FOCUSING METHOD FOR IMMUNIZATION AND ANTIBODY PRODUCTION

Abstract

The loss and / or dilution of one or more antibodies of interest, or part of an antibody, or defocusing, during antigen-related steps for: - immunization of B-cell stimulation, - selection of antibodies or antibody-producing cells, - isolation of antibodies or antibody-producing cells, - and antibody production, remains a limiting factor in antibody production technologies. To improve the focus of immunization and / or selection and / or isolation and / or production of one or more monoclonal or pseudomonoclonal antibodies (different antibodies directed against the same epitope), or of any part of one or more monoclonal or pseudomonoclonal antibodies recognizing an antigen of interest, we propose a versatile immunization and selection support and a new process using this support for the production of monoclonal and pseudomonoclonal antibodies.This versatile immunization and selection support comprises an SP3 diamond carbon phase that binds antigens, such that the bond between the support and the antigen is covalent, preferably a carbon-to-carbon bond between a carbon of the diamond phase and a carbon of the antigen. The diamond support is only weakly immunogenic; since the antigens are bound by a carbon-to-carbon bond (non-peptide or otherwise), the immune response is focused solely on the antigen. This support also improves antibody collection methods, particularly for phage display technologies.

Description

Antibody production and methods for developing new antibodies represent fundamental challenges in both biology and medicine. Thus, more and more biological tests involve steps using antibodies, but most innovative cancer treatments also involve antibody molecules or analogues of these molecules. In addition to these already known applications for antibodies, bacterial resistance to antibiotics opens the way to combined treatments, Antibody / Antibiotic, the action of antibodies aiming to block antibiotic resistance. All these applications require monoclonal or pseudo-monoclonal antibodies, i.e. a set of antibodies directed exactly against a given epitope with a similar affinity. The production of new antibodies is hampered by the complexity of handling antigens, whether for the immunization stage or for the antibody production and selection stages, regardless of the production method, hybridoma, phage display or other. For immunizing an animal or individual against a given epitope, large antigens generally have greater immunogenic power. However, unlike a smaller antigen, large antigens promote the production of antibodies and B lymphocytes with more varied paratopes. Indeed, the larger an antigen, the more potential epitopes it contains: the immune response may therefore not be solely focused on the desired epitope. In the worst case, paratopes produced may bind near the epitope of interest, mask it and therefore hinder the binding of antibodies directed against the epitope of interest. Conversely, the use of smaller antigens, whose hapten is centered on the epitope of the antigen, allows the production of antibodies and / or B lymphocytes that are less varied but better focused on the epitope. However, these smaller antigens are less immunogenic. To compensate for this lower immunogenicity, small antigens are often coupled to transport proteins or "carriers", responsible for promoting the immune response. However, these transport proteins represent an additional source of epitopes, which defocuses the immune response on themselves rather than on the coupled antigen. Another approach commonly used to circumvent these issues of focusing and boosting immunization is to compensate for the low immunogenicity of small antigens by co-injecting immunogenic adjuvants such as aluminum, lipid micelles, etc. However, vaccination protocols using such adjuvants can trigger unwanted immune responses such as macropathy, autoimmune diseases, significant inflammation, etc. These side effects complicate and defocus the immune response. They can therefore compromise the selection and clonal expansion of B lymphocytes expressing surface antibodies directed against the desired epitope. This defocusing is also problematic in the context of human or animal vaccination by generating undesirable immune responses, leading to the scarcity of adjuvants in vaccinations and the search for alternatives. 85 The question of focusing on the antigen also arises at the level of the selection of antibodies from a complex mixture, such as those encountered for example in extracts of activated B lymphocytes or plasma cells after immunization, or in phage banks, obtained by phage display methods. This last technique 90 consists of cloning into phagemids (vectors for bacteriophages) nucleotide sequences of heavy and light chains of antibodies derived from a mixture of B lymphocytes (activated or not by immunization) or plasma cells. Thanks to these constructions, it is possible to produce phages containing these DNA sequences and carrying on their surface the variable fragments 95 of antibodies corresponding to these sequences. The phages carrying antibodies specific to the antigen of interest are then selected and retained on the antigen, involving several cycles of screening and washing. Here again, the importance of focusing the selection on the antigen of interest is crucial. This depends on the selection method, the nature of the antigen used for screening, but also on the means of isolation. The latter includes: The support and the molecules (peptides, proteins or any other molecules) used to bind the antigen to the support. Indeed, to capture the antibodies of interest, these selection techniques require the immobilization of the antigen of interest on a support (e.g., magnetic beads). This immobilization is generally based on the strong interaction of a pair of molecules or peptide sequences, one fused to the antigen of interest, the other present on the surface of the support. Example: 6 histidines / Nickel, Streptavidin / Biotin, chitin-binding domain / Chitin.However, these molecules, as well as the support itself, also represent a potential source of parasitic epitopes, which can bias and defocus the selection by selecting antibodies against the isolation medium, rather than the antigen of interest. During selection, this defocusing can go as far as eliminating 115 the antibody targeting the antigen of interest in favor of antibodies directed against other regions of the antigen than that of the paratope or against the means of isolation. Due to the often incompressible sizes of the means of isolation such as those generally used to fix the antigen on a support (isolation or segregation) as well as the physical nature of the isolation supports 120 generally