WO2018109213A1

WO2018109213A1 - Aptamers directed against a kappa light chain-containing protein and uses thereof

Info

Publication number: WO2018109213A1
Application number: PCT/EP2017/083160
Authority: WO
Inventors: Gérald PERRET
Original assignee: Laboratoire Français Du Fractionnement Et Des Biotechnologies
Priority date: 2016-12-16
Filing date: 2017-12-15
Publication date: 2018-06-21

Abstract

The invention relates to aptamers which specifically bind to a kappa light chain-containing protein and their use in the purification, the removal or detection of said protein.

Description

APTAMERS DIRECTED AGAINST A KAPPA LIGHT CHAIN- CONTAINING

PROTEIN AND USES THEREOF

FIELD OF THE INVENTION

The invention relates to affinity ligands which specifically bind to kappa light chain- containing protein, such as kappa light chain-containing immunoglobulins, and their use in protein purification.

BACKGROUND OF THE INVENTION

Immunoglobulins, also known as antibodies, are Y-shaped proteins which play an important role in the immune system by recognizing and neutralizing pathogens. In most mammals, including human and mice, the structure of immunoglobulins is a tetramer. Said tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa).

In the case of human immunoglobulins, light chains are classified as kappa and lambda. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The enzymatic cleavage of immunoglobulin tetramer with papain enables to separate the Fab (fragment-antigen binding) fragments from the Fc (fragment constant) part of the protein. The Fab fragments contain the variable domains responsible for the antibody specificity.

In healthy human adults, IgG constitutes approximately 75% of total serum immunoglobulins. Human IgG has been subdivided into four subclasses on the basis of unique antigenic determinants. Relative subclass percentages of total IgG in serum are IgGl, 50-70%; IgG2, 20-40%; IgG3, 2-10%; and IgG4, 1-8%.

Immunoglobulins, in particular of IgG isotype, are widely studied for applications as therapeutic drugs, diagnostic reagents, and test reagents. Immunoglobulins are typically used in biochemical research and in the field of diagnosis as affinity ligands. As of today, monoclonal antibodies are more and more used as active ingredients in the treatments of cancers and autoimmune diseases or to prevent graft rejections. Antibodies can be also used as carriers for toxins, drugs, enzymes or radionuclides in therapies such as radioimmunotherapy, antibody-directed enzyme prodrug therapy (ADEPT) or antibody-drug (ADC) therapy. Monoclonal therapeutic antibodies are recombinantly produced in recombinant host cells or in transgenic non-human animals.

Since many of the monoclonal antibody therapies require high doses and/or continued administration, economical and quality-controlled large-scale production of these antibodies is of great importance. During purification of therapeutic antibodies, contaminants including host cell proteins, DNA, antibody variants, and small molecules, must be removed.

The common procedure used in purification of antibodies is protein A affinity chromatography because it efficiently and selectively binds to antibodies in complex solutions, such as harvested cell culture media. Protein A, which is a natural product of Staphylococcus aureus, binds to the Fc portion of a variety of mammalian IgG molecules. The main disadvantages of protein A chromatography include cost, quality control difficulties, resin stability, and acidic elution procedures which can impair the antibody's conformation and activity. Moreover, protein A is obtained from genetically modified bacteria through complex and expensive procedures explaining why protein A resin is over 30 times more expensive than ion exchange resin chromatography, and may account for >35 of the total raw material costs for large scale recovery of IgG. Also, since protein A molecules may cause immunogenic or other physiological responses in humans, any contaminating ligand leaked from the base matrix must be removed during processing. At last, protein A can cross react with IgGs from different species, which is a major drawback when the therapeutic antibody is expressed in a transgenic body fluid such as plasma which contains endogenous non-human IgG.

To overcome these disadvantages, several synthetic ligands have been proposed as replacements for protein A in the affinity purification of antibodies; these include the use of a thiophilic ligand, histidyl ligand, Avid Al, or peptides or nonpeptides designed to mimic protein A. However, none of these have become protein A alternatives at the industrial manufacturing level yet.

The production of IgG in body fluids of transgenic animals and its subsequent purification has been described, for instance in PCT applications W09517085 and W09419935. However, the IgG purification from body fluids is still a real challenge because the final product must be devoid of any non-human contaminating proteins, in particular non-human immunoglobulins, which may be antigenic.

Affinity media such as KappaSelect (GE Healthcare) have been developed for the purification of human Fab Kappa fragments using a recombinant protein produced in S. cerevisiae with affinity for the constant domain of immunoglobulin kappa light chain as a ligand. However these affinity media are expensive and thus cannot be used at industrial scale.

There is thus a need for alternative methods for the purification for immunoglobulins, in particular for monoclonal recombinant IgG, and which can be used at industrial scale.

SUMMARY OF THE INVENTION

The invention relates to an aptamer which specifically binds to a kappa light chain-containing protein. In some embodiments, the aptamer comprises a polynucleotide having at least 70% of sequence identity with SEQ ID NO: l. Said aptamer may comprise a polynucleotide which differs from the polynucleotide of SEQ ID NO: l in virtue of 1 to 14 nucleotide modifications. In some other embodiments, the aptamer is of formula (I) :

5'-[NUCl]_m-[Central]-[NUC2]n-3' (I), wherein

n and m are integers independently selected from 0 and 1,

- [NUC1] is a polynucleotide comprising from 2 to 40 nucleotides, preferably from 15 to 25 nucleotides,

[NUC2] is a polynucleotide comprising from 2 to 40 nucleotides, preferably from 15 to 25 nucleotides and

[Central] is a polynucleotide having at least 70%, preferably at least 80% of sequence identity with SEQ ID NO: 12.

For instance, the aptamer of the invention may have a nucleotide sequence selected from the group of SEQ ID NO: 1-11.

In some embodiments, the aptamer of the invention binds to a human kappa light chain- containing immunoglobulin. In some additional embodiments, the aptamer of the invention does not bind to a non-human immunoglobulin, such as a bovine immunoglobulin.

The invention also relates to an affinity ligand capable of specifically binding to a kappa light chain-containing immunoglobulin, which comprises an aptamer as defined above and at least one moiety selected from a mean for detection and a mean for immobilization onto a support. An object of the invention is also a solid affinity support comprising thereon a plurality of affinity ligands or a plurality of aptamers as defined above.

The aptamer, the affinity ligand or the affinity support of the invention can be used in the purification, the removal or the detection of a kappa light chain-containing protein. Said kappa light chain-containing protein may be, for instance, a full-length immunoglobulin, a ScFv, a Fab, a (Fab')2, or a Fc-fused ScFv. The invention also relates to a method for purifying a kappa light chain-containing protein from a starting sample comprising the steps of :

a) contacting said starting sample with an affinity support as defined above, in conditions suitable to form a complex between (i) the aptamers or the affinity ligands immobilized on said support and (ii) the kappa light chain-containing protein b) releasing the kappa light chain-containing protein from said complex, and

c) recovering the purified kappa light chain-containing protein.

In some embodiments, the kappa light chain-containing protein is a recombinant human kappa light chain-containing immunoglobulin. The starting sample may be obtained from a non-human animal transgenic for said human kappa light chain-containing protein. For instance, the starting composition may be a body fluid from a transgenic non-human animal or a derivative thereof.

A further object of the invention is a method for preparing a pharmaceutical composition comprising a purified kappa light chain-containing protein comprising the steps of :

a. Purifying a kappa light chain-containing protein from a starting sample as defined above,

b. Mixing the purified kappa light chain-containing protein with one or several pharmaceutically acceptable excipients. BRIEF DESCRIPTION OF THE FIGURES

Figure 1A shows the binding curves of human plasma derived kappa IgG and plasma derived human lambda IgG on aptamer of SEQ NO: 11 immobilized on a sensor chip, obtained by SPR technology. IgGs (1000 nM or 500 nM) were injected with a buffer comprising 50mM MES, 5mM MgCl₂, 150mM NaCl and having a pH of 5.5. A complex between kappa IgG and the aptamer was formed as evidenced by the increase of the signal. By contrast, lambda IgG did not bind to the aptamer. The injection of a buffer comprising 1 M NaCl did not induce the elution of kappa IgG, which show that the complex between the kappa IgG and the aptamer was stable, even in the presence of high salt concentration. X-axis: time in s. Y-axis: SPR response in arbitrary scale

Figure IB shows the binding curves of several IgGs on aptamer of SEQ NO: 11 immobilized on a sensor chip, obtained by SPR technology. The tested IgGs were (i) Ublituximab, a chimeric anti-hCD20 IgGl with kappa light chains, (ii) Rituximab, another chimeric anti- hCD20 IgGl with kappa light chains and a chimeric anti-CD71 IgGl with lambda light chains. IgGs (0.5 μΜ) were injected with a buffer comprising 50 mM MES, 5mM MgCl₂, 150 mM NaCl and having a pH of 5.5. Rituximab and Ublituximab bound to the immobilized aptamer. In contrast, no significant binding was observed for the lambda light chain- containing anti-CD71 IgGl. X-axis: time in s. Y-axis: SPR response in arbitrary scale

Figure 2A shows the alignments of the different tested trimmed sequences derived from the aptamer of SEQ ID NO: 11 (S 16-28).

