Patent Application
20230125698
The present invention relates to a composition comprising one or more citrullinated peptides, an apheresis column having a solid support comprising the citrullinated peptide composition and a method for treating an autoimmune disease with anti-citrullinated protein autoantibodies using the citrullinated peptide composition or the apheresis column comprising the composition.
The rheumatoid arthritis (RA) is the most common human autoimmune disease and the most common chronic inflammatory rheumatism. It affects 0.5 to 1% of the population in developed countries and is characterised by a chronic and destructive inflammation of the joints. It is accompanied by the production of anti-immunoglobulin antibodies, referred to as “rheumatoid factors”, as well as anti-citrullinated peptide/protein antibodies (ACPA). The ACPA often appear years before the beginning of the disease (Rantapää-Dahlqvist S, et al. Arthritis Rheum. 2003 October; 48(10):2741-9; Nielen M M, et al. Arthritis Rheum. 2004 February; 50(2):380-6) and are present in 70 to 80% of the established RA (De Rycke L, et al. Ann. Rheum. Dis. (2004 63:12, 1587-1593, 2004). Due to their high specificity and presence at the beginning of the disease, they have a high diagnostic value. Often associated with a more severe evolution, they have a relative prognostic value. Finally, they also have a predictive value, as they can predict the evolution of an undifferentiated arthritis to a RA. The ACPA can be detected by various ELISA tests (Enzyme-Linked ImmunoSorbent Assay: immunoenzymatic test on microtiter plate). The tests classically used in medical practice are commercial tests referred to as anti-CCP (anti-Cyclic citrullinated peptides). The test developed and used by the inventors is the AhFibA test (Anti-human Fibrinogen Antibodies), which detects the antibodies recognising citrullinated fibrinogen. The results of the AhFibA and anti-CCP tests are largely consistent but discordant in 5 to 10% of patients.
As soon as the disease is diagnosed, various more or less specific treatments are prescribed to reduce the risk of evolution to irreversible joint injuries.
After the corticosteroids, the immunosuppressive drugs have become the first-line treatment prescribed to the majority of patients. In forms resistant to these treatments, various biotherapies such as anti-TNF-alpha, anti-IL6R, anti-CD20 are instituted, sometimes successively. All of these treatments have to be continued chronically, are not always effective or are effective for only a limited time and, in addition, all have more or less serious side effects. Finally, in some patients, despite the implementation of this therapeutic arsenal, the disease resists and progresses.
Therefore, there is still a need to find new ways to treat the RA, that are still effective and do not cause disabling side effects for the patient.
In recent years, the role of ACPA in the initiation and maintenance of the arthritis, through the formation of immune complexes with the citrullinated fibrinogen present in the joints of the patients, as demonstrated by the inventors, has been widely recognised by the international community. These immune complexes induce, via various effector mechanisms, the secretion of pro-inflammatory cytokines, in particular TNF-alpha, which provoke and maintain the joint inflammation and the synthesis of the ACPA.
The inventors have identified from the citrullinated human fibrin, citrullinated peptides, referred to as immunodominant, which are the preferential targets of the ACPA and therefore capable of binding to them specifically. The inventors came up with the idea of ridding the body of the patients of the pathogenic ACPA with the help of these peptides. The inventors found that by using a column in which given citrullinated peptides are immobilised, they achieved near 100% purification rates of ACPA from patient plasma. They then designed a new device for the treatment of autoimmune diseases with anti-citrullinated protein autoantibodies such as rheumatoid arthritis.
An object of the present invention therefore relates to an apheresis column loaded with a solid support comprising a composition which comprises at least one peptide selected from the group consisting of:
whose peptide or peptides are immobilised directly or indirectly on the support.
The present invention also relates to a composition comprising at least two peptides selected from the group consisting of:
The present invention also relates to the use of the composition according to the invention in the manufacture of an apheresis column, preferably an apheresis column for the treatment of an autoimmune disease with anti-citrullinated protein autoantibodies such as the rheumatoid arthritis.
The present invention also relates to a composition according to the invention for its use in the treatment or the prevention, preferably by apheresis, of an autoimmune disease with anti-citrullinated protein autoantibodies and in particular the rheumatoid arthritis, wherein the peptide or the peptides of the composition are immobilised on a solid support.
