FR3025605A1 - FOCUSING METHOD FOR IMMUNIZATION AND PRODUCTION OF ANTIBODIES - Google Patents

Abstract

The loss and / or dilution of one or more antibodies of interest or part of an antibody, or defocusing, during the steps involving the antigen for: - Immunization of B lymphocyte stimulation, - Selection antibodies or antibody-producing cells; isolation of antibodies or antibody-producing cells; production of antibodies remains a limiting lock in antibody-producing technologies. To improve the focus of immunization and / or selection and / or isolation and / or production of one or more monoclonal or pseudo-monoclonal antibodies (different antibodies directed against the same epitope) or all parts of one or more monoclonal or pseudo-monoclonal antibodies recognizing an antigen of interest, we propose a polyvalent immunization and selection support and a new method using this support, for the production of monoclonal and pseudo-monoclonal antibodies. This polyvalent immunization and selection support comprises a diamond-fixing SP3 diamond carbon phase, so that the bond between the support and the antigen is via a covalent bond, preferably a carbon / carbon bond. carbon between a carbon of the diamond phase and a carbon of the antigen. The diamond support is only slightly immunogenic, the antigens being fixed by a carbon-carbon bond (non-peptide or otherwise), the immune response is focused on the antigen only. This support also improves antibody collection methods, in particular for phage display technologies.

Description

DESCRIPTION Antibody production and methods of developing new antibodies are fundamental issues in biology or medicine. Thus, more and more biology tests involve antibody-mediated steps, but also most innovative cancer treatments involve antibody molecules or analogs of these molecules. In addition to these already known applications for antibodies, the resistance of bacteria to antibiotics opens the way for combined treatments, Antibiotics / Antibiotics, the action of antibodies to block antibiotic resistance. All these applications involve having monoclonal or pseudo-monoclonal antibodies, ie 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 the antigens, whether for the immunization step or for the antibody production and selection steps, irrespective of the mode of production, hybridoma, phage display or other. For the immunization of an animal or an individual against a given epitope, large antigens generally have a higher immunogenicity. In contrast, unlike a smaller size antigen, large antigens promote the production of antibodies and B cells with more varied paratopes. Indeed, the larger the antigen, the more potential epitopes it presents: The immune response may therefore not be focused solely on the desired epitope. In the worst case, produced paratopes can attach to the epitope of interest, mask it, and thus hinder the binding of antibodies directed against the epitope of interest. In contrast, the use of smaller antigens, whose hapten is centered on the epitope of the antigen allows the production of antibodies and or B lymphocytes less varied but better focused on the epitope, in particular. 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 defocus the immune response on themselves rather than on the coupled antigen.

Another approach generally used to circumvent these problems of focusing and stimulating immunization is to compensate for the weak immunogenicity of small antigens by co-injecting immunogenic adjuvants such as aluminum, lipid micelles, etc. Nevertheless, the 75 vaccination protocols using such adjuvants can trigger undesirable immune responses such as: macropathy, autoimmune diseases, important inflammations, etc. These side effects complicate and defocus the immune response. They may therefore compromise the selection and clonal expansion of B-cells 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 rarefaction of adjuvants in vaccinations and the search for alternatives.