used (glass, latex, stiren...), focusing is not necessarily improved by reducing the size of the antigen, even reduced to that of a hapten (unlike immunization). Finally, since the sequences of the antibodies used in phage display are often derived from immunized lymphocytes, any defect in focusing during immunization has fatal repercussions on the phage display. To circumvent the focusing problems during the selection of the monoclonal antibody 130 resulting from the competition between, on the one hand, the epitope of the antigen, and on the other hand the other immunogenic targets present such as the rest of the antigen and the means of isolation, a commonly used approach consists on the one hand, of tightening the selection conditions by several screens comprising successive steps of affinity capture of antibodies in increasingly stringent conditions, and on the other hand by alternating different means of isolation: the antigen is then successively fixed on different supports by different peptides or molecules. These measures aim, during the screens, to eliminate antibodies directed against targets other than the antigen of interest. In addition, classic phage display requires several steps of precipitation followed by purifications and bacterial infections by phages, in order to preserve sufficient quantities of phages.Usually, isolation methods reduce the infectivity (the ability to infect and multiply in bacteria) captured there (via the antigen). Therefore, to recover the phages captured on the antigen, a step of cleavage of the antibody by a protease is usually carried out. These proteases having a relative cleavage specificity depending on the reaction conditions, they can damage the selected phages and harm the quality of the selection result and lead to a defocusing of the selection on the phages expressing the antibody of interest. Beyond the problems posed by the focus of the immune response on the epitope of the antigen and by the focus of the selection of monoclonal antibodies by the epitope, there also arises, for the phage display method, the question of a defocusing of the selection of the desired antibody at the time of cloning the coding sequences of the heavy and light chains of the antibodies in the phagemid. Indeed, the loss of the antibody directed against the desired epitope can occur even after successful immunization, during the steps of creating the library of coding sequences of the light (L) and heavy (H) chains. Namely, the reverse transcription of mRNAs into cDNA, and the cloning of the cDNAs of the light (L) and heavy (H) chains into a phagemid, thus constituting a cDNA library. Usually, the cDNAs of both the H and L chains are cloned independently so that the cDNAs of the two chains are randomly associated. This therefore creates random combinations of H and L chains within the antibodies produced on the surface of the phages during phage display. Therefore, after cloning, it is rare that the H and L chains (forming the antibody specific to the epitope of interest) are again combined together in the same phagemid during phage display. These random rematches can therefore lead to the loss of the antibody of interest. In the remainder of this document we define defocusing by the phenomena leading to the loss and / or dilution of one or more antibodies of interest or part of an antibody, in particular during the steps involving the antigen for: • stimulation or immunization, • production, • the selection, • isolation, cells or viruses producing said antibody(ies) or any part of said antibody(ies). We define focusing by the phenomena allowing the increase of the proportion of one or more antibodies of interest or part of an antibody in a mixture, through the selection, stimulation, amplification, isolation, of a clone expressing or secreting antibodies or fraction of antibodies, of cellular, viral, bacterial type, or of said antibody(ies) or fraction of antibodies. This focusing can be carried out in an organism in vivo, or outside an organism ex vivo, or even in vitro on cells or viruses. To improve the focus of immunization and / or the selection and / or the isolation and / or the production of one or more monoclonal or pseudo-monoclonal antibodies (different antibodies directed against the same epitope) or of any parts of one or more monoclonal or pseudo-monoclonal antibodies recognizing an antigen of interest, we propose a versatile immunization and selection support and a new process using this support, for the production of monoclonal and pseudo-monoclonal antibodies. 1-a) the polyvalent immunization and selection support, comprises a diamond carbon phase SP3 fixing antigens such as peptide, polysaccharide, lipid, alkaloid, nucleic acid, any combination of these compounds, or any other type of molecules capable of triggering an immune reaction or of being recognized by an antibody, so that the bond between the support and the antigen is made through a covalent bond, preferably a carbon / carbon bond between a carbon of the diamond phase and a carbon of the antigen. The diamond support is only very slightly immunogenic, the antigens being fixed by a carbon / carbon bond (non-peptidic or other), the immune response is focused on the antigen only. In certain embodiments the antigen is reduced to a hapten comprising only the motif recognized by the antibody. 1-b) In a preferred embodiment, the polyvalent immunization and selection support will be saturated by the antigen so that the entire grafting site accessible for the antigen is occupied. Indeed, as shown in Fig. 3: a nano-diamond support consisting of 2pm diamond particles functionalized by Poly A probes and deposited on a slide. After functionalization with unlabeled poly A probes, the addition of fluorescent poly A or fluorescent BSA shows that the diamond surface is saturated and that the non-specific absorption is very low (very weak fluorescent signal). On the other hand, the hybridization of a complementary fluorescent poly T strand on the grafted probes shows that the level of functionalization of the diamond particle by the poly A probe is very high, 1-c) In a preferred embodiment, the fixation of the antigens on the polyvalent diamond immunization and selection support is carried out through a hydrogenated carbon phase CHx (x=1,2,3) such that the hydrogenated carbon reacts with a carbon of the antigen adjacent to a nucleophilic function such as NH2, COOH, SH, OH (possibly electrophilic). In another embodiment, the covalent bond is carried out through a carbon SP2 on the surface of the diamond support. 