Figure 2B shows the competitive binding of immobilized aptamer of SEQ ID NO: 11 to kappa IgGs in the presence of trimmed polynucleotides derived from SEQ ID NO: 11, by SPR. The trimmed sequences S16-28.1, S16-28.2 and S16-28.3 (SEQ ID NO: 15-17) showed a low competitive binding. By contrast, aptamers S 16-28.6 and S 16-28.7 (SEQ ID NO:6-7) significantly inhibited the binding of the kappa IgG onto the immobilized aptamer of SEQ ID NO:l l. These aptamers comprise a polynucleotide of SEQ ID NO: l. X-axis: time in s. Y- axis: SPR response in arbitrary scale.

Figure 3A shows the dose-dependent binding of kappa light chain IgG Ublituximab to immobilized aptamer of SEQ ID NO: 1 on a sensor chip by SPR. Ublituximab were injected at different concentration with a buffer comprising 50mM MES, 5mM MgCl₂, 150mM NaCl and having a pH of 5.5. A complex between Ublituximab and the aptamer of SEQ ID NO: l was formed as evidenced by the increase of the signal. Higher the concentration of Ublituximab, higher the detected signal. X-axis: time in s. Y-axis: SPR response in arbitrary scale.

Figure 3B shows the binding curves of human kappa IgGs and bovine IgGs on aptamer of SEQ NO: l immobilized on a sensor chip, obtained by SPR technology. The bovine IgGs contained both kappa light chain IgGs and lambda light chain IgGs. The tested IgGs were injected at different concentrations with a buffer comprising 50mM MES, 5mM MgCl₂, 150mM NaCl and having a pH of 5.5. Human kappa IgGs bound to the immobilized aptamer. By contrast, no significant binding was observed for the bovine IgGs, which shows that the aptamer of SEQ ID NO: l is specific to human kappa IgGs as compared to bovine IgGs. X- axis: time in s. Y-axis: SPR response in arbitrary scale

DETAILED DESCRIPTION OF THE INVENTION

The Applicant performed his own research and identified a new family of aptamers which specifically bind to kappa light chain from immunoglobulin, and proteins containing said kappa light chain. This new family of aptamers were conceived by an in-house SELEX process conceived by the Applicant. These aptamers were shown to specifically bind to both human (Fab')₂ and human immunoglobulin IgG comprising kappa-light chain. Noteworthy, the aptamers were able to bind to both recombinant human IgGs and plasma human IgGs containing kappa light chain. The Applicant further showed that the aptamers had a low affinity for human immunoglobulins with lambda light chains and did not significantly cross-react with non- human immunoglobulins such as bovine immunoglobulins.

The aptamers of the invention can be used as affinity ligands in the purification, the removal or the detection of kappa light chain-containing proteins. For instance, the aptamers of the invention can be used in the purification of recombinant human kappa light chain IgGs from transgenic body fluids and derivatives thereof.

^■ Aptamers of the invention

In a first aspect, the invention relates to an aptamer which specifically binds to a kappa light chain-containing protein.

In particular, the invention relates to an aptamer which specifically binds to a kappa light chain from an immunoglobulin, preferably that of a human immunoglobulin.

As used herein, an "aptamer" (also called nucleic aptamer) refers to a synthetic single- stranded polynucleotide typically comprising from 20 to 150 nucleotides in length and able to bind with high affinity a target molecule. The aptamers are characterized by three- dimensional conformation(s) which may play a key role in their interactions with their target molecule. Accordingly, the aptamer of the invention is capable of forming a non-covalent complex with a protein containing a kappa light chain from an immunoglobulin. The interactions between an aptamer and its target molecule may include electrostatic interactions, hydrogen bonds, and aromatic stacking shape complementarity.

"An aptamer specifically binds to its target molecule" means that the aptamer displays a high affinity for the target molecule. The dissociation constant (Kd) of an aptamer for its target molecule is typically from 10^"6 to 10^"12 M. The Kd is preferably determined by surface plasmon resonance (SPR) assay in which the aptamer is immobilized on the biosensor chip and target molecule is passed over the immobilized aptamers, at a various concentrations, under flow conditions leading to measurement of K_on and K₀ff and thus Kd.

The term "specifically binding" is used herein to indicate that the aptamer has the capacity to recognize and interact specifically with its target molecule, while having relatively little detectable reactivity with other molecules which may be present in the sample. Preferably, the aptamer specifically binds to its target molecule if its affinity is significantly higher for the target molecule, as compared to other molecules, including molecules structurally close to the target molecule. For instance, an aptamer might be able to specifically bind to a human protein while displaying a lower affinity for a homolog of said human protein. As used herein, "aw aptamer displays a lower affinity for a given molecule as compared to its target molecule" or "aw aptamer is specific to its target molecule as compared to a given molecule" means that the Kd of the aptamer for said given molecule is at least 5-fold, preferably, at least 10, 20, 30, 40, 50, 100, 200, 500, or 1000-fold higher than the Kd of said aptamer for the target molecule.

The aptamers may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The aptamers can comprise one or several chemically- modified nucleotides. Chemically- modified nucleotides encompass, without being limited to, 2' -amino, or 2' fluoro nucleotides, 2'- ribopurine, phosphoramidite, locked nucleic acid (LNA), boronic acid-modified nucleotides, 5-iodo or 5-bromo-uracil, and 5-modified deoxyuridine such as benzyl-dU, isobutyl-dU, and naphtyl-dU. For 5-modified deoxyuridine, one can refer to Rohloff et al., Molecular Therapy- Nucleic acids, 2014, 3, e201 (see Figure 1 page 4), the disclosure of which being incorporated herein by reference. In certain embodiments, the aptamer may comprise a modified nucleotide at its 3 '-extremity or/and 5 '-extremity only (i.e. the first nucleotide and/or the last nucleotide of the aptamer is/are the sole chemically- modified nucleotide(s)). Preferably, said modified nucleotide may enable the grafting of the aptamer onto a solid support, or the coupling of said aptamer with any moiety of interest (e.g. useful for detection or immobilization).

Once the sequence of the aptamer is identified, the aptamer can be prepared by any routine method known by the skilled artisan, namely by chemical oligonucleotide synthesis, for instance in solid phase.

As used herein, "aw anti-kappa aptamer" , "an aptamer directed against a kappa light chain- containing protein" or "aw aptamer which specifically binds to a kappa light chain-containing protein" refers to a synthetic single- stranded polynucleotide which specifically binds to at least one protein comprising a kappa light chain from an immunoglobulin or a derivative or a fragment thereof. More precisely, an anti-kappa aptamer is an aptamer directed against a kappa light chain from an immunoglobulin and which is able to bind to a protein containing said kappa light chain or a fragment thereof. The aptamer of the invention may bind to the kappa light chain-containing protein on the kappa light chain domain of said protein or on a region of the protein involving the kappa light chain domain. In some embodiment, the aptamer of the invention is directed to a fragment of kappa light chain domain present in a protein. The fragment may refer to the constant region (CL) of the kappa light chain. In some embodiments, the aptamer of the invention is directed against an epitope present in the constant region of a kappa light chain, which is preferably human.

As used herein "a kappa light chain containing protein" refers to any protein comprising a kappa light chain from a immunoglobulin, preferably a human kappa light-chain, a variant, or a fragment of said kappa light chain. The kappa light chain-containing protein may be a naturally occurring protein, a variant of a naturally occurring protein, a fragment of a naturally occurring protein, a fusion protein or a protein conjugate. The kappa light chain- containing protein may be endogenously produced in an animal. Alternatively, the kappa light chain-containing protein is recombinantly produced. Preferably, the kappa light chain- containing protein comprises an antigen-binding domain. Said antigen-binding domain is preferably composed of the variable domain of the kappa light chain and the variable domain of a heavy chain from an immunoglobulin. The antigen of said protein can be of any type and includes, without being limited to, a membrane receptor, a cytokine, an interleukin, a hormone, a toxin, an enzyme, a co-factor enzyme, a viral or bacterial protein, a growth factor, a pathogen, a virus a plasma protein, DNA or RNA. For illustration, the antigen may be RhD, CD20, TNFcc, CD137, CD71 or CMV.

The kappa light chain-containing protein is preferably selected from the group consisting of full-length immunoglobulins, a fragment of a full-length immunoglobulin such as Fab, F(ab')2, Fab, chemically linked Fab, ScFv and fusion or conjugate proteins comprising an antigen-binding domain such as Fc-fused ScFv, di-ScFv, or tri-ScFv. In some embodiments, the kappa light chain is from a human immunoglobulin, such as a human IgG. A human immunoglobulin refers to a protein having the amino acid sequence of a human wild-type immunoglobulin, a fully-human immunoglobulin or a variant thereof, including chimeric immunoglobulin and humanized immunoglobulin. Said immunoglobulin may be a human plasma immunoglobulin, a recombinant or transgenic human immunoglobulin. For instance, an aptamer of the invention may be able to specifically bind to a human plasma kappa light chain-containing IgG and/or a recombinant human kappa light chain-containing IgG such as a fully human kappa light chain-containing IgG, a chimeric kappa light chain-containing IgG, and/or a humanized kappa light chain-containing IgG. The recombinant human IgG can be produced by a transgenic host cell or by a non-human transgenic animal. In some embodiments, the aptamers of the invention may bind to a kappa light chain-containing protein regardless the glycosylation of the kappa light chain. In some embodiments, the kappa light chain-containing protein can be a polyclonal or a monoclonal immunoglobulin. By "immunoglobulin" , "Ig" or "full-length antibody" as used herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions. "Full length antibody" covers monoclonal full-length antibodies, wild-type full-length antibodies, chimeric full-length antibodies, humanized full-length antibodies, the list not being limitative. In most mammals, including human and mice, the structure of full-length antibodies is generally a tetramer. Said tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). In the case of human immunoglobulins, light chains are classified as kappa and lambda. The kappa (κ) chain is encoded by the immunoglobulin kappa locus on chromosome 2 and the lambda (λ) chain is encoded by the immunoglobulin lambda locus on chromosome 22. The two light chains in a naturally-occurring antibody are identical. Each light chain is composed of one constant (CL) domain and one variable domain (VL) that is important for binding antigen. There are several allotypes for kappa light chain, namely Kml, Kml,2 and Km3. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgGl, IgG2, IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgMl, IgM2, IgD, and IgE. Each isotype can have either lambda light chains or kappa light chains.