The question of focusing on the antigen also arises in the selection of antibodies from a complex mixture, such as those encountered, for example, in activated lymphocyte B extracts or plasma cells after immunization, or in the phage libraries, obtained by phage display methods. The latter technique 90 consists of cloning in phagemids (vectors for bacteriophage) nucleotide sequences of heavy and light chains of antibodies from a mixture of lymphocyte B (activated or not by immunization) or plasma cells. With these constructs, it is possible to produce phage containing these DNA sequences and carrying on their surface the variable fragments of antibodies corresponding to these sequences. Phage carrying antibodies specific for 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 selection on the antigen of interest is crucial. This depends on the selection method, the nature of the antigen used for the screening, but also the means of isolation. The latter comprises: 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 (Ex: 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 selection by selecting antibodies against the isolation medium, rather than the antigen of interest. . During the selection, this defocusing can go as far as eliminating 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 antigen. 'isolation. Because of the often incompressible sizes of isolation means such as those generally used to fix the antigen on a support (isolation or segregation) and the physical nature of the isolation media 120 generally used (glass, latex, stiren ...), the focus 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 the immunization inevitably affects 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, 3025605 3 and, on the other hand, the other immunogenic targets present such as the remainder of antigen and isolation means, a commonly used approach is to firstly harden the selection conditions by several screens comprising successive steps of affinity capture of the antibodies under conditions of increasing stringency, and secondly by alternating different means of isolation: the antigen is then fixed successively on different supports by different peptides or molecules. The purpose of these measurements is to eliminate the antibodies against targets other than the antigen of interest in screens. In addition, the conventional phage display requires several 140 stages of precipitation followed by purifications and bacterial infections by phages, in order to retain sufficient amounts of phages. Usually, the isolation means reduce the infectivity (the ability to infect and multiply in bacteria) captured therein (via the antigen), therefore, to recover the phages captured on the antigen, a step of 145 Cleavage of the antibody by a protease is usually performed. Since these proteases have a relative cleavage specificity depending on the reaction conditions, they can damage the selected phages and impair the quality of the selection result and lead to a defocusing of the selection on the phage expressing the antibody of interest.

Beyond the problems raised by the focusing of the immune response on the epitope of the antigen and by the focusing of the selection of monoclonal antibodies by the epitope, the question of the phage display also raises the question of Defocussing the selection of the desired antibody 155 at the time of cloning the coding sequences of the heavy and light chains of the antibodies in the phagemid In fact, the loss of the antibody directed against the desired epitope can occur even after a successful immunization, during the steps of creation of the library 160 coding sequences of light chains (L) and heavy (H) Namely, the retrotranscription of mRNA cDNA, and the cloning of cDNAs of light (L) and heavy ( H) in a phagemid, thereby 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 phage surface during phage display. Therefore, after cloning, it is rare that the H and L chains (forming the specific antibody of the epitope of interest) are again combined together in the same phagemid during the phage display.

These random re-pairings can therefore lead to the loss of the antibody of interest.

In the remainder of this document we define the defocusing by the phenomena resulting in the loss and / or dilution of one or more antibodies of interest or part of an antibody, especially during the steps involving the antigen for: stimulation or immunization, - production, - selection, - isolation, of cells or viruses producing said antibody (s) or any part of said antibody (s).

185 We define the focus 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 antibody fraction, cell type, viral, bacterial, or said antibody or antibody fraction. This focusing can be carried out in an organism in vivo, or outside an organism ex vivo, or in vitro on cells or viruses.