1-d) In a particular embodiment, the grafting sites of the polyvalent immunization and selection support left free by the antigen may be saturated by a non-immunogenic molecule such as glycine, PEG molecules, etc. 1-e) In a particular embodiment, the polyvalent immunization and selection support is composed of or comprises diamond particles whose size is between nm and 500 pm, preferably 2 pm. The particles will preferably be hydrogenated CHx to fix the antigens in a saturating manner. 2-a) In a particular embodiment, the polyvalent immunization and selection support functionalized by an antigen is used to immunize an animal or a human by subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection and more generally by any means of administration, ingestion, injection or other means of penetration into the body. 2-b) in a particular embodiment, in the context of a subcutaneous or intramuscular injection, the injection area will be subjected to X-ray or gamma irradiation so as to generate local inflammation capable of stimulating the immune response. This irradiation will be used preferentially with a versatile immunization and selection support hydrogenated diamond CHx or possibly SP2, capable of generating free radicals likely to trigger local inflammation. 2-c) In a particular embodiment, the versatile diamond immunization and selection supports, typically particles of 10 nm to 500 pm, will be sensitive to a magnetic or electric field so that said fields exert on the particles a force capable of moving them or attracting them. In a particular embodiment, the particles constituting the support will be particles comprising a magnetic or paramagnetic core, for example a core of iron or iron oxide, nickel, boron, magnetic silica, neodymium, etc., or any combination of these elements. In certain embodiments, any combination of these elements may be directly integrated into the SP3 or SP2 crystal structure of the diamond particle. 2-d) in a particular embodiment, the vaccination will be carried out with versatile immunization and selection supports sensitive to a magnetic or electric field, such that the injection area is subjected to a field magnetic variant, possibly electric or a combination of both, so as to cause agitation of the particles generating heating capable of triggering an inflammation reaction, thus promoting the immune response. 2-e) in certain embodiments, the vaccination zone will be subjected to a constant magnetic field, possibly pulsed, so that the magnetic particles of the multipurpose immunization and selection support are maintained in the injection zone. Thus, magnetic particles may be, for example, injected into a tissue or organ, such as the spleen, bone marrow, or even a lymph node, etc., so that a magnetic field can retain said magnetic particles in the tissue or organ in order to trigger a specific immune reaction there. In certain embodiments, the particles may be guided and confined in a tissue or organ using a pulsed or continuous magnetic field. 3-a) After vaccination and the establishment of the immune response, the B lymphocytes and / or plasma cells may be recovered by any means already used in biology, such as Ficoll, sedimentation, etc. However, in a particular embodiment, the lymphocytes and / or plasma cells responding to the vaccination may be recovered using polyvalent immunization supports formed from diamond particles possibly sensitive to an electric or magnetic field and covered with the antigen linked by a covalent bond with the diamond phase. 4-a-1) Lymphocytes and plasma cells can be immortalized into hybridomas and undergo classic selection to obtain a monoclonal hybridoma. In other embodiments, the messenger RNAs of the lymphocytes and plasma cells will be extracted, reverse transcribed and cloned into phagemids, such that each phagemid comprises a random combination between a sequence of the variable heavy fragment H originating from one of the extracted lymphocytes and / or plasma cells and a sequence of the variable light fragment L originating from one of the extracted lymphocytes and plasma cells. The sequences are linked by the coding sequence of a linker peptide. Thus, after introduction of the phagemids into bacteria, a library of recombinant phages is produced such that the phages express all the combinations between the heavy and light sequences encountered between the lymphocytes and / or plasma cells of an extraction. The expression of the combined antibodies will be done on the surface of the phage capsid thanks to the fusion of the H-linker-L sequence of the recombinant antibody with all or part of the sequence of at least one protein of the phage capsid.This phage library can be screened in a traditional manner to select an antibody or can be screened in a vectorial manner using the focusing strategy described later in this document. An alternative to cloning with recombination, which risks defocusing the selection obtained after vaccination, consists of cloning the heavy chains and the light chains separately without recombination, so as to obtain two distinct phage libraries: one for the variable parts of the heavy chains and another for the parts. variables of the light chains. In certain embodiments, the variable part of the heavy chains will be cloned into phagemids such that the variable parts are linked to a linker peptide and / or to a stabilizing peptide such as the constant parts of the variable part of an antibody, for example of the IgG type. The stabilizing peptide may comprise all or part of the constant region of an antibody excluding the variable part of the antibody such that the recognition of the antigen is done by only one chain. The invention will therefore comprise a library of phages expressing on the capsid either only all or part of the antibody heavy chain H, or all or part of the antibody L chains. Said antibodies correspond, for example, to the antibodies encoded by the cDNAs derived from the messenger RNAs of an extract of Lymphocytes or plasma cells resulting from an immunization.The light or heavy chains are fused with all or part of at least one phage capsid protein and optionally present a chain-stabilizing peptide such as respectively all or part of the non-variable parts of the light or heavy chains. 