Composition
The present invention relates to a composition comprising at least one peptide selected from the group consisting of:
The different sequences are summarised in Table 1 below.
Ac-α36-50
Ac-α36-
The composition according to the invention also includes derivatives or fragments of the peptides defined above. The derivatives of these peptides may, for example, carry modifications intended to facilitate their synthesis and/or improve their stability.
Examples of such derivatives are peptides including amino acids whose carboxyl groups are esterified or transformed to amide groups. In the case of the β60-74Cit-NH2 peptide, a derivative of the latter may be the peptide referred to as β60-74Cit of amino acid sequences SEQ ID NO: 14 in which the C-terminal citrullyl residue is not amidated.
The derivatives of the peptides according to the invention may also comprise peptides including amino acids in which an amino group is for example methylated, homocitrullinated, carbamylated or acetylated.
An example is the Ac-α36-50Cit peptide which is an N-terminal acetylated derivative of the α36-50Cit peptide. The peptides α36-50Cit or Ac-α36-50Cit can be used as a variant of each other in the present invention. However, the Ac-α36-50Cit peptide is preferred.
The composition according to the invention may comprise at least 1, 2, 3, 4 or 5 peptides selected from the group consisting of α171-185Cit, α621-635Cit, β60-74Cit-NH2 and Ac-α36-50Cit and/or α36-50Cit, or derivatives or fragments thereof.
Preferably, the composition according to the invention may comprise at least 1, 2, 3 or 4 peptides selected from the group consisting of Ac-α36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2 or derivatives or fragments thereof.
The composition according to the invention may comprise 1, 2, 3, 4 or 5 peptides selected from the group consisting of α171-185Cit, α621-635Cit β60-74Cit-NH2 and α36-50Cit and/or Ac-α36-50Cit, or derivatives or fragments thereof.
Preferably, the composition according to the invention comprises 1, 2, 3, 4 or 5 peptides selected from the group consisting of Ac-α36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2 or derivatives or fragments thereof.
More preferably, the composition according to the invention comprises the 4 peptides: Ac-α36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2.
Even more preferably, the composition according to the invention consists of Ac-α36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2.
The composition according to the invention may also consist of 1, 2, 3 or 4 peptides selected from the group consisting of Ac-α36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2 or derivatives or fragments thereof.
The composition according to the invention may further comprise the peptide, referred to as α501-515Cit, having the amino acid sequence SGIGTLDGFX1HX2HPD (SEQ ID NO: 10) wherein X1 and X2 each represent a citrullyl residue or derivatives or fragments thereof. Also, according to one embodiment, the composition comprises and/or consists of at least 1, 2, 3, 4, 5 or 6 peptides or 1, 2, 3, 4, 5 or 6 peptides selected from the group consisting of Ac-α36-50Cit and/or α36-50Cit, α171-185Cit, α621-635Ci, β60-74Cit-NH2 and α501-515Cit or derivatives or fragments thereof.
The reactivity profile of the ACPA differs between patients. Thus the inventors have shown that most of the plasmas tested were reactive to the β60-74Cit-NH2 and/or Ac-α36-50Cit peptide or peptides.
Also, according to one embodiment, the composition according to the invention comprises at least the β60-74Cit-NH2 and/or Ac-α36-50Cit peptides (or alternatively α36-50Cit).
However, there are plasmas that are not reactive to these peptides but are reactive to other peptides of the invention. It is therefore preferable to combine as many peptides as possible to cover the maximum number of types of reactivity profiles of the plasmas.
Preferably, the composition according to the invention therefore comprises at least the α171-185Cit, α621-635Cit, β60-74Cit-NH2 and Ac-α36-50Cit peptides (or alternatively α36-50Cit). More preferably it comprises the α171-185Cit, α621-635Cit β60-74Cit-NH2 and Ac-α36-50Cit peptides (or alternatively α36-50Cit) and optionally the α501-515Cit peptide.