To improve focus of immunization and / or selection and / or isolation and / or production of one or more monoclonal or pseudo-monoclonal antibodies (different antibodies directed against the same epitope) or parts thereof One or more monoclonal or pseudomonoclonal antibodies recognizing an antigen of interest, we propose a polyvalent immunization and selection support 200 and a novel method using this support for the production of monoclonal and pseudo-monoclonal antibodies. 1-a) the polyvalent immunization and selection support, comprises a diamond carbon SP3 phase fixing antigens such as peptide, polysaccharide, lipid, alkaloid, nucleic acid, any combination of these compounds, or any other type of molecules likely to trigger an immune reaction or to be recognized by an antibody, so that the bond between the support and the antigen is through a covalent bond, preferably a carbon / carbon bond between a carbon of 210 the diamond phase and a carbon of the antigen. The diamond support is only slightly immunogenic, the antigens being fixed by a carbon / carbon bond (non-peptide or otherwise), the immune response is focused on the antigen only. In some embodiments the antigen is reduced to a hapten with only the pattern recognized by the antibody. 1-b) In a preferred embodiment, the polyvalent immunization and selection support will be saturated with the antigen so that all the accessible grafting site for the antigen is occupied. Indeed as the 220 shows Fig3: a nano-diamond carrier consisting of diamond particles of 2pm functionalized by Poly A probes and deposited on a slide. After functionalization with non-labeled poly A probes, the addition of fluorescent poly A or fluorescent BSA shows that the diamond surface is saturated 225 and that the non-specific absorption is very weak (very weak fluorescent signal). On the other hand, the hybridization of a complementary poly T fluorescent strand on the grafted probes, shows that the level of functionalization of the diamond particle by the poly A probe is very high, in one embodiment. Preferably, the attachment of the antigens to the polyvalent carrier of immunization and diamond selection is carried out through a hydrogenated carbon phase CHx (x = 1,2,3) such that the hydrogenated carbon reacts with a carbon of 1%. adjacent antigen of a nucleophilic function such as NH 2, COOH, SH, OH (optionally electrophilic). In another embodiment, the covalent bond is made 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 240 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 245 immunization and selection support is comprised of or comprises diamond particles whose size is between 10 Ω. nm and 500 μm, preferably 2 μm. The particles will preferably be hydrogenated CHx to fix the antigens in a saturating manner. 2-a) In a particular embodiment the polyvalent carrier of immunization and selection functionalized by an antigen, is used to immunize an animal or a human by a subcutaneous injection, an intramuscular injection, an intraperitoneal injection , an 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 zone will be irradiated with X-rays or gamma radiation so as to generate a local inflammation 260 that can stimulate the immune response. This irradiation will be used preferentially with a polyvalent support for immunization and selection of hydrogenated diamond CHx or possibly SP2, capable of generating free radicals capable of triggering local inflammation. 265 2-c) In a particular embodiment, the polyvalent immunization and diamond selection supports, typically particles of 10 nm to 500 μm, will be sensitive to a magnetic or electric field so that said fields exert on the particles a force 270 likely to move or attract 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 of iron oxide, nickel, boron, magnetic silica, neodymium, etc. or any combination of these elements. In some embodiments, any combination of these elements may be directly integrated with the crystal structure SP3 or SP2 of the diamond particle. 2-d) in a particular embodiment, the vaccination will be carried out with polyvalent means of immunization and selection sensitive to a magnetic or electric field 280, such that the injection zone is subjected to a magnetic variant field 3025605 , possibly electrically or a combination of both, so as to cause agitation of the particles generating a heating capable of triggering an inflammation reaction, thereby promoting the immune response. 285 2-e) in some embodiment, the vaccination zone will be subjected to a constant magnetic field possibly pulsed, so that the magnetic particles of the polyvalent carrier of immunization and selection, are maintained in the injection zone. Thus, magnetic particles may be, for example, injected into a tissue or an organ, such as the spleen, bone marrow, or a lymph node, etc., so that a magnetic field can retain said magnetic particles in the tissue or organ to trigger a specific immune response. In some embodiments, the particles may be guided and confined in a tissue or organ by a pulsed or continuous magnetic field. 3-a) After the vaccination and the establishment of the immune response, the B cells and or plasma cells can 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 vaccination may be recovered using polyvalent immunization carriers formed of diamond particles possibly sensitive to an electric or magnetic field and covered with the antigen 305 connected by a covalent bond with the diamond phase. 4-a-1) Lymphocytes and plasma cells can be immortalized in hybridomas and undergo classical selection to obtain a monoclonal hybridoma.