4-a-2) In a preferred embodiment, two single-chain phage banks originating from: • on the one hand, phagemids comprising the sequences of the H chains fused with a linker peptide and all or part of the constant chains of an L chain itself fused with all or part of the sequence of a capsid protein of a phage, for example the P3 protein, • and on the other hand, phagemids comprising the sequences of the L chains fused with a linker peptide and all or part of the constant parts of an H chain itself fused with all or part of the sequence of a capsid protein of a phage, for example the P3 protein (the order of the fusions may vary), Such that said libraries are screened successively by the antigen of interest against which an antibody is sought. In a particular embodiment, the targeted antigen is covalently fixed on a polyvalent immunization and selection support, such as diamond particles between 10nm and 500 pm, typically 2 pm. In an even more preferred embodiment, the fixation of the antigens is obtained by direct contact of the antigens with the hydrogenated diamond particles CHx so that a carbon-carbon bond is formed between at least one carbon of the antigen and at least one carbon of the surface. After contacting the single-chain H library and the diamond particles, the particles will be isolated and screened with washes of increasing stringency.In an even more preferred embodiment, the diamond particles will be sensitive to a magnetic field so that they can be isolated using a constant or induced magnet. The authors demonstrated that bacteria can be directly infected with phages retained on the particles of a versatile diamond immunization support Figure 2. This allows the captured phages to be amplified directly from capture on the particles. This method avoids the need for protease cleavage, which simplifies the manipulation of phages after selection of a . part, by eliminating the step of separating phages from the selection medium, and thus minimizes the defocusing usually caused by protease separation. Indeed, a peptide digestion step such as protease digestion is normally necessary to separate the phage from the antibody captured by the antigen attached to the isolation medium. To achieve infection with the selected phage-antibodies, the particles having captured phages by affinity are then directly incubated with a bacterial culture in the exponential growth phase at 37°C. The passivity of the diamond as well as the absence of binding peptide guarantee the collection of highly specific phages in a single screening cycle, with rinses of increasing stringency without having to change either the selection supports or the nature of the antigen attachment to the support. The heavy chain sequences with the best affinity are then cloned into the phagemids of the single-light chain libraries, so as to obtain one library per selected heavy chain sequence. Cloning is carried out in such a way that it allows the reconstitution of an H chain, linker, L chain antibody (the sequences being continuous) and to form a fusion with a total or partial sequence of a phage capsid protein. Each library is then screened in the same way as for the heavy chain in order to determine the HL pair with the best affinity. Selection can optionally start with the L chain library. 4-a-3) In a particular embodiment, parallel screening is used. It consists of archiving a 1 / X fraction (x=2...5,...10 etc.) of the particles having captured the specific phages at each particle rinsing step, with a stringency buffer that increases during screening. The archived fraction can be stored, for example, in a well plate, but is preferably used to infect a culture of bacteria, possibly in a well plate for phage amplification. When all the stringency conditions have been tested, a standardized sample from each well is then spotted or deposited on a preferably flat solid support, so as to obtain a matrix of spots or deposits whose XY coordinates make it possible to identify the well from which the plate originates and therefore the stringency level used.The surface of the support allows the phages of the deposit to be fixed: for example, hydrogenated diamond surface or covered with hydrogenated diamond particles, glass coated with epoxy, aldehyde, kapton, or any other support capable of fixing the phages in a stable manner. After the phages have been fixed, the surface of the support is passified to neutralize the functions that have not reacted, then the matrix is ​​complexed or hybridized with an antigen solution possibly labeled with a fluorescent, colorimetric marker or for any other means of detection. The intensity of the signal, fluorescent or any other physical measurement allowing the quantification of the captured antigens such as SPR, LIBS Etc, recorded at each spot of the matrix is ​​proportional to the affinity of the antibodies or the antibody fragment present on the surface of the capsids of the phages retained on the spot.The most intense spots allow the most efficient screening steps to be determined and these steps to be selected to isolate phages expressing the molecules. greater affinity. More generally, the screening of a library expressing all or part of an antibody such as said library, single chain phage, phage display, cellular mixture, bacterial, viral or any other eukaryotic or prokaryotic cell type, expresses all or part of an antibody includes: At least one contacting of the bank and a polyvalent immunization and selection support, functionalized by an antigen of interest, At least one isolation step, polyvalent immunization and selection supports which have reacted with the elements of the bank expressing an antibody or an antibody fraction recognizing the antigen and therefore fixed on the polyvalent immunization and selection support At least one step of culturing, bacterial infection, amplification, of the bank elements expressing an antibody or an antibody fraction recognizing the antigen and fixed on the polyvalent immunization support, At least one step of depositing on a preferably flat solid support, the isolated elements of the bank so as to obtain a matrix of spots or deposits whose XY coordinates make it possible to identify the isolated elements of the bank, At least one step of contacting, complexing, hybridizing the matrix with a solution of antigens optionally labeled with at least one fluorescent, colorimetric marker, allowing detection by any detection means without labeling such as signal intensity, fluorescent or any other physical measurement allows quantification of the antigens, at the level of each spot of the matrix At least one step of nucleic acid sequencing, quantitative, semi-quantitative, of the elements isolated from the bank, of their culture, of those resulting from bacterial infections by said isolated elements. In a particular embodiment, the polyvalent immunization and selection support will comprise, in addition to the antigen, a nucleic acid tag sequence or analogue such as PNA (peptide nucleic acid) or any other analogue. After capture of the phages, the particles can then be retained on a flat surface covered with a nucleic acid complementary to the tag, so as to allow, during the deposition of each particle spot, hybridization between the tag and the sequence complementary to the tag fixed on the surface. In a particular embodiment, the surface will be covered with hydrogenated diamond allowing a carbon-carbon bond to be established between the diamond and the sequence complementary to the tag by simple contact. After deposition of a particle spot having possibly captured phages, the nucleic acids complementary to the tag which have not hybridized with the tag of a particle will possibly be neutralized by free tag sequences. 