As used herein, "chimeric immunoglobulin" and "humanized immunoglobulin" refer to immunoglobulins that combine regions from more than one species. "Chimeric immunoglobulins" traditionally comprises variable region(s) from a non-human animal, generally the mouse (or rat, in some cases) and the constant region(s) from a human. Humanized immunoglobulins are chimeric immunoglobulins that contain minimal sequence derived from non-human IgG. Generally, in a humanized immunoglobulin, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to a human antibody except within its CDRs. Both chimeric and humanized immunoglobulin comprises a constant domain (CL) in their light chains which is of human origin. In the context of the invention, said constant domain (CL) is from a human kappa light chain.

As used herein, a variant of a wild-type or naturally-occurring protein refers to a protein having at least 80% of sequence identity, preferably at least 85%, 90%, or 95% of sequence identity with said wild-type or naturally- occurring protein and which displays a similar biological activity or binding affinity as compared to said wild-type protein. For instance, the kappa light chain-containing protein may be a variant of a kappa chain-containing IgG which may display an increased or a decreased biological activity, for instance in terms of CDC, or ADCC, or an increased half-life as compared to the corresponding wild-type IgG.

In some embodiments, the aptamer of the invention displays a higher affinity for a human kappa light chain as compared to a non-human kappa light chain. In some embodiments, the aptamer of the invention binds to a human kappa light chain-containing protein, without binding to a protein containing a non-human kappa light chain. In particular, the aptamer of the invention may be specific to human kappa chain-containing immunoglobulins such as kappa chain-containing IgGs, as compared to non-human immunoglobulins such as a bovine immunoglobulin. In other words, the aptamer of the invention may bind to human kappa immunoglobulins, in particular to a human kappa IgG, without cross -reacting with a non- human immunoglobulin.

In some other or additional embodiments, the aptamer of the invention is specific to kappa light chain as compared to lambda light chain. For instance, the aptamer of the invention may specifically bind to an immunoglobulin with kappa light chains without significantly binding to an immunoglobulin with lambda light chains. As another example, the aptamer of the invention may be specific to a kappa light chain-containing IgG, as compared to a lambda light chain containing IgG.

In some additional embodiments, the aptamer of the invention may specifically bind to kappa light-chain immunoglobulins, preferably human kappa light-chain immunoglobulins regardless their isotypes.

In some other embodiments, the aptamer of the invention has a higher affinity to kappa light chain-containing IgGs as compared to other immunoglobulins isotypes with kappa light chains. In some other embodiments, the aptamer of the invention may show an increased affinity for a specific kappa IgG subtype, such as kappa IgGl, as compared to other kappa IgG subclasses.

In some other or additional embodiments, the aptamer of the invention may be able to bind to several human kappa light chain allotypes. In a particular embodiment, the aptamer of the invention can show a higher affinity for a specific human kappa light chain allotype, as compared to other kappa light chain allotypes.

Typically, the aptamer of the invention forms a non-covalent complex with its target protein. This non-covalent complex can be dissociated in gentle conditions. In some other or additional embodiments, the binding affinity of the aptamer for its target protein is not impaired by high ionic strength, for instance in the presence of a NaCl concentration of about 0.5 M or 1 M. As mentioned above, the Applicant identified aptamers which specifically bind to kappa light chain containing-proteins, for instance kappa light chain-containing (Fab)2' and IgG, by performing an in-house SELEX on a ssDNA library.

More precisely, the Applicant identified a new family of aptamers displaying a high affinity for kappa light chain domain and comprising a polynucleotide domain having at least 70% of sequence identity with SEQ ID NO: 1.

Thus, in a certain aspect, the invention relates to an aptamer which specifically binds to a kappa light chain-containing protein and which comprises a polynucleotide having at least 70% of sequence identity with SEQ ID NO: l.

As used herein, a sequence identity of at least 70% encompasses a sequence identity of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%. The "percentage identity" between two nucleotide sequences (A) and (B) may be determined by comparing the two sequences aligned in an optimal manner, through a window of comparison. Said alignment of sequences can be carried out by well-known methods, for instance, using the algorithm for global alignment of Needleman-Wunsch. Once alignment is obtained, the percentage of identity can be obtained by dividing the full number of identical nucleotide residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Sequence identity is typically determined using sequence analysis software. For comparing two nucleic acid sequences, one can use, for example, the tool "Emboss needle" for pairwise sequence alignment of providing by EMBL-EBI and available on www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html using default settings : (I) Matrix : DNAfull, (ii) Gap open : 10, (iii) gap extend : 0.5, (iv) output format : pair, (v) end gap penalty : false, (vi) end gap open : 10, (vii) end gap extend : 0.5.

The aptamer of the invention typically comprises from 20 to 150 nucleotides in length, preferably from 30 to 100 nucleotides in length, for instance from 25 to 100 nucleotides in length. Accordingly, the aptamer of the invention may have 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 in length. The aptamer of the invention may have from 30 to 90 nucleotides in length, for instance from 35 to 70 nucleotides in length. In some embodiments, the aptamer of the invention comprises a polynucleotide having at least 75%, preferably at least 80%, and even at least 90% of sequence identity with SEQ ID NO:l.

In some other embodiments, the aptamer of the invention comprises a polynucleotide of SEQ ID NO: 1 or a polynucleotide which differs from the polynucleotide of SEQ ID NO: 1 in virtue of 1 to 14 nucleotide modifications.

As used herein, a "nucleotide modification" refers to the deletion of a nucleotide, the insertion of a nucleotide, or the substitution of a nucleotide by another nucleotide as compared to the reference sequence. 1 to 14 nucleotide modifications encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotide modifications.

In particular, the aptamer of the invention may comprise a polynucleotide having a sequence which differs from SEQ ID NO: l in virtue of 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide modifications. Preferred nucleotide modifications are deletions and substitutions. The aptamer of the invention may comprise a polynucleotide having a sequence which differs from SEQ ID NO:l in virtue of 1 to 8 nucleotide deletions. Deletions can be present at the 3' or 5' end of SEQ ID NO: l or inside SEQ ID NO: l. For instance, the aptamer of the invention can comprise a polynucleotide which differs from the polynucleotide of SEQ ID NO: 1 in virtue of 1, 2, 3, 4, 5, 6, 7 or 8 nucleotide deletions at the 3' and/or 5' end. As illustration, the deletion(s) can occur on nucleotide position(s) selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 39, 40, 41, 42, 43, 44, 45, and 46, the numbering referring to nucleotide numbering in the polynucleotide of SEQ ID NO: l.

Alternatively or additionally, one or several nucleotide substitutions can be present. The aptamer of the invention may comprise a polynucleotide with differs from the polynucleotide of SEQ ID NO: l, in virtue of 1 to 8, for instance 1, 2, 3, 4 or 5 nucleotide substitutions. For instance, the nucleotide substitutions can be present at nucleotide position(s) selected from 2, 4, 5, 6, 15, 16, 17 and 38 the numbering referring to the nucleotide numbering in SEQ ID NO:l.

In a particular embodiment of the invention, the aptamer of the invention comprises a polynucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10 and SEQ ID NO: 11 or which differs from a sequence selected from SEQ ID NO: 1-11 in virtue of 1 to 14 nucleotide modifications. In some embodiments, the aptamer of the invention comprises a polynucleotide having a sequence selected from SEQ ID NO: l-SEQ ID NO:7 and SEQ ID NO: 11. The aptamers of the invention may also comprise primers at its 3'- and 5'- terminus useful for its amplification by PCR. In some embodiments, these primer sequences can be included or partially included in the core sequence and thus participate in binding interactions with kappa light chain domain of the target protein. In some other embodiments, these primer sequences are outside the core sequence and may not play any role in the interaction of the aptamer with kappa light chain domain. In some further embodiments, the aptamer is devoid of primer sequences.

In a certain aspect, the aptamer of the invention is of formula (I) :

5'-[NUCl]_m-[Central]-[NUC2]n-3' (I), wherein n and m are integers independently selected from 0 and 1,

[NUC1] is a polynucleotide comprising from 2 to 40 nucleotides, preferably from 15 to 25 nucleotides,

When n=m=0, [NUC1] and [NUC2] are absent and the aptamer consists of the sequence

[CENTRAL].

When n=0 and m=l, [NUC2] is absent and [NUC1] is present, the aptamer is thus of formula (la): 5'-[NUCl]-[CENTRAL]-3'.

When n=l and m=0, [NUC1] is absent and [NUC2] is present, the aptamer is thus of formula (lb): 5'-[CENTRAL]-[NUC2]-3'.

In some embodiments, [NUC1] comprises, or consists of, a polynucleotide of SEQ ID N°13 or a polynucleotide which differs from SEQ ID N°13 in virtue of 1, 2, 3, or 4 nucleotide modifications.

In some other or additional embodiments, [NUC2] comprises, or consists of, a polynucleotide of SEQ ID N°14 or a polynucleotide which differs from SEQ ID N°14 in virtue of 1, 2, 3, or 4 nucleotide modifications.