In other embodiments, the messenger RNAs of the lymphocytes and plasma cells will be extracted, retrotranscribed and cloned into phagemids, such that each phagemid comprises a random combination between a sequence of the variable heavy H fragment from one of the lymphocytes and extracted plasmocytes and a sequence of the L light variable fragment from 315 of one of the extracted lymphocytes and plasma cells. The sequences are linked by the coding sequence of a peptide linker. Thus, after introduction of the phagemids into bacteria, a recombinant phage library is produced such that the phages express all the combinations between the heavy and light sequences encountered between the lymphocytes and / or the 320 plasmocytes of an extraction. The expression of the combined antibodies will be on the surface of the phage capsid by fusing the H-linker-L sequence of the recombinant antibody with all or part of the sequence of at least one protein of the capsid of the phage. phage. This phage library may be screened in a conventional manner for selecting an antibody or may be screened vectorially according to the focusing strategy described later in this document. An alternative to recombinant cloning, which risks defocusing the selection obtained after vaccination, is to clone separately the heavy and light chains without recombination, so as to obtain two separate phage banks: a 330 for the variable parts heavy chains and another for the variable parts of the light chains. In some embodiments, the variable portion of the heavy chains will be cloned into phagemids such that the variable portions are connected to a peptide linker and / or a stabilizing peptide such as the constant parts of the variable part of an antibody for example 335 of 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 made only by a single chain. The invention will therefore comprise a phage library expressing on the capsid either only all or part of the heavy chain H of antibodies, or all or part of the L chains of antibodies. Said antibodies correspond, for example, to the antibodies encoded by the cDNAs derived from the messenger RNAs of an extract of Lymphocytes or of plasmocytes resulting from an immunization. The light or heavy chains are fused with all or part of at least one phage capsid protein and optionally have a stabilizing peptide 345 of the chain such as all or part of the non-variable parts of the light or heavy chains, respectively. 4-a-2) In a preferred embodiment, two single-chain phage libraries from: on the one hand, phagemids comprising the H-chain sequences fused with a peptide linker and all or part of the constant chains; a L chain itself fused with all or part of the sequence of a phage capsid protein, for example the P3 protein, 355 - and secondly, phagemids comprising the L-chain sequences fused with a peptide linker and all or part of the constant parts of a chain H 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 360 fusions may vary), Such libraries are sequentially screened for the antigen of interest against which an antibody is being sought. In a particular embodiment, the targeted antigen is covalently attached to a polyvalent immunization and selection medium, such as diamond particles ranging from 10 nm to 500 μm, typically 2 μm. In a still more preferential mode the attachment of the antigens is obtained by putting the antigens in direct contact with the hydrogenated diamond particles CHx so that a carbon-carbon bond is formed between at least 370 carbon of the antigen. and at least one carbon of the surface. After contacting the single chain H-bank and the diamond particles, the particles will be isolated and screened with increasing stringency washes. 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 the fact of being able to infect bacteria directly with the phages retained on the particles of a polyvalent diamond immunization support FIG. 2. This makes it possible to amplify the captured phages directly from the capture on the cells. particles. This method avoids the use of protease-mediated cleavage, which simplifies phage manipulation after selection by removing the phage separation step of the selection medium, and thus minimizes the defocus usually caused by protease separation. Indeed, a peptide digestion step such as protease digestion 385 is normally required to separate the phage from the antibody captured by the antigen attached to the isolation means. To achieve the infection by the selected phage antibodies, the particles having captured affinity phages are then directly incubated with a bacterial culture in exponential growth phase at 37 ° C. The passivity of the diamond as well as the absence of binding peptide guarantee the collection of very specific phages in a single sieve cycle, with rinsing of increasing stringency without having to change either the selection supports or the nature of the fixation. antigen on the support. The highest affinity heavy chain sequences are then cloned into the phagemids of the single-chain light libraries to obtain a selected heavy sequence library. The cloning is carried out in such a way that it allows the reconstitution of a chain H, 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 bank is then screened in the same way as for the heavy chain to determine the HL pair with the best affinity. Selection may possibly start with the L chain library. 4-a-3) In a particular embodiment, parallel screening is used. It consists in each step of rinsing the particles, with a stringency buffer increasing during the screening, to archive a fraction 1 / X (x = 2 ... 5, ... 10 ect) particles having captured the phages specific. The archived fraction may be stored, for example, in a well plate, but 410 is preferably used to infect a bacterial culture optionally in a well plate for phage amplification. When all the stringency conditions have been tested, a standardized sample of each well is then spot or deposited on a preferentially flat solid support, so as to obtain a matrix of spots or deposits 415 whose XY coordinates make it possible to identify the well of from the plate and therefore the level of stringency used. The surface of the support makes it possible to fix the phages of the deposit: for example, a hydrogenated diamond surface or covered with hydrogenated diamond particles, glass covered with epoxy, aldehyde, kapton, or any other medium capable of fixing the phages of 420. stable. After phage binding, the surface of the support is passivated to neutralize unreacted functions, and then the matrix is complexed or hybridized with an antigen solution optionally labeled with a fluorescent marker, colorimetric marker or for any other detection means. The signal intensity, fluorescent or any other physical measurement making it possible to quantify the captured antigens such as SPR, LIBS Etc, recorded at each spot of the matrix is proportional to the affinity of the antibodies or of the antibody fragment. present on the surface of the capsids of the phages retained on the spot. The more intense spots make it possible to determine the most efficient screening steps and to select these steps to isolate the phages expressing the higher affinity molecules. More generally, the screening of a library expressing all or part of an antibody such as the said library, single chain phage, phage display, cell mixture, bacterial, viral or any other eukaryotic or prokaryotic cell type, 435 expresses all or part of an antibody comprises: at least one contacting of the library with a polyvalent immunization and selection support, functionalized with an antigen of interest, at least one isolation step, polyvalent immunization carriers and which have reacted with the elements of the library expressing a 440 antibody or an antibody fraction recognizing the antigen and thus attached to the polyvalent carrier of immunization and selection at least one step of culturing, of infection bacterial, amplification, library elements expressing an antibody or antibody fraction recognizing the antigen and fixed on the polyvalent immunization support, 445 At least one step of de deposit on a preferably flat solid support, elements isolated from the bank so as to obtain a matrix of spots or deposits whose XY coordinates make it possible to identify the elements isolated from the bank, At least one step of contacting, complexing, hybridization of the matrix 450 with a solution of antigens optionally labeled with at least one fluorescent, colorimetric marker, allowing detection by any detection means without labeling such that the signal intensity, fluorescent or any other physical measurement makes it possible to quantify the antigens, at each spot of the matrix 455 At least one nucleic acid sequencing step, quantitative, semiquantitative, isolated elements of the library, of their culture, resulting from bacterial infections by said isolated elements. In a particular embodiment, the polyvalent immunization and selection support 460 will comprise in addition to the antigen a nucleic acid tag sequence or the like such as PNA (nucleic acid peptide) or any other analog. 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, the hybridization between the tag and the complementary sequence of the tag. fixed on the surface. In a particular embodiment the surface will be coated with hydrogenated diamond to establish a carbon-carbon bond between the diamond and the complementary sequence of the tag by simple contact. After deposition 470 of particle spot having optionally captured phages, nucleic acids complementary to the tag which have not hybridized with the tag of a particle will be optionally neutralized by free tag sequences. 5) The phages of the wells of the storage or culture plate corresponding to the spot with the most intense signals are sequenced by vector blade methods such as those sold by the companies Ion Torent or Illumina. For each sequenced well, the abundance of each H and L sequence of said well makes it possible to determine the antibodies or the antibody fragment having 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 mat such as those made in the cell chips allows the transfection directly by the spot blade with the capture particles. phages. 11) The identified candidate H and L sequences may be cloned into eukaryotic, prokaryotic organisms, or may be used in so-called cell free acellular devices to produce the antibody. In some embodiments, the sequences will be cloned into algae either at the nuclear or chloroplast level to obtain the H, L, H and L sequences linked by a linker, optionally secreted by the algae. 495 10) In some embodiments and in addition to the patent No. FR 1001516, after selection the H and L chains will be produced separately, an LPXTG peptide motif (or x = one of the 20 amino acids) will be grafted in C terminal of each Chains. The C-terminal LPXTG motif of the H and L chains will allow the enzymatic fusion of these chains, by virtue of the C 2 and L-terminal sortase, of two PNAs, or any other complementary nucleic acid analogue comprising a peptide sequence. at least one glycine bound at its C end to the N 'end of a PNA and at the C' end of the other PNA, respectively. For example, the linkage between the peptide sequence of at least one glycine and the PNA will be on the one hand, 505 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 'terminus of the peptide sequence of at least one glycine and PNA. NAPs will be called structure NAPs. 11) In some embodiments, the focused search of an antibody will utilize two independent single chain L and H phage libraries from different immunizations. The combination of H / L chains will therefore be heterogeneous, possibly interspecies. In a still more particular embodiment, the focused search of an antibody 515 will use two independent single chain H and H phage libraries from different immunizations. The combination of H / H chains will therefore be heterogeneous, possibly interspecies. In other embodiments, the focused search of an antibody will use a single chain phage library cloning nanobodies and a single chain 520 phage library 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 interspecies. 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 530 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) is continuous by a sequence tag_a (j) of its own so that a said PNA_a (j) is identifiable by this tag_a (j). A second single-chain phage library 535 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 b_ chains (j) are the most affine for said antigen. After production of these m chains, each of them is merged antiparallel to a PNA_b (j) selected from m PNA structure 540 such that for each PNA_a (j) there is a complementary PNA_b (j) and that said PNA_b (j) ) is specific to the string_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) ) is paired with its combination 545 complementary string_b (j) -PNA_b (j) to form m complexes between a said string 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 550 functionalization on the one hand one of the m sequences (j) complementary to the tag_a and secondly a PNA sequence or the like of a single nucleic acid selected from nxm PNA sequences and to formally identify the particle. Thus each particle can set a complex chain_a (i) -PNA_a (j) / chain_b (j) -PNA_b (j) given that will be uniquely identifiable by its particle tag. The operation is repeated for the n chains (a) selected during the simple phage step a, in order to obtain nxm particles functionalized by nxm combinations of chains a and b such that each particle has a unique combination of chain a ( i) -PNA_a (j) / string_b (j) -PNA_b (j) identifiable by a single particle tag. The complex nxm are then brought into contact with the labeled antigen of interest. After optional elimination of the free antigens, the complexes fixed to their particle are hybridized on a spot matrix or each spot represents a nucleic acid, PNA, or nucleic acid analogue complementary to one of the single particle tag sequences, so 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 the particles. 575 7) In some embodiments the selection of the antibody of interest will use the antigen of interest optionally covalently attached to polyvalent immunization and selection media such as diamond particles, said particles will possess less a tag sequence, 580 which may be a PNA sequence, nucleic acid, or any other nucleic acid analogue. The specific tag sequence will unambiguously identify the particle as well as the peptide of interest. Antigens altered with the antigen of interest will be attached to other polyvalent immunizing and screening particles or carriers such that said particles are identifiable by a single nucleic acid tag or PNA for alteration or type of alteration. given, 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 some embodiments a mixture of particles, possibly sensitive to a magnetic field, and functionalized either by the antigen of interest, or by altered antigens such as said particles 595 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 possibly a phage display library. The particles are then isolated by specific hybridization of their tag, for example on a chip comprising a sequence spot array or probes 600 nucleic acid or analog complementary to the tag, a comb where each tooth is functionalized by a type of probe or sequence nucleic acid or analogue complementary to a type of tag or by the successive action of bars or rods functionalized with a type of probe or nucleic acid sequence or analog complementary to a type of tag. After 605 sequencing and quantification of 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 affine and most specific chain for the antigen of interest and for these alterations can be determined. A consensus sequence may also be determined. 610 11-b) In other embodiments of the altered antigens and antigens bound to beads having a tag specifically identifying the antigen or altered antigens, are used to select B cells and / or plasma cells. a cellular mixture.