5) The phages in the wells of the storage or culture plate corresponding to the spot with the most intense signals are sequenced using vector-based methods on slides such as those marketed by Ion Torent or Illumina. For each sequenced well, the abundance of each H and L sequence in said well makes it possible to determine the antibodies or antibody fragment with the greatest abundance and therefore the greatest affinity. 6) In particular applications, a transfer by application of the spot matrix of particles having captured phages on a bacterial lawn such as those carried out in cell chips allows transfection directly by the slide spotted with the phage capture particles. 11) The identified candidate H and L sequences may be cloned into eukaryotic or prokaryotic organisms, or may be used in cell-free devices to produce the antibody. In certain embodiments, the sequences will be cloned into algae either at the nuclear level or at the chloroplast level to obtain the H, L, H and L sequences linked by a linker, possibly secreted by the algae. 10) In certain embodiments and in addition to patent No. FR 1001516, after selection the H and L chains will be produced separately, a LPXTG peptide motif (where x = one of the 20 amino acids) will be grafted at the C terminal of each of the chains. The LPXTG motif at the C terminal of the H and L chains will allow the enzymatic fusion of these chains, thanks to sortase, at the C and N' terminal of two PNAs, or any other complementary nucleic acid analogue comprising a peptide sequence of at least one glycine linked by its C terminal end to the N' end of one PNA and respectively to the C end of the other PNA. For example, the bond between the peptide sequence of at least one glycine and the PNA will be made on the one hand, thanks to a peptide bond between the C terminal of the peptide sequence of at least one glycine and the N' terminal of the PNA and on the other hand an imide bond between the C and C termini of the peptide sequence of at least one glycine and the PNA.The PNAs will be called structural PNAs. 11) In certain embodiments, the focused search for an antibody will use two independent banks of single-chain L and H phages from different immunizations. The combination of H / L chains will therefore be heterogeneous, possibly inter-species. In an even more particular embodiment, the focused search for an antibody will use two independent banks of single-chain H and H phages from different immunizations. The combination of H / H chains will therefore be heterogeneous, possibly inter-species. In other embodiments, the focused search for an antibody will use a single-chain phage bank cloning nanobodies and a single-chain phage bank cloning either L chains, or H chains, or both, possibly another nanobody chain. The combination of nanobody / H or L chains will therefore be heterogeneous, possibly inter-species. 8) In some embodiments, a first single-chain phage library corresponding to peptides comprising all or part of the H, L or nanobody chains will be screened by an antigen of interest as described in chapter 4 in order to select the n sequences whose respective chain has the greatest affinities for the given antigen. After production of these chains, each chain a(i) is fused to m PNA a(j) of structure such that each PNA_a(j) has a different and specific sequence, and that each of the PNA_a(j) continues with a tag_a(j) sequence which is specific to it so that a said PNA_a(j) is identifiable by this tag_a(j). A second library b of single-chain phage corresponding to peptides comprising all or part of H, L or nanobody chains is screened against the same antigen, as described in chapter 4, in order to select the m sequences whose chains_b(j) are the most affinity for said antigen. After production of these m chains, each of them is fused antiparallel to a PNA_b(j) chosen from m PNAs of structure such that for each PNA_a(j) there exists a complementary PNA_b(j) and that said PNA_b(j) is specific to the chain_b(j) and to it alone.A combination of m chain_a(i)-PNA_a(j) of structure are then mixed with the m chains b(j) to be associated with their respective m PNA b(j), such that each combination of chain_a(i)-PNA_a(j) pairs with its complementary combination chain_b(j)-PNA_b(j) to form m complexes between a said chain_a(i) and the m chains_b(j). The m chain_a(i)-PNA_a(j) / chain_b(j)-PNA_b(j) complexes are then mixed with m different polyvalent immunization supports chosen from nxm, such that each diamond particle forming said supports carries a double functionalization on the one hand one of the m sequences (j) complementary to the tag a and on the other hand a PNA sequence or unique nucleic acid analogue chosen from nxm PNA sequences and allowing the particle to be formally identified. Thus each particle will be able to fix a given chain_a(i)-PNA_a(j) / chain_b(j)-PNA_b(j) complex which will be uniquely identifiable by its particle tag. The operation is repeated for the n a(i) chains selected during the single a-chain phage step, in order to obtain nxm particles functionalized by nxm combinations of a and b chains such that each particle has a unique a(i)-PNA_a(j) / b(j)-PNA_b(j) chain combination identifiable by a unique particle tag. The nxm complexes are then brought into contact with the labeled antigen of interest. After possible removal of free antigens, the complexes attached to their particle are hybridized on a matrix of spots where each spot represents a nucleic acid, PNA, or nucleic acid analog complementary to one of the unique particle tag sequences, so that the particles present can be captured on the spot complementary to their particle tag sequence. Once rinsed, the signal level makes it possible to determine the relative affinity of the complexes. In some embodiments an identical strategy may be used for strings produced by the same bank. For a complexity n=1 the complexes can directly hybridize on a matrix of spots without the use of particles. 7) In certain embodiments, the selection of the antibody of interest will use the antigen of interest optionally fixed covalently on polyvalent immunization and selection supports such as diamond particles, said particles will possess at least one tag sequence, which may be a PNA sequence, nucleic acid, or any other nucleic acid analogue. The specific tag sequence will allow unambiguous identification of the particle as well as the peptide of interest. Altered antigens of the antigen of interest will be attached to other particles or polyvalent immunization and selection supports such that said particles are identifiable by a unique nucleic acid or PNA tag for a given alteration or type of alteration, or by alteration class. For example, the alterations may be modifications of the antigen of interest by glycosylations, modifications or deletions or additions of chemical functions, substitutions or deletions of amino acids or residues, or a combination of these modifications. 