It goes without saying that the aptamer of formula (I) preferably comprises a polynucleotide domain having at least 70% of sequence identity with SEQ ID NO: l.

^■ Affinity ligands and affinity supports of the invention The invention also relates to affinity ligands comprising an aptamer directed against a kappa light chain containing-protein. Said affinity ligands may be immobilized onto a solid support for the detection, the quantification, or the purification of a kappa light chain containing- protein. Alternatively or additionally, the affinity ligands may comprise a mean of detection. A mean of detection may be any compound generating a signal quantifiable, preferably by instrumented reading. Suitable detectable labels may be selected, for example, from the group consisting of colloidal metals such as gold or silver; non-metallic colloids such as colloidal selenium, tellurium or sulphur particles; fluorescent, luminescent and chemiluminescent dyes, fluorescent proteins such as GFP, magnetic particles, radioactive elements, and enzymes such as horseradish peroxidase.

Typically, the affinity ligand of the invention comprises (i) an aptamer moiety, i.e. an aptamer which specifically binds to a kappa light chain-containing protein, as defined above, linked to at least one (ii) non-aptamer entity, which can be a mean of detection or a mean useful for immobilization on an appropriate substrate. Preferably, the non-aptamer entity is linked to the 5'- or the 3 '-end of the aptamer.

In certain embodiments, the affinity ligand may comprise a mean for immobilization linked to the aptamer moiety directly or by a spacer group, preferably at its 3' or 5' end. Accordingly, the affinity ligand may comprise, or consist of, a compound of formula (Ii):

[IMM] -([SPACER] )_p- [APTAMER] wherein

- [APTAMER] denotes an aptamer as defined above,

[SPACER] is a spacer group,

[EVIM] is a moiety for the immobilization of the aptamer onto a support and p is 0 or 1.

p is 0 means that the spacer is absent and that [EVIM] is directly linked to [APTAMER], preferably at the 3' or the 5 '-end of aptamer.

p is 1 means that the spacer is present and links to [EVIM] and [APTAMER] .

The spacer group is typically selected to decrease the steric hindrance of the aptamer moiety and improve its accessibility while preserving the aptamer capability of specifically binding to kappa light chain domain. The spacer group may be of any type. The spacer may be a non- specific single-stranded nucleotide, i.e. which is not able to bind to a protein, including a kappa light chain domain. Typically the spacer may comprise from 2 to 20 nucleotides in length. Examples of appropriate nucleic spacers are polyA and polyT. In some other embodiments, the spacer may be a non-nucleic chemical entity. For instance, the spacer may be selected from the group consisting of a peptide, a polypeptide, an oligo- or polysaccharide, a hydrocarbon chain optionally interrupted by one or several heteroatoms and optionally substituted by one or several substituents such as hydroxyl, halogens, or C1-C3 alkyl ; polymers including homopolymers, copolymers and block polymers, and combinations thereof. For instance, the spacer may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) or polypropylene glycol, polyvinyl alcool, polyacrylate, polymethacrylate, polysilicone, and combination thereof. For instance, the spacer may comprise several hydrocarbon chains, oligomers or polymers linked by any appropriate group, such as a heteroatom, preferably -O- or -S-, -NHC(O)-, -OC(O)-, -NH-, -NH-CO-NH-, -0- CO-NH-, phosphodiester or phosphorothioate. Such spacer chains may comprise from 2 to 200 carbon atoms, such as from 5 to 50 carbon atoms. Preferably, the spacer is selected from non-specific oligonucleotides, hydrocarbon chains, polyethers, in particular polyethylene glycol and combinations thereof.

For instance, the spacer comprises at least one polyethylene glycol moiety comprising from 2 to 20 monomers. For illustration, the spacer may comprise from 1 to 4 triethylene glycol blocks linked together by appropriate linkers. For example, the spacer may be a C₁₂ hydrophilic triethylene glycol ethylamine derivative. Alternatively, the spacer may be a C₂-

C20 hydrocarbon chain, in particular a C2-C20 alkyl chain such as a C12 methylene chain.

The spacer is preferably linked to the 3 '-end or the 5-end of the aptamer moiety, preferably linked to the 5 '-end of the aptamer moiety.

[EVIM] refers to any suitable moiety enabling to immobilize the affinity ligand onto a substrate, preferably a solid support. [IMM] depends on the type of interactions sought to immobilize the affinity ligand on the substrate.

For instance, the affinity ligand may be immobilized thanks to specific non-covalent interactions including hydrogen bonds electrostatic forces or Van der Waals forces. For example, the immobilization of the affinity ligand onto the support may rely ligand/anti- ligand couples (e.g. antibody/antigen such as biotin/anti-biotin antibody and digoxygenine/anti-digoxigenin antibody, or ligand/receptor) and protein binding tags. A multitude of protein tags are well-known by the skilled person and include, for example, biotin (for binding to streptavidin or avidin derivatives), glutathione (for binding to proteins or other substances linked to glutathione-S-transferase), maltose (for binding to proteins or other substances linked to maltose binding protein), lectins (for binding to sugar moieties), c- myc tag, hemaglutinin antigen (HA) tag, thioredoxin tag, FLAG tag, polyArg tag, polyHis tag, Strep-tag, chitin-binding domain, cellulose-binding domain, and the like. In some embodiments, [EVIM] denotes biotin. Accordingly, the affinity ligand of the invention is suitable to be immobilized on supports grafted with avidin or streptavidin.

Alternatively, the affinity ligand may be suitable for covalent grafting on a solid support. [EVIM] may thus refer to a chemical entity comprising a reactive chemical group. The chemical entity has typically a molecular weight below than 1000 g.mol^"1, preferably less than 800 g.mol^"1 such as less than 700, 600, 500 or 400 g.mol^"1. The reactive groups can be of any type and encompasses primary amine, maleimide group, sulfhydryl group, and the likes.

For instance, the chemical entity may derive from SIAB compound, SMCC compound or derivatives thereof. The use of sulfo-SIAB to immobilize oligonucleotides is for instance described in Allerson et al., RNA, 2003, 9:364-374

In some embodiments, [IMM] comprises a primary amino group. For instance, [IMM] may be -NFh or a Ci-30 aminoalkyl preferably a C1-C6 aminoalkyl. An affinity ligand wherein [IMM] comprises a primary suitable group is suitable for immobilization on support having thereon activated carboxylic acid groups. Activated carboxylic acid groups encompass, without being limited to, acid chloride, mixed anhydride and ester groups. A preferred activated carboxylic acid group is N-hydroxysuccinimide ester.

As mentioned above, [IMM]-([SPACER])p is preferably links to the 3'-end or the 5'-end of the aptamer. The terminus of the aptamer moiety which is not linked to [IMM]-([SPACER])p may comprise a chemically modified nucleotide such as 2'-o-methyl or 2' fluoropyrimidine, 2'-ribopurine, phosphoramidite, an inverted nucleotide or a chemical group such as PEG or cholesterol. Such modifications may prevent the degradation, in particular the enzymatic degradation of the ligands. In other embodiments, said free terminus of the aptamer (i.e. which is not bound to [IMM] or to [SPACER]) may be linked to a mean of detection as described above.

A further object of the invention is an affinity support capable of selectively binding to kappa light chain-containing protein, which comprises thereon a plurality of affinity ligands as defined above.

The affinity ligands can be immobilized onto the solid support by non-covalent interactions or by a covalent bond(s).

In some embodiments, the affinity ligands are covalently grafted on said support. Typically, the grafting is performed by reacting the chemical reactive group present in the moiety [EVIM] of the ligand with a chemical reactive group present on the surface of the solid support.

Preferably, the chemical reactive group of the ligand is a primary amine group and that present on the solid support is an activated carboxylic acid group such as a NHS-activated carboxylic group (namely N-hydroxysuccimidyle ester). In this case, the grafting reaction can be performed at a pH lower than 6, for instance at a pH from 3.5 to 4.5 as illustrated in Example 2 and described in WO2012090183, the disclosure of which being incorporated herein by reference.

The solid support of the affinity support may be of any type and is selected depending on the contemplated use.

For instance, the solid support may be selected among plastic, metal, and inorganic support such as glass, nickel/nickel oxide, titanium, zirconia, silicon, strained silicon, polycrystalline silicon, silicon dioxide, or ceramic. The said support may be contained in a device such as microelectronic device, microfluidic device, a captor, a biosensor or a chip for instance suitable for use in SPR. Alternatively, the support may be in the form of beads, such as polymeric, metallic or magnetic beads. Such supports may be suitable for detection and diagnostic purposes.

In other embodiments, the solid support may be a polymeric gel, filter or membrane. In particular, the solid support may be composed of agarose, cross-linked agarose, cellulose or synthetic polymers such as polyacrylamide, polyethylene, polyamide, polysulfone, and derivatives thereof. Such supports may be suitable for the purification of kappa light chain- containing protein. For instance, the solid support may be a support for chromatography, in particular for liquid affinity chromatography. The affinity support of the invention may be appropriate for carrying out affinity chromatography at the industrial scale. The affinity support of the invention may thus be used as stationary phase in chromatography process, for instance, in column chromatography process or in batch chromatography process.

^■ Uses of the aptamers and affinity ligands according to the invention in protein purification and in other fields

In an additional aspect, the aptamers and the affinity ligands of the invention may be used in the diagnostic and detection field. In particular, the aptamers and the affinity ligands of the invention may be useful for the diagnostic or the prognostic of diseases or disorders associated with the presence of free kappa light chains in blood.