The sequencing of the mRNAs or cDNAs from the L and H chains of the isolated lymphocytes or plasmocytes makes it possible to identify the most affine chains. The consensus sequence analysis makes it possible to define new antibodies. 620 12) in some embodiments a mixture of different antigens are attached to beads, possibly sensitive to a magnetic field and having a tag such that each tag specifically identifies a given antigen. The particle mixture is used to screen a single chain phage library or possibly a display phage library. The 625 particles are then isolated by means of their tag as described in Chapter 11, each type of particles is then treated separately so as to identify by sequencing the H and L sequences of the phage retained on each of the particles. In certain embodiments, the particles segregated by their tag through a probe matrix of sequences complementary to the tag 630 or by combs are used to infect phages in cell mats 3025605 13 or 96-well plates. 13) In a particular embodiment, a mixture of antigens from, for example, 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 carrier of tagged immunization B such that hydrogenated diamond particles labeled with a nucleic acid tag B or equivalent such as a PNA tag makes it possible to identify the particles, hence the origin of the antigens. A second 640 mixture of antigens from, for example, 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 645 or equivalent such as a PNA tag to identify the particles thus the origin of the antigens. Untagged C-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 encoding the H and L chains optionally H nanobody. A display phage library or two single-phage L and 655 H phage libraries or a H nanobody single-strand phage library are or are made such that the phages of the library or banks express on their capsid all or part of the H and / or or L or H nanobody encoded by the mRNAs of B lymphocytes or extracted plasma cells. The bank or libraries are then screened by particles B and C tagged by their specific 660 PNAs functionalized with the antigen mixtures, successively or preferably with an equimolar or "equiparticular" mixture of the two types of tagged particles, so that the Antibody or antibody fraction expressed on the surface of phages and having an affinity for antigens attached to the tagged particles can be specifically captured by the tagged particles, together with the phages supporting said antibodies or antibody fragment. The antigens on particles B and C will capture antibodies competitively between type B and type C particles. After isolation of B particles and C particles using different PNA tags B and C, the DNA of the 670 phage captured on the particles by affinity of antibodies or antibody fragment for functionalizing antigens is sequenced quantitatively. The difference in abundance and the nature of the H and / or L sequences originating from the phages of the B particles and the phages of the C particles makes it possible to determine the relative abundance between the antigens present on the surface 675 of the two types of particles B and C. The most relevant antibody sequences or antibody portions, for example those with the highest abundance differential between the two types of particles tagged between the two cell types, can be transformed into antibodies or antibody fragments. then grafted specifically on tagged particles 680 such as an antibody or antibody fragment corresponds to one tag and one.