The present invention also relates to the use of the composition as defined above in the manufacture of an apheresis column, preferably an apheresis column for the treatment or prevention of any autoimmune disease with anti-citrullinated protein autoantibodies. The autoimmune disease with anti-citrullinated protein autoantibodies can be selected from the group consisting of the syndrome of Sjögren, juvenile idiopathic arthritis and rheumatoid arthritis. The autoimmune disease with anti-citrullinated protein autoantibodies is preferably the rheumatoid arthritis.
Apheresis Column
The present invention also relates to an apheresis column loaded with a solid support comprising the composition as defined above whose peptide or peptides are immobilised directly or indirectly on the support.
The term “loaded” means that the column carries or contains the solid support in such a way that a liquid such as blood or a blood product can flow through the column in contact with the solid support.
Generally speaking, the apheresis is a technique in which blood or a blood product from a subject is circulated ex-vivo (extracorporeally) through a medical device that modifies the latter by adding, removing and/or replacing component or components before reinjecting it into the subject. More specifically, plasmapheresis is when the product treated by apheresis is plasma. In particular, plasmapheresis has been described to desensitize ABO-incompatible kidney transplant candidates; these candidates had anti-ABO antibodies that compromised the tolerance of the ABO-incompatible graft (L. Rostaing et al., Treatment of large plasma volumes using specific immunoadsorption to desensitize ABO-incompatible kidney transplant candidates, J. Nephropathology. 2016; 5(3):90-97).
Thus, the apheresis column according to the invention allows the removal of the ACPA from the blood or a blood product such as the plasma of a subject.
In a conventional apheresis method, the blood is collected directly at the level of a vein or an artery of a subject. In some embodiments, the blood is separated into one or more blood products (e.g. a solid fraction comprising the red and white blood cells, platelets and a liquid fraction such as plasma). A component (e.g. autoantibodies) is removed from one of the blood products (e.g. plasma). Eventually, the blood products are combined. The blood can then be reinjected into the vein or the artery of the subject.
The present invention relates in particular to an apheresis column loaded with a solid support comprising at least 1, at least 2, at least 3, at least 4 or at least 5 or 1, 2, 3, 4 or 5 peptide or peptides selected from the group consisting of:
or derivatives or fragments thereof,
whose peptide or peptides are immobilised directly or indirectly on the support.
The solid support of the apheresis column may also comprise the peptide, referred to as α501-515Cit, having the amino acid sequence SGIGTLDGFX1HX2HPD (SEQ ID NO: 10) wherein X1 and X2 each represent a citrullyl residue or derivatives or fragments thereof.
More preferably, the solid support of the apheresis column comprises the 4 peptides: Ac-36-50Cit, α171-185Cit, α621-635Cit and β60-74Cit-NH2.
The present invention also relates to an assembly of columns comprising at least 1, at least 2, at least 3, at least 4 or at least 5 or 1, 2, 3, 4 or 5 apheresis columns, each apheresis column loaded with a solid support comprising a peptide selected from the group consisting of:
or derivatives or fragments thereof,
whose peptide or peptides are immobilised directly or indirectly on the support.
According to an embodiment, the assembly of apheresis column comprises 4 apheresis columns, each loaded with a solid support comprising the Ac-α36-50Cit peptide, the α171-185Cit peptide, the α621-635Cit peptide and the β60-74Cit-NH2 peptide respectively. According to an embodiment, the assembly of apheresis column may further comprise the α501-515Cit peptide.
The peptides according to the invention are preferably synthetic peptides.
The peptides according to the invention can be biotinylated by methods known in the prior art. In specific embodiments, the peptide or peptides are biotinylated via a spacer group. The spacer allows the citrullinated peptide to be moved away from the solid support and facilitates its recognition by the ACPA. Any suitable spacer that allows the binding properties of the peptide according to the invention to the ACPA to be maintained can be used in the invention. In specific embodiments, the spacer is a polyethylene glycol (PEG) or an amino hexanoic acid (AHX).
The solid support for immobilising the peptides of the invention is known in the prior art. The solid support is preferably made of a material that does not activate blood cells. It is preferable to use a support treated with an anticoagulant agent, for example a heparinised support. Alternatively, the blood of the patient may be treated with an anticoagulant such as heparin prior to application to the support.