For instance, the aptamers or the ligands of the invention may be used in the diagnostic or the prognostic of disorders involving the presence of free light chains from immunoglobulins in blood such certain plasma cell disorders e.g. as multiple myeloma. The aptamers or the ligands of the invention may be used in the diagnostic or the prognostic of disorders wherein the plasma level of free kappa light chains is a biomarker of the occurrence or the outcome of the disorders.

In another aspect, the invention relates to a method for capturing a kappa light chain- containing protein, said method comprising:

- providing a solid support having an aptamer or an affinity ligand of the invention immobilized thereon,

contacting said solid support with a solution containing a kappa light chain-containing protein, whereby the kappa light chain-containing protein is captured by the formation of a complex between the kappa light chain-containing protein and said aptamer or said affinity ligand immobilized on the solid support.

In some embodiments, the method may comprise one or several additional steps such as: a step of releasing kappa light chain-containing protein from said complex, a step of recovering kappa light chain-containing protein from said complex a step of detecting the formation of the complex between kappa light chain-containing protein and said aptamer or affinity ligand

a step of quantifying kappa light chain-containing protein,

The detection of the complex and the quantification of kappa light chain-containing protein (or that of the complex) may be performed by any method known by the skilled artisan. For instance, the detection and the quantification may be performed by SPR as illustrated in the Examples.

Alternatively, one may use an ELISA-type assay wherein a labelled antibody directed against the target protein is used for detecting or quantifying the complex formed between said protein and the affinity ligands. Said antibody may be labelled with a fluorophore or coupled to an enzyme suitable for the detection, such as the horseradish peroxidase.

The invention also relates to a complex comprising (i) kappa light chain-containing protein and (ii) an aptamer or an affinity ligand of the invention, as described above.

In a further aspect, the invention relates to a method for removing kappa light chain- containing protein from a sample, said method comprising:

providing a solid support having an aptamer or an affinity ligand of the invention immobilized thereon,

contacting said solid support with the sample, whereby the kappa light chain-containing protein is removed from the sample, by the formation of a complex between the kappa light chain-containing protein and said aptamer or said affinity ligand immobilized on the solid support, and optionally recovering the sample deprived of the kappa light-chain containing protein. As fully illustrated in Example below, the aptamers of the invention are particularly suitable for use in the purification of proteins comprising a kappa light chain from immunoglobulins. The aptamers of the invention may be also used as an agent for removing kappa light chain- containing proteins, such as antibody fragments containing a kappa light chain.

The invention also relates to the use of an aptamer, an affinity ligand or an affinity support of the invention for purifying or removing an antibody fragment comprising a kappa light chain. Such antibody fragments may be obtained by enzymatic cleavage and include Fabs. The purification or the removal of the antibody fragment containing a kappa light chain protein is preferably performed by chromatography.

For instance, the aptamer of the invention may be used in a method of purification of a Fc fragment produced by enzymatic cleavage of an immunoglobulin, wherein said aptamer is used as an agent for removing the Fab fragments produced by the enzymatic cleavage.

In a particular embodiment, the invention relates to the use of an aptamer, an affinity ligand or an affinity support of the invention for the purification of a protein comprising a kappa light chain.

A further object of the invention is thus a method for purifying a kappa light chain-containing protein from a starting sample comprising:

a. contacting said starting sample with an affinity support as defined above, in conditions suitable to form a complex between (i) the aptamers or the affinity ligands immobilized on said support and (ii) a kappa light chain-containing protein, b. releasing kappa light chain-containing protein from said complex, and

c. recovering kappa light chain-containing protein in purified form.

A further object of the invention is a method for preparing a purified kappa light chain- containing protein composition from a starting sample comprising said protein comprising: a. contacting said starting sample with an affinity support as defined above, in conditions suitable to form a complex between (i) the aptamers or the affinity ligands immobilized on said support and (ii) kappa light chain-containing protein,

b. releasing kappa light chain-containing protein from said complex, and

c. recovering a purified kappa light chain-containing protein composition.

As used herein, the starting sample may be any composition which potentially comprises a kappa light chain-containing protein. Typically, the starting sample may be, or may derive from, a cell culture, a fermentation broth, a cell lysate, a tissue, an organ, or a body fluid. As used herein, a "starting sample derives from an entity of interest, such as milk, blood or cell culture" means that the starting sample is obtained from said entity by subjecting said entity to one or several treatment steps. For instance, the entity of interest may be subjected to one or several treatments such as cell lysis, a precipitation step such as salt precipitation, cryo-precipitation or flocculation, a filtration step such as depth filtration or ultrafiltration, centrifugation, clarification, chromatography, an extraction step such as a liquid-liquid or a solid-liquid extraction, viral inactivation, pasteurization, freezing/thawing steps and the like. The starting sample typically comprises contaminants from which a kappa light chain- containing protein is to be separated. The contaminants may be of any type and depend on the nature of the starting composition. The contaminants encompass proteins, salts, hormones, vitamins, nutriments, lipids, cell debris such as cell membrane fragments and the like.

The kappa light chain-containing protein can be of any type, as described above.

In some embodiment, the kappa light chain-containing protein is a full-length immunoglobulin, preferably containing a kappa light chain with a constant domain of human origin. Said immunoglobulin may be a human immunoglobulin, which includes a human protein occurring in blood plasma, a recombinant human immunoglobulin and variants thereof. Recombinant immunoglobulins encompass humanized immunoglobulins, chimeric immunoglobulins, full human immunoglobulins and variants thereof. A variant of a human immunoglobulin may comprise one or several amino acid mutations in Fc region, as compared to a human wild- type Fc.

In some preferred embodiments, the full-length immunoglobulin is of human IgG isotype, such as IgGl, IgG2, IgG3 and IgG4.

In some embodiments, the kappa light chain-containing protein is a naturally-occurring human immunoglobulin, which is purified from a starting sample selected from human blood and derivatives thereof.

In a preferred embodiment, the kappa light chain-containing protein is a recombinant immunoglobulin, wherein the kappa light chain preferably contains a constant region (CL) from a human immunoglobulin. As mentioned above, said recombinant immunoglobulin can be selected from humanized immunoglobulins, chimeric immunoglobulins, full human immunoglobulins and variants thereof.

The recombinant immunoglobulin can be produced in a recombinant cell line, bacterium or yeast or in a non-human transgenic animal. Preferably, said immunoglobulin is produced in a non-human transgenic animal. The transgenic animal is any animal which does not endogenously express the kappa light chain-containing protein of interest and which has been genetically modified so as to express said protein of interest.

Preferably, the transgenic animal is a non-human mammal such as cow, pig, sheep, rabbit, primate, goat, mice, horse and the like.

In some embodiments, the transgenic animal is a trans-chromosomic animal. In said embodiment, the kappa light chain-containing protein is preferably a full-length immunoglobulin, such as a human IgG.

The kappa light chain-containing protein of interest is typically produced in a body fluid of said animal. Body fluids encompass, without being limited to, blood, milk, lymph, tears, and urine.

Methods for producing a transgenic animal able to secrete a protein of interest in a body fluid are well-known in the state of art. For instance, a method for producing a non-human transgenic mammal which expresses the transgenic protein of interest in milk encompass introducing a genetic construct comprising a gene coding for said protein operably linked to a promoter from a protein which is naturally secreted in milk (such as casein promoter or WHAP promoter) in an embryo of the non-human mammal. The embryo is then transferring in the uterus of a female from the same animal species and which has been hormonally prepared for pregnancy. Another method for producing human immunoglobulin within animal blood is the trans-chromosomic approach. This approach is based on the use of a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hlg) heavy-chain (H) and (Lambda or Kappa) light-chain loci. The HAC vector was introduced into mammalian primary fetal fibroblasts using a microcell- mediated chromosome transfer (MMCT) approach [Nat Biotechnol. 2002 Sep;20(9):889-94]. Accordingly, in the methods of the invention, the starting sample can be a body fluid or a derivative thereof obtained from a non-human animal transgenic, for instance trans- chromosomic, for said kappa chain-containing protein. As indicated above, "a derivative of a body fluid" refers to a composition obtained by subjecting said body fluid to one or several treatments. The treatments encompass without being limited to, precipitation step such as salt precipitation, caprylic acid or caprylate precipitation, cryo-precipitation or flocculation, filtration such as depth filtration or ultrafiltration, centrifugation, clarification, chromatography, extraction step, viral inactivation, pasteurization, freezing/thawing steps and their combinations. Derivatives of blood encompass, without being limited to, a plasma, a plasma fraction such as fraction II +III obtained by Cohn's ethanol precipitation, blood cryoprecipitate, a plasma fraction obtained by caprylic/caprylate precipitation and the likes.

Derivatives of milk encompass, without being limited to, clarified milk, defatted milk, and micelle-depleted milk.

In some embodiments, the kappa chain-containing protein to purify is a human recombinant immunoglobulin, in particular a human recombinant IgG and the starting sample comprises a non-human immunoglobulin and/or a mutant immunoglobulin as contaminants to remove. A mutant immunoglobulin refers to an immunoglobulin combining light or heavy chains of human origins with light or heavy chains endogenously produced by the transgenic animal. For instance, the starting sample may be a plasma from a non-human transgenic animal such as transgenic bovine plasma and derivatives thereof. Such starting sample may comprise (i) a recombinant human kappa light chain-containing immunoglobulin to purify and (ii) endogenous immunoglobulins and mutant immunoglobulins as contaminants to remove.