Said tagged particles can then be used to capture and isolate in the antigen mixtures, their specific antigen for example according to one of the embodiments described above, such as segregation on a matrix of probes directed against the tag, comb or rod. comprising 685 probes directed against tags or any other embodiment involving said antibodies or fragments. The antigens isolated by the antibody-functionalized particles can be analyzed and identified by conventional analytical methods such as mass spectrometry, NMR, etc. 690 13-a) in some embodiments the method described in 13) can 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. In certain embodiments, the antigens isolated by the method may be used to carry out an optionally curative vaccination in association or not with a polyvalent immunization support. 700 13-C) The method described in 13) can 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 some embodiments, the set of antigens is reduced to a single antigen. In some embodiments, a polyvalent immunization and selection support, such as hydrogenated diamond particles, possibly sensitive to a magnetic field, is functionalized by an antigen 710 of interest, while another polyvalent carrier Immunization and selection techniques, such as hydrogenated diamond particles, are functionalized with altered antigens such that the alteration suppresses the hapten or possibly the epitope against which an antibody is sought. Both types of functionalized particles will then be injected into different body areas of a subject to initiate antagonistic reactions. For example, the particles containing the altered antigens will be injected into the spleen or bone marrow of the subject or into any organ where the presentation of the antigens of the ego occurs, in other words where the B lymphocytes directed against the ego are eliminated, whereas the Functionalized particles 720 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 strange body triggers an immune response. 1525) In certain embodiments phages of the phage display or single-chain phage libraries will comprise at least one fluorescent protein among the proteins of their capsid making it possible to identify them by quantifying them directly during their selection by the polyvalent carriers of the phage display. immunization and selection. This fluorescence will be obtained for example by the presence of at least one P9 protein or part of the P9 protein 3025605 fused with all or part of a fluorescent protein such as GFP. These fluorescent phages will make it possible to immediately evaluate the amount of phages present on the surface of a polyvalent immunization support. 735 15-a) In some embodiments of the different phage libraries respectively comprise phages having a fusion protein of their capsid with own fluorescence and different for each library allowing the simultaneous screening of the libraries 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 some embodiments a single H-chain phage library will have phages with a fluorescence of a given color for example green whereas the phages of a second single-chain library L will have phages of another color, for example. red example. During successive washings, the variation of the fluorescence 745 retained on the particles makes it possible to estimate the variation in the affinity of the antibodies or antibody fragments retained on the particles. 16) In some embodiments phages expressing a given antibody or antibody fragment and having at least one fusion protein 750 of their fluorescent capsid, can directly be used to fluoresce immunogenic labeling. 17) In some embodiments, the polyvalent immunization and diamond particle selection media are attached to magnetic, electrical, field-sensitive, field-like beads 755 or microparticles, such as silicon polystyrene latex beads, having magnetic so that said fields exert on the balls a force likely to move them, attract them, immobilize them. In some embodiments, the planar surfaces used to make the spot matrices will be a PMMA, Kapton, styrene, vinyl polymer surface including diamond particles from 10 nm to 500 μm. FIG. Phage display - affinity of phages-ScFv for fluorescent cadE measured by chip.