The solid support may be made of polymers such as polysaccharides, preferably of high molecular weight e.g. 100 kDa or more, such as agarose or cellulose. The polysaccharide of the support may be cross-linked or non-cross-linked. Other polymers such as carboxylated polystyrene can be used. The solid supports can be in the form of magnetic beads or glass.
The solid support can be porous or non-porous. It may be in the form of particles which may be spherical or irregular. The average particle size can range from 10 μm to 2 mm, preferably from 30 μm to 100 μm.
The support can be treated with an anticoagulant agent such as heparin.
Methods for immobilising peptides on a solid support are known. Thus a peptide according to the invention can be immobilised on the support directly or indirectly.
Advantageously, the peptide or the peptides selected from the group consisting of α171-185Cit, α621-635Cit and β60-74Cit-NH2, and optionally the α501-515Cit peptide, are immobilised directly or indirectly on the solid support by their N-terminal end and/or
Preferably, the peptides in α171-185Cit, α621-635Cit and β60-74Cit-NH2, and optionally the α501-515Cit peptide, are immobilised directly or indirectly on the solid support by their N-terminal end and the Ac-α36-50Cit peptide (or alternatively α36-50Cit) is immobilised directly or indirectly on the solid support through its C-terminal end.
The indirect immobilisation can be made by means of a suitable intermediate compound. A preferred method for indirectly immobilising a peptide is based on the interaction between the biotin and an intermediate compound such as the avidin or the streptavidin. Thus, the biotinylating of the peptide and the use of the avidin or the streptavidin immobilised on the solid support allows a reliable attachment of the peptides to the solid support. Specifically, the method may comprise providing the peptide according to the invention in biotinylated form, providing a solid support having streptavidin (or alternatively avidin) immobilised on its surface, contacting the support with an aqueous solution of biotinylated peptide or peptides according to the invention and rinsing the support with an aqueous solvent.
Also, in a preferred embodiment, the solid support then comprises the streptavidin or the avidin immobilised directly thereon. The Ac-α36-50Cit peptide or peptides (or alternatively α36-50Cit), α171-185Cit, α621-635Cit and β60-74Cit-NH2, and optionally the α501-515Cit peptide, are biotinylated and linked to the streptavidin (or the avidin). The citrullinated peptides can be biotinylated in C-terminal or in N-terminal. Preferably, the α171-185Cit, α621-635Cit and β60-74Cit-NH2 peptides, and optionally the α501-515Cit peptide, are biotinylated in N-terminal and the Ac-α36-50Cit peptide (or alternatively α36-50Cit) is biotinylated in C-terminal. The Ac-α36-50Cit peptides (or alternatively α36-50Cit), α171-185Cit, α621-635Cit and β60-74Cit-NH2, and optionally the α501-515Cit peptide, may be biotinylated via a spacer group, for example amino hexanoic acid (AHX) or alternatively PEG. In addition to the affinity between the biotin and the avidin or the streptavidin, the antibody-antigen interactions can also be used for the indirect immobilisation of peptides on a support.
Alternatively, the peptides can be immobilised directly onto a solid support using bio-conjugation techniques such as, for example, the direct immobilisation of peptides onto solid supports activated with cyanogen bromide via amino functionalities in the primary sequence of the peptide or the direct immobilisation of peptides onto solid supports containing activated epoxides via carboxyl functionalities in the primary sequence of the peptide. Alternatively, the “Click” chemistry can be used to immobilise peptides on solid supports, the peptide and the support being modified with the appropriate mutually reactive chemical functionalities (azides and alkynes). In other embodiments, the Staudinger ligation chemistry can be used to immobilise suitably modified peptides on the suitably derivatives solid supports.
Treatment Method
The present invention also relates to a composition as hereinbefore defined for use in an apheresis column, preferably an apheresis column for the treatment or the prevention of an autoimmune disease with anti-citrullinated protein autoantibodies.
Autoimmune disease with anti-citrullinated protein autoantibodies is a disease in which the body of a person develops antibodies against citrullinated proteins in that same person. Autoimmune disease with anti-citrullinated protein autoantibodies can be selected in particular from the group consisting of the syndrome of Sjögren, juvenile idiopathic arthritis and rheumatoid arthritis. Preferably, the autoimmune disease with anti-citrullinated protein autoantibodies is rheumatoid arthritis.