11-a) in certain embodiments a mixture of particles, optionally sensitive to a magnetic field, and functionalized either by the antigen of interest or by altered antigens such that said particles, perfectly identifiable through tags for the antigen of interest and for each different type of alteration, are brought into contact with a single-chain phage library or optionally a phage display library. Then the particles are isolated by specific hybridization of their tag, for example on a chip comprising a matrix of spots of nucleic acid sequences or probes or analogues complementary to the tags, a comb where each tooth is functionalized by a type of probe or nucleic acid sequence or analogue complementary to a type of tag or by the successive action of bars or rods functionalized by a type of probe or nucleic acid sequence or analogue complementary to a type of tag.After sequencing and quantifying the sequences of the different types of chains retained through the phages and the study of the alterations and their position in the sequence, the most refined and specific chain for the antigen of interest and for these alterations can be determined. A consensus sequence can also be determined. 11-b) In other embodiments, antigens and altered antigens fixed on beads having a tag making it possible to specifically identify the altered antigen or antigens, are used to select B lymphocytes and / or plasma cells from a cell mixture. Sequencing of mRNA or cDNA originating from the L and H chains of isolated lymphocytes or plasma cells makes it possible to identify the most affinity chains. Analysis of consensus sequences makes it possible to define new antibodies. 12) in certain embodiments a mixture of different antigens are fixed on beads, possibly sensitive to a magnetic field and having a tag such that each tag makes it possible to specifically identify a given antigen. The mixture of particles is used to screen a single-chain phage library or possibly a display phage library. The particles are then isolated using their tag as described in chapter 11, each type of particle is then treated separately so as to identify by sequencing the H and L sequences of the phages retained on each of the particles. In certain embodiments the particles segregated by their tag using a matrix of probes with sequences complementary to the tag or by combs are used to infect phages in a cell lawn or in 96-well plates. 13) in a particular embodiment, a mixture of antigens originating for example from a cell type 1 is used on the one hand, to functionalize a polyvalent immunization and selection support A such as hydrogenated diamond particles and on the other hand to functionalize a polyvalent tagged immunization support B such as hydrogenated diamond particles labeled with a nucleic acid tag B or equivalent such as a PNA tag makes it possible to identify the particles and therefore the origin of the antigens.A second mixture of antigens originating for example from another cell type 2 is used on the one hand to functionalize a polyvalent immunization and selection support C such as hydrogenated diamond particles and on the other hand to functionalize a polyvalent immunization support tagged D such as hydrogenated diamond particles labeled with another nucleic acid tag D or equivalent such as a PNA tag making it possible to identify the particles and therefore the origin of the antigens. The untagged AC functionalized diamond particles are then mixed and used to vaccinate at least one animal. After isolation of B lymphocytes and plasma cells from at least one animal and extraction, amplification and cloning of all or part of the sequences coding for the H and L chains, optionally H of the nanobody. A display phage bank or two single-stranded L and H phage banks or a single-stranded H nanobody phage bank are or are produced such that the phages of the bank(s) express on their capsid all or part of the H and / or L or H nanobody chains encoded by the mRNAs of the extracted B lymphocytes or plasma cells. The library(ies) are then screened by the B and C particles tagged with their specific PNA functionalized by the antigen mixtures, successively or preferably with an equimolar or "equiparticular" mixture of the two types of tagged particles, so that the antibodies or antibody fraction expressed on the surface of the phages and having an affinity for antigens fixed on the tagged particles can be specifically captured by the tagged particles, at the same time as the phages supporting said antibodies or antibody fragments. The antigens present on the B and C particles will capture the antibodies competitively between the B-type and C-type particles. After isolation of the B particles and the C particles using the different B and C PNA tags, the DNA of the phages captured on the particles thanks to the affinity of the antibodies or antibody fragments for the functionalizing antigens is quantitatively sequenced.The difference in abundance and the nature of the H and or L sequences from the B particle phages and the C particle phages makes it possible to determine the relative abundance between the antigens present on the surface of the two types of B and C particles. The most relevant antibody sequences or antibody portions, for example those which present a highest abundance differential between the two types of tagged particles, therefore between the two cell types, can be transformed into antibodies or antibody fragments and then specifically grafted onto tagged particles such that one antibody or antibody fragment corresponds to one and only one tag. Said tagged particles can then be used to capture and isolate in the mixtures of antigens, their specific antigen for example according to one of the embodiments described above, such as segregation on a matrix of probes directed against the tags, comb or rod comprising probes directed against the tags or any other embodiment involving said antibodies or fragments. The antigens isolated by the particles functionalized by antibodies can be analyzed and identified by conventional analysis methods such as mass spectrometry, NMR, etc. 13-a) in certain embodiments the method described in 13) may be used to search for disease markers such as cancer, degenerative diseases, etc. By comparing the antibody signature or antibody fragment obtained between a healthy cell and a diseased cell. 13-b) In certain embodiments, the antigens isolated by the method may be used to carry out a possibly curative vaccination in association or not with a polyvalent immunization support. 