770 Figure-1.1: Phages-ScFv resulting from phage display selection by polyvalent immunization and selection media coated with Peptide Cadherin E. These phages are amplified, deposited on a slide and incubated with fluorescent peptide E-peptide. The amount of cadherin E peptides retained on the 775 spots is evaluated by a fluorescence scanner. Figure-1.2: Phages-ScFv from the phage display display by magnetic beads (without selection peptide). These phages are amplified, deposited on a slide and incubated with fluorescent peptide E-peptide. The quantity of 780 peptides E-peptide retained on the spots is evaluated by a fluorescence scanner. FIG. 1.3: Graphical representation of the quantity of fluorescence (bound to the amount of fixed fluorescent cadherin E peptides) by phage deposition in 785 depending on the stringency during washings of the phage display selection of the polyvalent immunization carriers and Eclectin E peptide-coated selection cells with phage capture. Figurel.4: Graphical representation of the amount of fluorescence (bound to the amount of fixed fluorescent Eglenerin E peptides) by phage deposition as a function of stringency during washings of phage display selection by magnetic beads (without peptide). selection). Figurel.5: Schematic representation of the 795 increase in stringency performed during the phage display steps. Figure-2: Infectivity of phages-ScFv associated with polyvalent media of immunization and selection particles of nanodiamonds 2pm.