The present invention also relates to a composition as hereinbefore defined for use in the treatment or the prevention, preferably by apheresis, of an autoimmune disease with anti-citrullinated protein autoantibodies, preferably rheumatoid arthritis, wherein the peptide or the peptides of the composition are immobilised on a solid support.
Preferably, the solid support is loaded onto an apheresis column.
Preferably, the treatment or the prevention of an autoimmune disease with anti-citrullinated protein autoantibodies comprises treating in vitro the blood of a patient suffering from said autoimmune disease with an apheresis column loaded with a solid support comprising the composition as hereinbefore defined.
The present invention also relates to a composition as hereinbefore defined for use in an apheresis column, preferably an apheresis column for the treatment of rheumatoid arthritis.
The present invention also relates to the use of an apheresis column as defined above, in particular to the use of this apheresis column in the treatment or the prevention of an autoimmune disease with anti-citrullinated protein autoantibodies and more preferably rheumatoid arthritis.
The present invention also relates to a method for treatment or prevention by apheresis, in a subject requiring it in which an apheresis column as defined above is used.
The present invention also relates to a method for purifying anti-citrullinated peptide antibodies (ACPA) from the blood or a blood product of a subject, comprising:
The subject of the invention is preferably a mammal, more preferably a human being.
In a preferred embodiment, the subject treated according to the invention is a subject with a severe form of rheumatoid arthritis. The subject may also be a subject with high titer of anti-citrullinated protein/peptide antibodies (ACPA). High titer ACPA subjects are defined as subjects with an OD≥0.9 in a dosing of the ACPA by an AhFibA test. An OD≥20.9 in AhFibA test corresponds to the 40% of rheumatoid arthritis patients with the highest rates of ACPA (
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be construed as limiting the scope of the present invention.
Materials and Methods
Serum Samples
Serum samples were obtained from 202 rheumatoid arthritis patients positive for the “AhFibA” ELISA test, which allows to detect and titer the ACPA using the citrullinated human fibrinogen as an immunosorbent.
Synthetic Peptides
Peptides corresponding to residues 36 to 50, 171 to 185 and 621 to 635 of the α-chain of the human fibrinogen (NP_068657—Isoform 2) and residues 60 to 74 of its β-chain (AAA18024) were synthesised.
The peptides were synthesised in their citrullinated form (by systematic substitution of all the arginyl residues by a citrullyl residue) or in their native non-citrullinated form (arginyl residues).
The β60-74 and β60-74Cit peptides were synthesised with a carboxamide in place of the terminal carboxyl group to give the β60-74NH2 and β60-74Cit-NH2 peptides.
The α36-50, and α36-50Cit peptides were synthesised with an acetyl on the N-terminal side to obtain the Ac-α36-50and Ac-α36-50Cit peptides.
The peptides were biotinylated at the C-terminal of an amino hexanoyl spacer for the Ac-α36-50Cit peptide and at the N-terminal of the same spacer, for the α171-185Cit, α621-635Cit and β60-74Cit-NH2 peptides.
Tests ELISA
The antigens or immunosorbents (citrullinated fibrinogen or citrullinated and non-citrullinated peptides), solubilised in PBS (1.5 mM KH2PO4 SIGMA 795488; 7 mM K2HPO4 SIGMA P3786; 0.15M NaCl SIGMA 31434; pH 7.2) at concentrations of 5 and 10 μg/mL, are adsorbed in the microtiter plate wells (MAXISORP NUNC 2023-09) by incubation overnight at 4° C., or used in avidin-biotin system: avidin 5 μg/mL in PBS incubated overnight at 4° C. and then, after washing, biotinylated peptide at 10 μg/mL in PBS, incubated 1 h at 4° C. After blocking in PBS 2% BSA (Sigma A3059) for 1 hour at 4° C. and washing in PBS 0.1% Tween®20 (SIGMA P1379), the samples to be tested are deposited diluted 1:50 (1:100 in the Avidin-Biotin system) or at equivalent corrected dilutions, in PBS 2% BSA 2MNaCl. After washing, the secondary antibody (goat anti-human IgG Fc fragment coupled to horseradish peroxidase; Southern Biotech 2040-05) is incubated for 1 hour at 4° C. After washing, the revelation is performed with a solution of Ortho-Phenylene Diamine dihydrochloride (Sigma P2788), 0.03% H2O2 (Sigma H1009), in citrate/H3PO4 buffer pH5 (0.05 M citric acid Sigma C0759; 0.1 M Na2HPO4 Prolabo 28026292) for 5 minutes at room temperature, and then stopped with a solution of H2SO4 6N (Sigma 07208). The optical density (OD) is read at 492 nm with a Multiskan Fc plate reader (ThermoScientific).
