 20h later. These results also show that the binding of hybrid FcSDHFSTIE-N3-DBCO-β60-74Cit is greater than that of the wild form FcWT-N3-DBCO-β60-74Cit.
Human B lymphocytes freshly purified from peripheral blood of healthy donors by CD19+ magnetic sorting were labelled with green PKH-67 (Paul Karl Horan/fluorescent lipid marker) then incubated with purified ACPAs (pH3 fraction, obtained as in Example 2) on β60-74cit peptide (SEQ ID NO: 19) at a concentration of 5 μg/ml for 30 min at 37° C. Subsequently, the cells were washed and then placed in the presence of the FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit hybrid (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one spacer PEGn that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a PEGn spacer itself bound to a peptide represented by SEQ ID NO: 19). This construction is referred to as CIT in Table 4 below). The cells were also placed in the presence of the FcSDHFSTIE-N3-DBCO-PEG-β60-74Arg hybrid (that corresponds to the FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit construct in which the β60-74 peptide is not citrullinated). This construction is referred to as NCIT in Table 4 below). The cells were placed in the presence of the hybrids at 10 μg/ml for 30 min at 37° C. After washing, the cells were brought into contact with macrophages (marked PKH-26 red), at a ratio of 1 B lymphocyte to 1 macrophage, for 2 h at 37° C. The cells were then separated and analysed by flow cytometry. The cells showing double fluorescence (PKH-67 green/PKH-26 red) correspond to the macrophages that have phagocytosed lymphocytes. The different cell populations are expressed as a percentage of the total cell population: percentage of phagocytosis and percentage of B lymphocytes. In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The results are presented in Table 4 as four lines that correspond to four independent experiments.
The results obtained confirm that phagocytosis activity is increased in the presence of citrullinated hybrids (increase in phagocytosis associated with a reduction in the population of B lymphocytes). Indeed, in the 4 independent experiments, a significant increase in the percentage of phagocytosis, always associated with a significant decrease in the population of B lymphocytes, was observed when the B lymphocytes were coated with the hybrid FcSDHFSTIE-N3-DBCO-PEG3-β60-74Cit (CIT) versus FcSDHFSTIE-N3-DBCO-PEG3-β60-74Arg (NCIT).
Human B lymphocytes freshly purified from peripheral blood of healthy donors by CD19+ magnetic sorting were labelled with green PKH-67 (Paul Karl Horan/fluorescent lipid marker), then incubated for 1h at 37° C., with human recombinant monoclonal 2H06 ACPA whose Fc has an SDH mutation at a concentration of 10 μg/ml. Such antibodies can be obtained as in Example 3. In parallel, macrophages labelled with PKH-26 red were placed in the presence of Fc-WT-PEG-α621-635cit hybrids (a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO that is coupled to a PEGn spacer that is itself bound to the peptide represented by SEQ ID NO: 18) and Fc-WT-PEG-621-635arg (this is the Fc-WT-PEG-α621-635cit hybrid, with the difference that the peptide is not citrullinated), at 5 g/ml for 1h at 37° C. After washing, the cells were brought into contact for 2 h at 37° C. at A ratio of 1 B lymphocyte to 1 macrophage. The cells were then separated and analysed by flow cytometry. The percentage of phagocytosis corresponds to the percentage of cells marked by the 2 fluorochromes (PKH-67 green/PKH-26 red). In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The results are presented in Table 5 as six lines that correspond to five independent experiments. NCIT represents the result with the Fc-WT-PEG-α621-635arg peptide, CIT represents the result with the Fc-WT-PEG-α621-635cit peptide, and (ΔCIT-NCIT) represents the difference between the OD obtained in the presence of Fc-WT-PEG-α621-635cit and the OD obtained in the presence of Fc-WT-PEG-α621-635arg, divided by the OD obtained in the presence of Fc-WT-PEG-α621-635arg.