13-C) The method described in 13) may be used to simultaneously analyze several sets or fractions of antigens, each set of antigens being used to functionalize a particle with a tag specific to this set. In certain embodiments, the set of antigens is reduced to a single antigen. 14) In certain embodiments, a polyvalent immunization and selection support, such as hydrogenated diamond particles, optionally sensitive to a magnetic field, are functionalized with an antigen of interest, while another polyvalent immunization and selection support, such as hydrogenated diamond particles, are functionalized with altered antigens such that the alteration removes the hapten or possibly the epitope against which an antibody is sought. The two types of functionalized particles will then be injected into different body areas of a subject in order to trigger antagonistic reactions. For example, particles containing altered antigens will be injected into the spleen or bone marrow of the subject or into any organ where the presentation of self antigens occurs, in other words where B lymphocytes directed against the self are eliminated, while particles functionalized with the antigen of interest will be injected into an area where the immune response will be triggered such as a subcutaneous area, muscle, peritoneum, blood, or any body area for which a foreign body triggers an immune reaction. 15) In certain embodiments, the phages of the phage display or single-chain phage libraries will comprise at least one fluorescent protein among the proteins of their capsid allowing them to be identified and quantified directly during their selection by the polyvalent immunization and selection supports. This fluorescence will be obtained, for example, by the presence of at least one P9 protein or part of the P9 protein. fused with all or part of a fluorescent protein such as GFP. These fluorescent phages will allow immediate assessment of the quantity of phages present on the surface of a polyvalent immunization support. 15-a) In certain embodiments, different phage banks respectively comprise phages having a fusion protein of their capsid with their own fluorescence and different for each bank allowing the simultaneous screening of the banks with an antigen of interest, the analysis of the fluorescence retained on the diamond particles thus makes it possible to identify the origin of the phages. Thus, in certain embodiments, a single H chain phage bank will have phages with a fluorescence of a given color, for example green, while the phages of a second single L chain bank will have phages of another color, for example red. During successive washes, the variation of the fluorescence retained on the particles makes it possible to estimate the variation of the affinity of the antibodies or antibody fragments retained on the particles. 16) In certain embodiments, the phages expressing a given antibody or antibody fragment and having at least one fusion protein of their fluorescent capsid, can be directly used to make a fluorescent immunogenic labeling. 17) In certain embodiments, the versatile diamond particle immunization and selection supports are fixed on beads or microparticles sensitive to a magnetic or electric field, such as latex or silicon polystyrene beads comprising magnetic elements so that said fields exert on the beads a force capable of moving them, attracting them, or immobilizing them. 18) In certain embodiments the flat surfaces used to make the spot matrices will be a PMMA, Kapton, styrene, vinyl polymer surface, etc. including diamond particles from 10 nm to 500 pm. LEGENDS: Figure-1: Phage display selection - affinity of ScFv phages for fluorescent CadE measured by chip. Figure-1.1: ScFv phages from phage display selection using polyvalent immunization and selection supports coated with Cadherin E peptide. These phages are amplified, deposited on a slide and incubated with fluorescent Cadherin E peptide. The quantity of Cadherin E peptides retained on the spots is evaluated by a fluorescence scanner. Figure-1.2: ScFv phages resulting from phage display selection using magnetic beads (without selection peptide). These phages are amplified, deposited on a slide and incubated with fluorescent Cadherin E peptide. The quantity of Cadherin E peptides retained on the spots is evaluated by a scanner at fluorescence. Figure 1.3: Graphical representation of the amount of fluorescence (related to the amount of fluorescent Cadherin E peptides bound) per phage deposition as a function of stringency during washes of phage display selection of polyvalent immunization and selection supports coated with Cadherin E peptide having captured phages. Figure 1.4: Graphical representation of the amount of fluorescence (related to the amount of fluorescent Cadherin E peptides bound) per phage deposition as a function of stringency during washes of phage display selection by magnetic beads (without selection peptide). Figure 1.5: Schematic representation of the increase in stringency achieved during the phage display steps. Figure-2: Infectivity of ScFv-phages associated with polyvalent immunization and selection supports 2pm nanodiamond particles. Figure 2.6: ScFv phages captured by CadE-coated nanodiamonds, directly used to infect bacteria. The phages are amplified, purified, normalized, deposited on a slide and revealed by fluorescent CadE. Figure-2.7: ScFv phages captured by nanodiamonds coated with triGlycine peptide (non-immunogenic), directly used to infect bacteria. The phages are amplified, purified, normalized, deposited on a slide and revealed by CadE-fluorescence. Figure-2.8: Quantification of CadE binding on deposit 6 by scanner. Fluorescence intensity expressed in arbitrary units (AU). Figure-2.9: Quantification of CadE binding on deposit 7 by scanner. Fluorescence intensity expressed in arbitrary units (AU). Figure-3: Example of a solution of 2pm nano-diamonds functionalized by Poly A probes and deposited on a slide. 10: Unlabeled poly A probes 12: fluorescent poly T complementary strand 14: saturating fluorescent signal 13: very weak fluorescent signal Figure-3.10: After functionalization by unlabeled poly A probes (10), Figure-3.11: Addition of fluorescent Poly A (11) or fluorescent BSA shows that the diamond surface is saturated and that the non-specific absorption is very low (very weak fluorescent signal 3 ). Figure-3.12: On the other hand, the hybridization of a complementary fluorescent poly T 835 strand (12) on the grafted probes shows a very high level of functionalization of the particles (saturating fluorescent signal) (14).