Conditions for Carrying ELISA Tests
Expression of the results of the ELISA tests: for the peptides, the variation in OD between the reactivity of the citrullinated peptide and the reactivity of the non-citrullinated peptide is taken into account, each reactivity being previously subtracted from its blank (OD in the absence of sample). This gives the following formula:
ΔDO=(DOcit−DOblank cit)−(DOnoncit−DOblank noncit) [Math 1]
Note that for the fibrinogen, the non-citrullinated form never shows any reactivity and therefore does not need to be evaluated. For the AhFibA test the OD value is therefore simply subtracted from its blank (OD in the absence of sample).
The ΔDO for the peptides and the OD for the citrullinated fibrinogen are considered to express the titer of the samples tested.
The inter-assay variations are corrected for by using a range of dilutions of a pool of patient serums tested in each microtiter plate. This reference pool, named P97, was made up of 97 samples of the same volume from 97 patients with a high ACPA titer. The OD values of this internal range are compared to that of reference values where each point in the range is the average of 30 determinations previously performed from this same pool. The least squares method then allows to obtain a correction factor which is applied to all the OD values obtained on the corresponding plate. This corrected OD corresponds to the titer of the sample.
With: X internal values, Y: reference values
Construction of Chromatography Columns
Four 1 mL HiTrap Streptavidin HP columns (GE; 29-0513-24) are independently loaded with each of the 4 biotinylated β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit and α171-185Cit peptides. After equilibration of the column in PBS, a column volume (1 mL) of saturating solution of biotinylated peptides is injected at 0.2 mL/min, incubated for 15 min and then recovered from the column and lyophilised.
The amount of peptide remaining in the lyophilized fraction is determined by high performance liquid chromatography (UPLC); this amount is subtracted from the starting amount to estimate the amount of peptide bound in the column.
Four columns were loaded with the respective amounts of peptides shown in the table above.
After use for the serial purification tests, the 4 matrices of the columns loaded with the β60-74Cit-NH2, α36Ac-50 Cit, α621-635Cit and α171-185Cit peptides were recovered and mixed and then packed into an empty 10 mL column (GE, C10/10) with volume adapter (GE, AC10), to form a column loaded with the 4 mixed peptides of a final volume of 4 mL.
Purification
Two representative pools, P97 (previously described) and P27, a pool of 27 patient serums with an ACPA titer greater than 1.5, as well as individual serums selected on the basis of their reactivity to the 4 β60-74Cit-NH2, α36Ac-50 Cit, α621-635Cit and α171-185Cit peptides were diluted to ¼ in PBS before chromatography.
After equilibration of the columns in PBS at a flow rate of 0.5 mL/min, a volume of 6 mL of pool or serum thus diluted was injected onto the 4 columns mounted in series in the following order: column loaded with β60-74Cit-NH2 peptide, then α36Ac-50 Cit, α621-635Cit and α171-185Cit, and subsequently onto the column containing the 4 peptides. Fractions not retained were recovered. After washing in PBS, the elution of the antibodies specific for the 4 peptides was made with a 0.2M pH3 glycine-HCl buffer (Invitrogen; 15527-013). The eluted fractions were recovered and brought to pH 7 with a 2M Tris solution (Euromedex; 77-86-1). Starting serums, non-retained fractions and eluates were then tested in ELISA after correction of the dilution factor induced by chromatography.