The affinity support used in the methods of the invention may be any affinity support described hereabove. Preferably, the affinity support is an affinity support for performing preparative affinity chromatography. Indeed, the methods for purifying the kappa light chain- containing protein or for preparing a purified composition of said protein are preferably based on chromatography technologies, for instance in batch mode, column mode, or Sequential Multi Column Chromatography (SMCC) mode, wherein the affinity support plays the role of the stationary phase.

In step a), an appropriate volume of the starting sample containing the kappa light chain- containing protein is contacting with an affinity support in conditions suitable to promote the specific interactions of the aptamer moieties present on the surface of the affinity support with said protein, whereby a complex is formed between said protein molecules and said aptamer moieties. In step a), the kappa light chain-containing protein is thus retained on the affinity support. The conditions of step a), in particular in terms of pH and salinity, may be selected so as to promote the binding of the kappa chain-containing protein of interest onto the affinity support while minimizing the binding of the other molecules present in the starting sample onto the affinity support.

Typically, step a) is performed in the presence of a buffer solution (called hereafter a "binding buffer"). The binding buffer can be mixed with the starting composition prior to step a) or can be added during step a). The binding buffer is typically an aqueous solution containing a buffer agent. The buffer agent may be selected so as to be compatible with the protein to purify and the affinity support. The buffer agent should also have a pH enabling the formation of the complex between the aptamers and the protein to purify.

In the present case, a buffer having a pH from 4.5 to 6.5 may be convenient. The buffering agent may be present at a concentration of about 5 mM to 500 mM.

Without to be bound by any theory, the concentration of salts may promote the formation of the complex between the protein to purify and the aptamer moieties of the solid support and/or prevent the binding of the other molecules present in the starting composition. Typically, step a) may be performed in the presence of sodium chloride, for instance at a concentration ranging from 10 mM to 500 mM.

A divalent cation, such as Mg²⁺, may be also present in the binding buffer, for instance at a concentration from 0.01 mM to 100 mM.

At the end of step a), and prior to step b), the affinity support may be washed with an appropriate washing buffer so as to remove the substances which are not specifically bound, but adsorbed onto the support. It goes without saying that the washing buffer does not significantly impair the complex between the kappa chain-containing protein and the aptamer moiety while promoting desorption of the substances which do not specifically bind to the affinity support.

Thus, in some embodiments, the method of the invention comprises a step of washing the affinity support at the end of step a) and before step b). Any conventional washing buffer, well known to those skilled in the art, may be used. In some embodiments, the washing buffer as the same composition as that of the binding buffer used in step a). In other embodiments, the washing buffer may comprise the same components, but at different concentrations, as compared to the binding buffer used in step a). In some additional or alternative embodiments, the pH of the washing buffer is the same as that of the binding buffer.

In other embodiments, the washing buffer may further comprise NaCl. The ionic strength of the washing buffer may be higher than that of the binding buffer. Indeed, the Applicants showed that, for certain aptamers of the invention, high ionic strength may not significantly impair the binding of the protein to purify to the aptamer moieties. In other words, the complex between the kappa chain-containing protein and certain aptamers of the invention may be stable, even in the presence of high ionic strength. Thus, in some embodiments, the washing solution has a ionic strength higher than that of the binding buffer used in step a). In alternate or additional embodiments, the washing buffer may comprise a concentration of NaCl of at least 100 Mm, for instance of at least 0.5 M. In some other or additional embodiments, the washing buffer may comprise at least one additional component, preferably selected among alkyl diols, in particular among ethylene glycol or propylene glycol. Indeed, for certain aptamers of the invention, the presence of alkyl diols such as ethylene glycol in the washing solution do not impair the complex between IgG and the aptamer. The washing buffer may thus comprise an alkyl diol such as ethylene glycol or propylene glycol in an amount from 1% to 70% in weight, preferably from 10% to 60% in weight, such as 50% in weight.

Step b) aims at releasing the protein from the complex formed in step a). This release may be obtained by destabilizing the complex between the protein and the aptamer moieties, i.e. by using conditions which decrease the affinity of the aptamers to the kappa light chain- containing protein of interest. Noteworthy, the complex between the aptamer moiety and the protein may be destabilized in mild conditions which are not susceptible to alter said protein. For instance, the dissociation of the complex between the aptamer and the kappa light chain- containing protein may be performed by increasing or decreasing the pH, by modulating the concentration of divalent cations, for instance by using a divalent cation chelating agent such as EDTA, and/or by modulating the ionic strength in the elution buffer as compared to the binding buffer. In an alternate or additional embodiment, a detergent agent may be present in the elution buffer to promote the dissociation of said complex.

At the end of step c), the purified kappa light chain-containing protein is typically obtained in the form of a liquid purified composition. This liquid purified composition may undergo one or several additional steps. Said liquid composition may be concentrated, and/or subjected to virus inactivation or removal, for instance by sterile filtration or by a detergent, diafiltration, formulation step with one or several pharmaceutically acceptable excipients, lyophilization, packaging, preferably under sterile conditions, and combinations thereof.

In a more general aspect, the method for purifying the kappa light chain-containing protein or the method for preparing a purified composition of said protein may comprise one or several additional steps including, without being limited to, chromatography step(s) such as exclusion chromatography, ion-exchange chromatography, multimodal chromatography, reversed-phase chromatography, hydroxyapatite chromatography, or affinity chromatography, precipitation step, one or several steps of filtration such as depth filtration, ultrafiltration, tangential ultrafiltration, nanofiltration, and reverse osmosis, clarification step, viral inactivation or removal step, sterilization, formulation, freeze-drying, packaging and combinations thereof. Said additional steps can be performed prior to or after the affinity chromatography with the affinity support according to the invention. In a particular embodiment, the method of purification or the method for preparing a purified composition of the invention comprises one or several of the following features:

the kappa light chain-containing protein is a human recombinant immunoglobulin or a variant thereof,

the starting sample is a body fluid from a non-human animal transgenic for said kappa light chain-containing protein, or a derivative thereof.

the starting sample is a blood plasma from a non-human animal transgenic for said kappa light chain-containing protein, or a derivative thereof.

the kappa light chain-containing protein is a human recombinant immunoglobulin of IgG isotype

the starting sample contains a non-human immunoglobulin as contaminant.

The invention also relates to a purified composition of the kappa chain-containing protein obtainable or obtained by the method for preparing a purified composition of said protein according to the invention.

A further object of the invention is a purified composition of a kappa light chain containing protein which comprises at least 90% by weight, preferably at least 91%, 92%, 93%, 94, 95%, 96%, 97% 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of said protein as compared to the total weight of proteins present in said composition. In some embodiments, the kappa light chain-containing protein is a human immunoglobulin, preferably a recombinant human immunoglobulin, or a variant thereof. In some embodiments, the kappa light chain-containing protein is a human immunoglobulin of IgG recombinantly produced in a recombinant host cell or in a non-human transgenic animal. The composition comprises at most 10%, preferably at most 9%, 8%, 7%, 6%, 5%, 4% 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% by weight of other proteins, in particular of non-human proteins. In some embodiments, the composition is substantially devoid of any non-human immunoglobulin, in particular non-human IgG, including non-human IgG with non-human kappa light chain.

The invention also relates to a pharmaceutical composition comprising a purified composition of a kappa light chain containing protein as defined above, in combination with one or more pharmaceutically acceptable excipients. Said pharmaceutical composition as well as the purified composition of according to the invention can be used in the treatment of several disease, depending on the biological target of the kappa light chain-containing protein. For instance, the kappa light chain-containing protein may be a chimeric anti-CD20 antibody which may be used in the treatment of leukemias or lymphomas.

^■ Method for obtaining the aptamers of the invention

The Applicants carried-out several SELEX strategies described in the prior art to identify aptamers against human IgG. None of these strategies succeeded in the identification of an aptamer against a common region of IgGs. For example, a standard SELEX performed on the Fc fragment derived from a monoclonal IgG led to the identification of aptamers against the hypervariable region of the monoclonal IgG, which was present in trace amounts in the Fc preparation.

In that context, the Applicant performed extensive researches to develop a new method for obtaining aptamers directed against "SELEX-resistant" proteins such as IgG, in particular kappa light chain-containing proteins.

The Applicant conceived a new SELEX process which enables to obtain aptamers displaying high binding affinity for "SELEX-resistant" proteins, and which may be used as affinity ligands in purification process. This new SELEX process is characterized by a selection step which is performed in conditions of pH suitable to create "positive patches" on the surface of the protein target. In other words, the process conceived by the Applicant is based on the enhancement of the local interactions between the potential aptamers and the targeted protein by promoting positive charges on a surface domain of the protein. This method can be implemented for proteins having one or several surface histidines. The pH of the selection step (.i.e. the step wherein the protein target is contacted with the candidate mixture of nucleic acids) should be selected so as to promote the protonation of at least one surface histidine of the protein target.