Claims

CLAIMS 1) Focusing device for the selection, isolation, amplification or stimulation of a cellular or viral clone or of an antibody or part of an antibody, characterized in that it comprises a polyvalent support comprising a diamond carbon phase SP3 linked to an antigen by a carbon / carbon bond between a carbon of the diamond phase and a carbon of the antigen, all of the grafting sites of said carbon phase being occupied or saturated by the antigen, supplemented by a non-immunogenic molecule. 2) Device according to claim 1, characterized in that the multipurpose immunization and selection support comprises diamond particles with a size between 10 nanometers and 500 microns. 3) Device according to any one of claims 1 to 2, characterized in that a fixation of at least one antigen on the polyvalent diamond immunization and selection support is carried out through a hydrogenated carbon phase CHx (x=1,2,3) such that the grafting site comprises a hydrogenated carbon, possibly an SP2 carbon which reacts with a carbon of the antigen adjacent to a nucleophilic function such as NH2, COOH, OH, possibly an electrophilic function to join an SP3 carbon. 4) Device according to any one of claims 1 to 3, characterized in that the multipurpose diamond immunization and selection support is fixed on beads or microparticles sensitive to a magnetic or electric field, so that said fields exert on the beads a force capable of moving them, attracting them, immobilizing them, or repelling them. 5) Device according to any one of claims 1 to 4, for use in immunizing an animal or a human. 6) In vitro focusing method for the selection, isolation, amplification, stimulation of a cellular, viral clone, of an antibody or part of an antibody using a device according to any one of claims 1 to 5, characterized in that the selection, isolation, amplification of at least one clone from a clone bank, the clones of said bank being chosen from: • single chain phage, • phage display, • cellular mixture, • bacteria, • viruses, • any other eukaryotic or prokaryotic cell type, such that the clones express all or part of an antibody, understand : • At least one step of bringing the bank into contact with a versatile immunization and selection support, functionalized by an antigen of interest, • At least one isolation step, polyvalent immunization and selection supports and library elements expressing an antibody or an antibody fraction recognizing the antigen, fixed on the polyvalent immunization and selection support, • At least one step of culturing, bacterial infection, amplification, of the bank elements expressing an antibody or an antibody fraction recognizing the antigen and fixed on the polyvalent immunization support, • At least one step of depositing isolated elements of the bank on a preferably flat solid support in order to obtain a matrix of spots or deposits whose XY coordinates make it possible to identify the isolated elements of the bank, • At least one step of contacting, complexing, hybridizing the matrix with a solution of antigens possibly labeled with a fluorescent, colorimetric marker, detectable by any physical detection means such as signal intensity, fluorescent, or any other physical measurement allowing the antigens to be quantified, at the level of each spot of the matrix, • At least one step of nucleic acid sequencing, quantitative semi-quantitative, of isolated element, of their culture, of the results of bacterial infections by said isolated elements. 7) In vitro focusing method for the selection, isolation, amplification, stimulation of a cellular, viral clone, of an antibody or part of an antibody using a device according to any one of claims 1 to 5, characterized in that the polyvalent immunization and selection support functionalized by an antigen, makes it possible to select at least one phage clone expressing a type of antibody such that at least one phage fixed on the polyvalent immunization support retains a capacity to infect bacteria. 8) Method according to any one of claims 6 and 7, characterized in that a polyvalent immunization and selection support will comprise, in addition to the antigen of interest, at least one nucleic acid tag sequence or analogue such as PNA (peptide nucleic acid) or any other analogue making it possible to identify, specifically address the polyvalent immunization and selection support on a flat surface, a plate, combs, any object comprising nucleic acids complementary to the tag, antigens comprising the tag. 9) Method according to any one of claims 6 to 8, characterized in that a polyvalent immunization support comprises particles functionalized by different antigens, such as an antigen of interest and variants of this same altered antigen, in certain embodiment each antigen or type of antigen attached to a particle is identifiable by a nucleic acid tag attached to said particle. 10) Method according to any one of claims 6 to 9, characterized in that at least two libraries coding for different antibody fragments are screened to produce an antibody or antibody fragment directed against an antigen of interest, the two libraries being able to be screened independently, such that the independent results are combined to produce a molecule, molecule subunits forming an antibody, antibody fraction, synergistic so that the first selection from the screening of a first library is used in the screening of a second library. 11) Method according to any one of claims 6 to 10, characterized in that the banks, phages, cells used comprise elements such as phages or cells expressing fluorescent proteins. 12) Method according to any one of claims 6 to 11, characterized in that phage banks are focused from cellular extract, optionally fractionated, fixed on a polyvalent immunization and selection support.

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