Determination of the Percentage or Rate of Purification
For each pool or serum treated, the percentage of purification was calculated from the residual immunoreactivity in ACPA (AhFibA ELISA test) of the purified, non-retained fractions and a range of dilutions of the corresponding pool or starting serum.
A fictitious example of the calculation of percentage purification is shown in
Reactivity Profile of the Serum Samples
The reactivity profiles of the 202 samples of patient serums with respect to the citrullinated fibrinogen and the 4 citrullinated peptides were determined by ELISA in at least two independent assays, each being done in duplicate. For the sake of reproducibility, only the microtiter plates whose internal range required the application of a correction factor between 0.5 and 2 with respect to the reference range were considered.
Results
ELISA Reactivity Profiles of Serums from Patients with Rheumatoid Arthritis (RA) with Respect to the Citrullinated Fibrinogen (AhFibA Test) and with Respect to the 4 Immunodominant Peptides.
202 serums from RA patients were tested in AhFibA and ranked in order of increasing reactivity (OD or titer) (
The individual reactivity of the serums with respect to the 4 peptides is highly variable and allows the definition of numerous reactivity profiles. The frequency of the different profiles observed is shown in
The search for correlations between the reactivities of the serums with respect to the different peptides reveals significant correlations between the β60-74Cit-NH2, α621-635Cit and α171-185Cit peptides; these correlations reflect a certain degree of cross-reactivity between these peptides, but their relative weakness shows that each peptide has its own reactivity. However, no correlation was found between α36Ac-50Cit and the other 3 peptides. The double-entry table in
Purification of Serum Pools from RA Patients on 4 Chromatography Columns Loaded with β60-74Cit-NH2, Ac-α36-50 Cit, α621-635Cit and α171-185Cit Peptides Respectively and Mounted in Series, or on a Column Containing a Mixture of the 4 Peptides.
Two chromatography systems, shown in
The efficacy of the two systems was compared using two pools of serums from RA patients. These conditions were chosen because they are more demanding than those for testing an individual serum. Indeed, in a pool containing many serums with different reactivity profiles, the mixture of ACPA is highly polyclonal and composed of ACPA of different antigenic specificity.
A first pool, P97, consists of 97 serums with a distribution of the AhFibA titres representative of the general population of RA patients. A second pool, P27, consists of 27 serums from patients with high AhFibA titers (OD>1.5).
In addition, 10 ACPA-positive serums were selected based on their reactivity profiles with respect to the 4 β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit and α171-185Cit peptides (Table 4 below).
Ac-α36-50Cit
These serums as well as the two pools P97 and P27 were run on 4 serial columns loaded with the 4 β60-74Cit-NH2, Ac-α36-50Cit, α621-635Cit and α171-185Cit peptides respectively. For all the samples, the ACPA titer decreased sharply after passing through the columns (
An efficiency indicator of the purification was needed to compare the results between the different chromatographic methods and samples. As the optical density of a sample in ELISA is not linearly but exponentially correlated with the amount of ACPA contained in this sample, a simple ratio of optical densities between the titer of the starting serum and that of the fraction not retained on the columns could not allow to calculate a purification rate.
We therefore used a graphical method which consists of determining in the AhFibA ELISA the OD of a series of dilutions of the starting serum, allowing us to establish a reference curve. The OD of the non-retained fraction is determined in the same way, after correction for the chromatography-related dilution factor. The graphical reading on the reference curve indicates to which dilution of the starting serum the OD of the non-retained fraction corresponds. The percentage of purification calculated results from the ratio between the experimental dilution of the non-retained fraction and the corresponding dilution of the starting serum.
This graphical method of determining the purification rate is illustrated in
The purification rates calculated for the 2 pools and for the 10 individual serums after a single pass through the columns are all above 65% and 7 out of 12, including the 2 pools, are above 85%. For the serum n° 41, this rate reaches 98%.
The relationship between the ACPA titer of the starting serum and the purification rate obtained after passage through the 4 columns mounted in series was studied.
Throughout this application, various references describe the prior art to which the invention belongs. The disclosures of these references are incorporated by reference into this application.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2002992 | Mar 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057849 | 3/25/2021 | WO |