The results show that phagocytosis activity increases in the presence of citrullinated hybrids: increase in phagocytosis (from 21% to 63%) associated with a decrease in the population of B lymphocytes (from 3% to 17%). In each of the six experiments the percentage of double-positives was higher, so phagocytosis is increased and population B decreases when macrophages are armed with Fc-WT-PEG-α621-635cit (CIT) hybrids versus Fc-WT-PEG-α621-635arg (NCIT) hybrids. The results are also shown in
The NK cells were incubated for 30 min, 24h or 48h at 37° C., in the presence of FcSDHFSTIE-N3-DBCO-A488 (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO, the molecule being labelled with the fluorochrome A488) or FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysineA488 (an Fc fragment comprising the S239D, H268F, S324T and 1332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO that is coupled to a PEGn spacer bound to a peptide of SEQ ID NO: 19, the molecule being labelled with an A488 fluorochrome that is bound to the molecule via a lysine). The binding of these 2 fluorescent probes to NK cells was analysed by cytofluorimetry. In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The results are presented below in Tables 6 (30 minutes), 7 (24 hours) and 8 (48 hours). NM represents unlabelled NK cells, without antibodies.
The results presented in Tables 6 to 8 show that FcSDHFSTIE-N3-DBCO-A488 and FcSDHFSTIE-N3-DBCO-PEG3-β60-74cit-lysine A488 remain attached to the NK cell membrane for at least 48h at physiological temperature and that the attachment of the peptide to the Fc fragment does not prevent its interaction with the Fc gamma receptors of NK cells.
The NK cells were incubated for 30 min at 37° C. either in the presence of the FcSDHFSTIE-N3-DBCO-A647 fragment (an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself even coupled to an azide covalently bound to a DBCO, the molecule being labelled with fluorochrome A647) at 10 μg/ml, or in the presence of this same FcSDHFSTIE-N3-DBCO-A647 fragment at 10 g/ml, co-incubated with human IgG at increasing concentrations (1, 10, 100, 1,000, 10,000 μg/ml). In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8. Binding of the FcSDHFSTIE-N3-DBCO-A647 fragment (known as SDH) to NK cells was analysed by cytofluorometry.
The results are presented in Table 9.
The results show a dose-dependent decrease in the binding of FcSDHFSTIE-N3-DBCO-A647 to NK cells from 100 μg/ml of IgG, which remains effective and high even in the presence of a concentration of IgG 1,000 times greater than that of the Fc fragment (10,000 μg/ml), which corresponds to the physiological serum concentration of IgG in humans.
WTαCIT represents the hybrid comprising a wild-type Fc fragment that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a PEGn spacer that is itself bound to the peptide represented by SEQ ID NO: 18; WTαArg corresponds to the hybrid WTαCIT, except for the peptide of SEQ ID NO: 18 is non-citrullinated; SDHαCIT represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to at least one PEGn spacer that is itself coupled to an azide covalently bound to a DBCO cyclooctyne that is coupled to a PEGn spacer that is itself bound to the peptide represented by SEQ ID NO: 18; and SDHαArg corresponds to the SDHαCIT hybrid, except for the peptide of SEQ ID NO: 18 is not citrullinated. In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The experiment was carried out in 96-well round bottom plates. Cells of the Nalm6 line (transduced for membrane expression of the recombinant human monoclonal 2H06 ACPA) were used as target cells. 100,000 cells/well were incubated in the presence, on the one hand, of WTαCit or SDHαCit hybrid molecules used at 15 μg/ml and, on the other hand, freshly purified NK cells (marked with the purple cell tracker) used as effector cells, also at 100,000 cells/well, for 17h at 37° C. The WTαArg and SDHαArg hybrid molecules, used as specificity controls, were incubated under the same conditions. As negative controls, Nalm6 cells were incubated in the absence of hybrids, alone or in the presence of NK cells. Cell lysis was measured by flow cytometry by labelling with the fluorescent probe 7-AAD. The results show the percentage of cells positive for this marker (% dead cells). (%) (ΔCIT-Arg) represents the difference between the percentages of 7-AAD-positive dead cells in the presence of hybrids carrying WT and SDH citrullinated peptides versus their non-citrullinated counterparts, divided by the percentage of 7-AAD-positive cells in the presence of the corresponding non-citrullinated hybrid.