Accordingly, the invention also relates to a method for obtaining an aptamer which is directed against a kappa light chain from an immunoglobulin, said method comprising:

a) contacting a kappa light chain-containing protein with a candidate mixture of nucleic acids at a pH of 4.0 to 8.0 , preferably from 4.5 to 6.0,

b) recovering nucleic acids which bind to said kappa light chain-containing protein, while removing unbound nucleic acids,

c) amplifying the nucleic acids obtained in step (b) to yield to a candidate mixture of nucleic acids with increased affinity to said kappa light chain, and

d) repeated steps (a), (b), (c) until obtaining one or several aptamers directed against said kappa light chain. In step (a), the candidate mixture of nucleic acids is generally a mixture of chemically synthesized random nucleic acid. The candidate mixture may comprise from 10⁸ to 10¹⁸, typically about 10¹⁵ nucleic acids. The candidate mixture may be a mixture of DNA nucleic acids or a mixture of RNA nucleic acids. In some embodiments, the candidate mixture consists of a multitude of single- stranded DNAs (ssDNA), wherein each ssDNA comprises a central random sequence of about 20 to 100 nucleotides flanked by specific sequences of about 15 to 40 nucleotides which function as primers for PCR amplification. In some other embodiments, the candidate mixture consists of a multitude of RNA nucleic acids, wherein each RNA comprises a central random sequence of about 20 to 100 nucleotides flanked by primer sequences of about 15 to 40 nucleotides for RT-PCR amplification. In some embodiments, the candidate mixture of nucleic acids consists of unmodified nucleic acids, this means that the nucleic acids comprise naturally- occurring nucleotides only. In some other embodiments, the candidate mixture may comprise chemically-modified nucleic acids. In other words, the nucleic acids may comprise one or several chemically-modified nucleotides. In preferred embodiments, the candidate mixture consists of single- stranded DNAs.

The kappa light chain-containing protein can be of any type. Preferably, the first rounds of the method is performed with a kappa light chain-containing immunoglobulin fragment selected from F(ab')2, Fab', Fab, Fv, and ScFv, preferably F(ab')2. The following steps of the method can be performed with a full-length immunoglobulin or a mixture of full-length immunoglobulin, with kappa light chains. For instance, to obtain an aptamer able to bind to human IgG with kappa light chains, the first step of the method can be performed with a F(ab')2 from a kappa light chain human IgG and the following steps can be performed with a mixture of human IgGs with kappa light chains.

Step a) is performed in conditions favourable for the binding of the kappa light chain containing-proteins with nucleic acids having affinity for said IgG. Preferably, the pH of step a) is from 5.0 to 6.0, such as 5.1, 5.2, 5.3, 5.4 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0. An appropriate pH for step a) is for instance, 5.5 + 0.1. Such pH enables to protonate at least one surface histidine present in the kappa light chain domain of the protein.

Step (a) may be performed in a buffered aqueous solution. The buffering agent may be selected from any buffer agents enabling to obtain the desired pH, and compatible with the protein targets and the nucleic acids mixture. The buffer agent may be selected from, without being limited to, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N- morpholino)ethanesulfonic acid (MES), HEPES, Bis-TRIS, citrate and acetate. The buffering agent may be present at a concentration of about 5 mM to 1 M, for instance from 10 mM to 500 mM, for instance from lOmM to 200mM such as about 50 mM.

The binding buffer used in step (a) may further contain a divalent cation such as Mg²⁺, for instance at a concentration from 0.1 mM to 500 mM.

In some embodiments, the kappa light chain-containing protein may be present in free-state in step (a). In some other embodiments, said protein may be immobilized on a solid support in order to make easier the subsequent separation of the complex formed by the protein target with certain nucleic acids and the unbound nucleic acids in step (b). For instance, said protein may be immobilized onto magnetic beads, on solid support for chromatography such as sepharose or agarose, on microplate wells and the like. Alternatively, the kappa light chain- containing protein may be tagged with molecules useful for capturing of the complex in step (b). For instance, said protein may be biotinylated.

Step (b) aims at recovering nucleic acids which bind to the kappa light chain-containing protein in step (a), while removing unbound nucleic acids. Typically, step (b) comprises separating the complex formed in step (a) from unbound nucleotides, and then releasing the nucleic acids from the complex whereby a new mixture of nucleic acids with increased affinity to the target protein is obtained.

The separation of the complex from the unbound nucleic acids may be performed by various methods and may depend on the features of the kappa light chain-containing protein. These methods include, without being limited to, affinity chromatography, capillary electrophoresis, flow cytometry, electrophoretic mobility shift, Surface Plasmon resonance (SPR), centrifugation, ultrafiltration and the like. The skilled artisan may refer to any separation methods described in the state in the art for SELEX processes, and for instance described in Stoltenburg et al. Biomolecular Engineering, 2007, 24, 381-403, the disclosure of which being incorporating herein by reference. As illustration only, if the kappa light chain- containing protein is immobilized on a support, the separation may be performed by recovering the support, washing the support with an appropriate solution and then releasing nucleic acids from the complex immobilized on the support. If said protein has been incubated in free-state with the candidate mixture, the separation of the nucleic acid-protein complex from unbound nucleic acids can be performed by chromatography by using a stationary support able to specifically bind to said protein or the possible tag introduced on said protein, whereby the complexes are retained on the support and the unbound nucleic acids flow out. For instance, one may use a stationary phase having thereon antibodies directed against the target protein. Alternatively, the partitioning may be performed by ultrafiltration on nitrocellulose filters with appropriate molecular weight cut-offs. Once the complexes separated from unbound nucleic acids, the nucleic acids which bind to the kappa light chain-containing protein are released from the complexes. The release can be performed by denaturing treatments such as heat treatment or by elution. Preferably, said nucleic acids are recovered by using an elution buffer able to dissociate the complex. The dissociation may occur by increasing the ionic strength, by modulating the pH in the elution buffer, by introducing a divalent chelating agent such as EDTA, and/or by introducing a detergent such as urea as compared to the buffered solution used in step a). The dissociation conditions to use depend on the final binding properties which are sought for the aptamers. For instance, the elution buffer may have a pH of at least 7.0, such as about 8.0, and/or may comprise a divalent chelating agent and/or a detergent. The detergent may be urea and the chelating agent may be EDTA.

In step (c), the nucleic acids recovered in step (b) are amplified so as to generate a new mixture of nucleic acids. This new mixture is characterized by an increased affinity to the target protein as compared to the starting candidate mixture.

Step (a), (b) and (c) form together a round of selection. As indicated in step (d), this round of selection can be repeated several times, typically 6-20 times until obtaining an aptamer or a pool of aptamers directed against the target protein. It goes without saying that the step (a) of round "N" is performed with the mixture of nucleic acids obtained in step (c) of the round "N-l". At the end of each selection round, the complexity of the mixture obtained in step (c) is reduced and the enrichment in nucleic acids which specifically bind to the target protein is increased.

The conditions for implementing step (a), (b) and (c) may be the same or may be different from one round of selection to another. In particular, the conditions of step (a) (e.g. the incubation conditions of the target protein with the mixture of nucleic acids) can change. For instance, step (a) of round "N" can be performed in more drastic conditions than in round "N+l" in order to direct the selection to aptamers having the highest affinity for the target protein. Typically, such result can be obtained by increasing the ionic strength of the buffer used in step (a).

The method of the invention may comprise one or several additional steps. The method of the invention may comprise counter- selection or subtractive selection rounds. The counter- selection rounds may aim at eliminating nucleic acids which cross-react with other entities or directing the selection to aptamers binding to a specific epitope in the kappa light chain domain. For instance, when aptamers directed against a human kappa light chain, the method of the invention can comprise a step of counter- selection against a protein containing a non- human kappa light chain-containing protein, such as a bovine immunoglobulin.

The method of the invention may comprise one or several of the following steps:

a step of cloning the aptamer pool,

a step of sequencing an aptamer,

a step of producing an aptamer, for instance by chemical synthesis,

a step of identifying consensus sequences in the pool of aptamers, for instance by sequence alignment,

a step of optimizing the sequence of an aptamer,

In some embodiments, the method of the invention may comprise the following additional steps:

sequencing an aptamer obtained in step (c)

optimizing said aptamer, and

producing the optimized aptamer, preferably by chemical synthesis.

The optimization of the aptamer may comprise the determination of the core sequence of the aptamer, i.e. the determination of the minimal nucleotide moiety able to specifically bind to the kappa light chain-containing protein. Typically, truncated versions of the aptamer are prepared so as to determine the regions which are not important in the direct interaction with the target protein.

The binding capacity of the starting aptamer and the truncated versions may be assessed by any appropriate methods such as SPR.

Alternatively or additionally, the sequence of the aptamer may be subjected to mutagenesis in order to obtain aptamer mutants, for instance with improved affinity or specificity as compared to their parent aptamer. Typically one or several nucleotide modifications are introduced in the sequence of the aptamer. The resulting mutants are then tested for their ability to specifically bind to the target protein, for example by SPR or ELISA-type assay. In additional or alternate embodiments, the optimization may comprise introducing one or several chemical modifications in the aptamer. Typically, such modifications encompass replacing nucleotide(s) of the aptamer by corresponding chemically-modified nucleotides. The modifications may be performed in order to increase the stability of the aptamers or to introduce chemical moiety enabling functionalization or immobilization on a support. Further aspects and advantages of the present invention are disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application. EXAMPLES

EXAMPLE 1: Identification of aptamers by the method of the invention

1. Material and method

• Oligonucleotide library

The ssDNA library used in the SELEX process of the invention consisted of a 40-base random region flanked by two constant 23-base primer regions.

• Target proteins

F(ab')2 fragment or full-length human IgGs.

• SELEX protocol

A SELEX method was performed using as target alternatively human plasma derived IgG F(ab')2 fragment (Jackson Immuno Research) and human plasma derived whole IgG (ClairYg, LFB). The target and the oligonucleotide library were incubated within a buffer comprising 50mM MES, 5mM MgCl₂, 150mM NaCl and having a pH of 5.5. Different partitioning steps, including filtration on nitrocellulose membranes and affinity chromatography, were applied over 12 rounds. For all rounds a generic elution buffer was used comprising 50 mM Tris-HCl, 200 mM, EDTA, 7 M Urea and having a pH of 8.0. The evolved library was analysed with a NGS approach in order to identify aptamers leads.