The results are presented in
The results show that the citrullinated hybrids (WT and SDH) are able to specifically increase the lysis by ADCC of the Nalm6 ACPA-positive target cells caused by the NK cells. This increase in mortality is 62% for the WT hybrids and 83% for the SDH hybrids.
The hybrids used here are the following: FcWT-β60-74 represents a wild-type Fc fragment that is bound to a PEGn spacer that itself is coupled to an azide covalently bound to a cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74). FcSDH-β60-74 represents an Fc fragment comprising the S239D, H268F, S324T and I332E mutations that is bound to a PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74). FcLALAPG-β60-74 represents an Fc fragment comprising the L234A, L235A and P329G mutations that is bound to a PEGn spacer that is itself coupled to an azide covalently bound to a cyclooctyne coupled to a PEGn spacer that is itself bound to a peptide represented by SEQ ID NO: 19 (β60-74cit60,72,74). In the constructions, n represents a number between 1 and 10 when a PEGn spacer is used, preferably 1, 2, 3, 4 or 8.
The NK cells were incubated for 30 min at 37° C., in the presence of hybrid molecules Fc-WT-β60-74cit, Fc-SDH-β60-74cit, Fc-LALAPG-β60-74cit at 10 μg/ml. After washing, the cells were incubated for 30 min at 4° C., in the presence of an anti-CD107a fluorescent antibody (degranulation marker). After washing, the membrane expression of CD107a was analysed by flow cytometry. The results are presented in Table 11.
The results presented in Table 11 show that the membrane expression of the CD107a degranulation marker is equivalent (between 1.8% and 3%), whether or not the NK cell is coated with the hybrid molecules according to the invention. Attachment of the hybrid molecules therefore does not induce degranulation of the NK cells.
A—The human B lymphoblastic cell line “Nalm6” was transduced to induce membrane expression of the recombinant monoclonal 2H06 ACPA. The transduction efficiency was analysed by flow cytometry using double labelling with an IgG anti-Fab antibody and a tetramer of the biotinylated α621-635cit peptide attached to fluorescent Avidin. B—A sorting carried out on 70 million Nalm6 cells made it possible to recover the Nalm6 Fab+ cells, from which a pool of 300,000 cells was returned to culture. After proliferation, the enrichment of the transduced 2H06 Nalm6 line was confirmed by flow cytometry. C—The Fc-WT-PEG-α621-635 cit and arg hybrids were labelled with Alexa Fluor 647 using the “Lightning Link” kit. The non-transduced (NT) Nalm6 and 2H06 Nalm6 lines were incubated with the Fc-WT-PEG-α621-635 hybrids. After washing, the membrane binding of the Fc-WT-PEG-α621-635 hybrids was analysed by flow cytometry.
The results are presented in
Table 12 represents the mean fluorescence intensities (MFI) and the ratio of MFI between the condition in the presence of fluorescent hybrid and the control condition where the cells are unmarked. um represents unmarked Nalm6 cells, without fluorescent hybrids.
The results show that after cell sorting and proliferation, 89.1% of the Nalm6 cells express an IgG Fab, that of 2H06 ACPA, validating the success of the transduction and the establishment of the 2H06 Nalm6 line (B). The results show a strong interaction between the 2H06 Nalm6 line and the fluorescent Fc-WT-PEG-α621-635 cit hybrid, whereas no interaction exists with the non-citrullinated Fc-WT-PEG-α621-635 arg hybrid (C). Double labelling with the fluorescent Fc-WT-PEG-α621-635 cit hybrid and the fluorescent IgG anti-Fab antibody shows a perfect correlation of the 2 labels on the 2H06 Nalm6 line, whereas neither of the two labels the non-transduced Nalm6 line (A). These results show that the binding of the hybrid Fc-WT-PEG-α621-635 cit is strictly bound to the membrane expression of 2H06 ACPA.
The macrophages were labelled with the green PKH-67 membrane fluorochrome and then incubated with the Fc-WT-PEG-α621-635, Fc-SDH-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, at a concentration of 5 μg/ml for 60 min at 37° C. The cells of the Nalm6 human B lymphoblastic line transduced for expression of the monoclonal 2H06 ACPA on its membrane, were labelled with red PKH-26 (fluorescent lipid marker), then placed in the presence of macrophages at a ratio of 2 Nalm6 target cells to 1 macrophage. After 2h at 37° C., the macrophages were detached then the cell populations analysed by flow cytometry. The results in
The synthesis of 3 independent experiments (see
The macrophages were labelled with the green PKH-67 membrane fluorochrome, then armed with hybrids Fc-WT-PEG-α621-635, Fc-SDH-PEG-α621-635 Fc-GASDIE-PEG-α621-635 or Fc-GASDALIE-PEG-α621-635, in citrullinated (Cit) or non-citrullinated (Arg) form, at different concentrations (5 μg/ml; 1 μg/ml; 100 ng/ml; 50 ng/ml; 10 ng/ml; 5 ng/ml) for 60 min at 37° C. The cells of the Nalm6 B line transduced for the expression of the monoclonal 2H06 ACPA were marked with red PKH-26 (fluorescent lipid marker) then placed in the presence of armed macrophages at a ratio of 1 Nalm6 target cell to 1 macrophage. After 2 h at 37° C., the macrophages were detached, then the cell populations analysed by flow cytometry. The results presented in
To confirm that the destruction of 2H06 Nalm6 B cells results from a phagocytosis process, 2H06 Nalm6 B cells were labelled with a pH-sensitive dye, pHRodoSE, that exhibits weak fluorescence intensity at neutral pH but emits strong fluorescence in an acidic medium. However, the pH in the intra-cytoplasmic vesicles (lysosomes) in which the phagocytosed elements are found is acidic. Non-phagocytosed cells are therefore not detectable, whereas phagocytosed cells, internalised in the lysosomal compartment, become fluorescent.
Thus, 2H06 Nalm6 cells were labelled with pHRodoSE (Thermofisher) at 20 ng/ml. The labelled Nalm6 cells were incubated with macrophages armed or not with Fc-WT-PEG-621-635 Cit or Arg hybrids, at 5 μg/ml for 1h at 37° C., treated or not with cytochalasin D for 30 min at 2 μg/ml then 1 μg/ml during phagocytosis. The cells were incubated for 2 h at 37° C., then the macrophages were separated and the cell populations analysed by flow cytometry or stained with May Grunwald Giemsa (MGG) for morphological analysis by optical microscopy. The results presented in
The cells of the 2H06 Nalm6 B line were labelled with the green PKH-67 membrane fluorochrome and the macrophages with the red PKH-26. The Fc-WT-PEG-α621-635 and Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, were either incubated for 1h at 37° C., with the macrophages at concentration of 5 μg/ml (arming followed by washing), before bringing them into contact with the target cells at a ratio of 1 Nalm6 target cell to 1 macrophage, for 2 h at 37° C., or incubated at the same concentration in the medium containing Nalm6 target cells and macrophages at the same 1/1 ratio, for 2 h at 37° C. The macrophages were then separated and the cell populations analysed by flow cytometry.
The results presented in
The cells of the 2H06 Nalm6 line were labelled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) then incubated with macrophages pre-armed with hybrids Fc-WT-PEG-α621-635 and Fc-GASDIE-PEG-α621-635 in citrullinated (Cit) or non-citrullinated (Arg) form, by incubation for 1h with the hybrids at a concentration of 1 mg/ml then washing. The phagocytosis experiment took place over 2 h at 37° C., in the presence or absence of competing human IgGs at a physiological concentration of 10 mg/ml. The macrophages were detached, then labelled with an anti-CD11b BV421 antibody and the cell populations were analysed by flow cytometry The results presented in
Cells of the 2H06 Nalm6 line were labelled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) then incubated with macrophages in the presence of Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids under citrullinated (cit) or non-citrullinated (arg) form, at a concentration of 10 μg/ml. The experiment took place over 2 h at 37° C., in the presence or absence of competing human IgGs at a concentration of 10 mg/ml or of a mixture of human serum having the same IgG concentration. The concentration ratio here between hybrids and IgG is therefore 1/1000. The cells were removed and marked with an anti-CD11b BV421 antibody in order to identify the macrophages then analysed by flow cytometry.