• Specificity of the aptamers of the invention against different types of immunoglobulins

The binding capacity of the aptamers of SEQ ID NO: 1 and SEQ ID NO: 11 was tested with respect to different types of immunoglobulins, by SPR assay as described above:

Human plasma derived Kappa and Lambda Immunoglobulins separated from ClairYg® (therapeutic plasma derived IgG) by affinity chromatography on Protein L A chimeric anti-CD71 IgGl with lambda light chains produced in YB20

- A chimeric anti-hCD20 IgGl with Kappa light chains produced in YB20

(Ublituximad)

Rituximab a chimeric anti-hCD20 IgGl Kappa light chains produced in CHO

Bovine plasma derived IgGs. The S 16-28 (SEQ ID NO: 11) and Gammapta S 16-28.1 (SEQ ID NO: l) were synthetized with a biotin and immobilized at approximately 1700 RU on a Streptavidin Chip (sensor chip SA, GE healthcare Life science). All the immunoglobulins tested were diluted in a buffer comprising 50 mM MES, 5mM MgC12, 150 mM NaCl and having a pH of 5.5. The different concentrations used for the injections are mentioned in both Figures and Figures descriptions.

• Preparation of the trimmed sequences from aptamer S16-28 and assessment by SPR

SI 6-28 aptamer (SEQ ID NO: 11) and derived trimmed sequences were chemically synthetized. The SI 6-28 aptamer was biotinylated to be immobilized on a Streptavidin Chip (sensor chip SA, GE healthcare Life science). All trimmed S 16-28 derivatives sequences were obtained by the overlap of 50 nucleotides with the SI 6-28 5 '-extremity (S 16-28.1) and for each followed sequence, a step forward of 4 nucleotides by keeping the same length from the 5 'extremity to the 3 'extremity (S 16-28.10). The SI 6-28 was immobilized at approximately 1700 RU. In order to evaluate the capacity of each trimmed sequence to compete with the S16-28, the sequences were incubated independently at a concentration of 5μΜ within a solution containing Kappa IgGs at a concentration of ΙμΜ. After the incubation each mixture was injected on the SI 6-28 immobilized aptamer and the signal was recorded.

2. Results

Among all the aptamers leads identified by SELEX process, the aptamer Gammapta 16-28 (also called S 16-28) (SEQ ID NO: 11) displayed surprising and interesting properties. As shown in Figure 1A, this aptamer was able to discriminate Human Kappa and Lamba Immunoglobulins. In addition, the binding to human Kappa immunoglobulins is very strong since even a buffer comprising 1 M NaCl did not induce any dissociation. This capacity to selectively bind to Kappa immunoglobulins was checked with monoclonal antibodies (mAbs). In the Figure IB, Gammapta 16-28 bound efficiently to two different mAbs IgGl-Kappa Rituximab and Ublituximab with similar affinity. By contrast, no significant affinity was observed for an IgGl-Lambda mAb (anti-CD71). The core-sequence of Gammapta 16-28 was identified with a competitive approach. This approach, shown in Figures 2B, was based on the immobilization of the Gammapta 16-28 full length of SEQ ID NO: 11 to a Biacore chip and the injection of either Kappa Immunoglobulins or a mixture of Kappa immunoglobulins incubated with one of the oligonucleotides described in Figure 2B. The binding observed with each mixture was compared with the sample containing only Kappa Immunoglobulin. In this experiment S 16-28.6 (SEQ ID NO:6) and S 16-28.7 (SEQ ID NO:7) provided a similar competitive effect than the Gammapta S 16-28 full length. The overlap between S 16-28.6 and SI 6-28.7 was slightly decreased to generate the core sequence Gammapta S 16-28.1 (SEQ ID NO:l).

The binding of Gammapta S 16-28.1 was evaluated with Ublituximab as shown in Figure 3 A and was similar to Gammapta 16-28 full length. In the Figure 3B, the selectivity of the core sequence Gammapta S 16-28.1 of SEQ ID NO: l was assessed with the injection of high levels of bovine plasma derived IgGs containing both Kappa and Lambda IgGs. Even with the highest concentration no significant binding on Gammapta S 16-28.1 was observed with bovine IgGs. This experiment illustrated the high selectivity of Gammapta S 16-28.1 for human IgG containing kappa light chain.

Table 1 (sequences)

NO of SEQ Description

ID

1 Gammapta 16-28.1 (core sequence)

2 Gammapta 16-28.2

3 Gammapta 16-28.3

4 Aptamer SI 6-28-4

5 Aptamer S 16-28.5

6 Aptamer SI 6-28.6

7 Aptamer SI 6-28.7

8 Aptamer SI 6-28.8

9 Aptamer SI 6-28.9

10 Aptamer SI 6-28.10

11 Aptamer SI 6-28 (full length)

12 Central part of SI 6-28

13 Primer region 5'

14 Primer region 3'

15 S 16-28.1 (comparative)

16 S 16-28.2 (comparative)

17 S 16-28.3 (comparative) Example 2: Performance of the anti-IgG kappa chromatography using an aptamer resin of the invention

1. Material and methods:

The performance of the anti-IgG kappa chromatography using the aptamer resin of the invention was compared to a chromatography using the KappaSelect resin of GE healthcare. The ligand in the Kappaselect resin is based on a single-chain antibody fragment that is screened for human Ig kappa.

First the two resins (KappaSelect resin and aptamer resin) were each packed in 4mL columns having a diameter of 11 mm leading to a height of 4,2 cm. The throughput was ImL/minute (63 cm/hour).

Purified human immunoglobulins (ClairYg consisting of 66% of kappa immunoglobulins and 33% of lambda immunoglobulins) were injected into the columns and spiked with 10% (applied to the kappa immunoglobulins) of bovine IgG. The loading rate was 5mg of IgG kappa/mL of resin.

Two runs were applied for each resin.

The compositions of the buffers are given in Table 2 below :

Table 2:

Designation Composition Volume

Aptamer purification

Equilibration 150mM NaCl, 5mM MgCl, 50mM MES pH 5,5 20CV

Washing 150mM NaCl, 5mM MgCl, 50mM MES pH 5,5 10CV

Elution lOOmM Triethanoalamine, lOOmM EDTA, pHIO 6CV

Regeneration NaOH 0,1M 5CV

KappaSelect purification

Equilibration PBS pH 7,4 20CV

Washing PBS pH 7,4 10CV

Elution lOOmM glycine pH2,8 6CV

Regeneration NaOH 0,1M 5CV 2. Results:

A good reproducibility was observed for both resins (KappaSelect and aptamer resin of the invention).

A better elution was observed with the aptamer resin of the invention compared to the KappaSelect resin. This leads to an improved purification yield: 78% of the captured immunoglobulins were purified with the aptamer resin, while the purification yield with the KappaSelect resin was 57%.

Thus the aptamer resin of the invention leads to a better purification yield and is particularly suitable to an industrial scale.

Claims

CLAIMS An aptamer which specifically binds to a kappa light chain-containing protein. The aptamer of claim 1, which comprises a polynucleotide having at least 70% of sequence identity with SEQ ID NO: l. The aptamer of claim 1 or 2 which comprises a polynucleotide which differs from the polynucleotide of SEQ ID NO: l in virtue of 1 to 14 nucleotide modifications. The aptamer of anyone of claims 1-3, wherein the aptamer if of formula (I) : 5'-[NUCl]m-[Central]-[NUC2]n-3' (I), wherein n and m are integers independently selected from 0 and 1,

The aptamer of any one of claims 1-4, which has a nucleotide sequence selected from the group of SEQ ID NO: 1-11.

The aptamer of any one of claims 1-5, which binds to a human kappa light chain- containing immunoglobulin.

The aptamer of claim 6, which does not bind to a non-human immunoglobulin, preferably a bovine immunoglobulin.

An affinity ligand capable of specifically binding kappa light chain-containing

immunoglobulin, which comprises an aptamer in anyone of claims 1-7 and at least one moiety selected from a mean for detection and a mean for immobilization onto a support.

9. A solid affinity support comprising thereon a plurality of affinity ligands as defined in claims 8 or a plurality of aptamers as defined in any one of claims 1-7.

10. Use of an aptamer as defined in any one of claims 1-7, the affinity ligand as defined in claims 8 or the affinity support of claim 9 in the purification, the removal, or the detection of a kappa light chain-containing protein such as full-length immunoglobulins, ScFv, Fab, (Fab')2, and Fc-fused ScFv.

11. A method for purifying a kappa light chain-containing protein from a starting sample comprising the steps of :

a) contacting said starting sample with an affinity support as defined in claim 9, in conditions suitable to form a complex between (i) the aptamers or the affinity ligands immobilized on said support and (ii) the kappa light chain-containing protein b) releasing the kappa light chain-containing protein from said complex, and

c) recovering the purified kappa light chain-containing protein.

12. The method of claim 11, wherein the kappa light chain-containing protein is a recombinant human kappa light chain-containing immunoglobulin.

13. The method of claims 11 or 12, wherein the starting sample is obtained from a non-human animal transgenic for said human kappa light chain-containing protein.

14. The method of any one of claims 11-13, wherein the starting composition is selected from a body fluid from a transgenic non-human animal and derivatives thereof.

15. A method for preparing a pharmaceutical composition comprising a purified kappa light chain-containing protein comprising the step of :

a. Purifying a kappa light chain-containing protein from a starting sample as defined in any one of claims 11-14

b. Mixing the purified kappa light chain-containing protein with one or several pharmaceutically acceptable excipients.