The results presented in
The cells of the 2H06 Nalm6 line were labelled with the fluorescent dye CFSE (CarboxyFluorescein Succinimidyl Ester) then mixed with non-transduced (NT) Nalm6 cells at a ratio of ¼ (1 transduced cell to 4 non-transduced cells). The macrophages were pre-armed with the Fc-WT-PEG-α621-635 or Fc-GASDIE-PEG-α621-635 hybrids, in citrullinated (Cit) or non-citrullinated (Arg) form, by incubation for 1h at a concentration of 1 g/ml at 37° C. The Nalm6 cells were then placed in the presence of the macrophages for 2 h at 37° C., then the macrophages were detached, the total cell population sampled and marked with an anti-CD19 APC antibody, in order to discriminate 2H06 Nalm6 cells and non-transduced NT Nalm6 cells. The total cell population was then analysed by flow cytometry.
To confirm the specificity of phagocytosis for ACPA+ cells in the presence of citrullinated hybrids, we co-incubated 2H06 Nalm6 target cells (ACPA+) and non-transduced NT Nalm6 cells (ACPA−). The results presented in
The membrane expression of 2H06 ACPA on the 2H06 Nalm6 target cells was checked by labelling with an anti-Fab antibody and the lytic activity (ADCC) of the effector human NK cells verified in vitro. The 2H06 Nalm6 target cells were labelled with the CTV fluorescent marker (CellTrace Violet) at a high concentration (5 μM) whereas non-transduced NT Nalm6 cells were labelled with the same marker but at a ten times lower concentration (0.5 μM). In flow cytometry, strongly labelled cells (2H06 Nalm6 CTVhigh) can thus be distinguished from weakly labelled cells (NT Nalm6 CTVlow). SCID/BEIGE immunodeficient mice received 50 μg of anti-FcR antibodies intraperitoneally (IP) in order to block the FcRs of murine peritoneal macrophages then, 30 minutes later, the 2H06 Nalm6 target cells and non-transduced NT Nalm6 cells were injected IP, in the presence or absence of freshly prepared human NK cells and Fc-WT-PEG-621-635 hybrids either citrullinated (cit) or not (arg).
Four groups of 7 SCID/BEIGE mice received by intraperitoneal injection (IP) the following combinations of cells and hybrids:
Five hours after the injection, the cells were recovered by peritoneal lavage with PBS 2% FCS then analysed by flow cytometry after labelling with 7AAD and Annexin (Becton Dickinson kit). The results presented in
This very significant result (*** p<0.001) provides in vivo proof of the efficacy and specificity of ADCC lysis of ACPA+ cells by human NK cells in the presence of Fc-citrullinated peptide hybrids carrying ACPA target epitopes.
The membrane expression of 2H06 ACPA on the 2H06 Nalm6 target cells was checked by labelling with an anti-Fab antibody. The purification of monocytes into macrophages was carried out using peripheral blood from a healthy donor. The differentiation of the monocytes was induced in the presence of 100 ng/ml of M-CSF and the phagocytic activity of the macrophages thus generated was verified in vitro.
Before intraperitoneal (IP) injection, the macrophages were armed with the various citrullinated (cit) or not (arg) Fc-WT-PEG-α621-635 hybrids and the 2H06 Nalm6 target cells were labelled with the fluorescent marker CTV (CellTrace Violet) at 0.5 μM: strongly fluorescent when they are free (2H06 Nalm6 CTVhigh), these cells show a much weaker fluorescence (Nalm6-2H06 CTVlow) after phagocytosis by macrophages.
Four groups of 5 SCID/BEIGE mice received the target cells by IP injection, in the presence or absence of fresh pre-armed macrophages and the presence or absence of FcWT-PEG-α621-635 cit or arg hybrids, according to the following combinations:
Two hours after the injection, the cells were recovered by peritoneal lavage with PBS 5% FCS, the erythrocytes were removed by gentle lysis and the cells were labelled with an anti-human CD45-FITC antibody then with 7AAD (Becton Dickinson kit) before flow cytometry analysis.
The results presented in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2102805 | Mar 2021 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2022/050506 | 3/18/2022 | WO |