800 Figure-2.6: Phages-ScFv captured by nanodiamonds coated with CadE, directly used to infect bacteria. The phages are amplified, purified, normalized, deposited on a slide and revealed by fluorescent CadE.

805 Figure-2.7: Phages-ScFv captured by nanodiamonds coated with triGlycine peptide (non-immunogenic), directly used to infect bacteria. The phages are amplified, purified, normalized, deposited on slides and revealed by CadE-fluorescent.

810 Figure-2.8: Quantification of the fixation of CadE on the deposit 6 by scanner. Fluorescence intensity expressed in arbitrary units (AU). Figure-2.9: Quantification of the fixation of CadE on the deposit 7 by scanner. Fluorescence intensity expressed in arbitrary units (AU).

815 Figure-3: Example of a solution of nano-diamonds of 2pm functionalized by Poly A probes and deposited on a slide. 10: non-labeled poly A probes 820 12: complementary fluorescent poly T strand 14: saturating fluorescent signal 825 13: very weak fluorescent signal Figure-3.10: After functionalization with unlabelled poly A probes (10), 830 Figure-3.11: l addition of fluorescent Poly A (11) or fluorescent BSA shows that the diamond surface is saturated and that the non-specific absorption is very weak (very weak fluorescent signal 3). Figure-3.12: In contrast, the hybridization of a complementary poly T 835 fluorescent strand (12) on the grafted probes, shows a very high level of functionalization of the particles (saturating fluorescent signal) (14).

Claims (14)

CLAIMS1) Focusing device for the selection, isolation, amplification or stimulation of a cellular or viral clone or an antibody or antibody part, characterized in that it comprises a polyvalent support comprising a phase of diamond carbon SP3 bound to an antigen by a covalent bond such as carbon / carbon, between a carbon of the diamond phase and an element of the antigen. 2) Device according to claim 1, characterized in that the support lOpolyvalent immunization and selection, 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 carrier 15immunization and diamond selection is achieved through a hydrogenated carbon phase CHx (x = 1,2,3) such that the hydrogenated carbon, optionally a carbon SP2, reacts with a carbon of the adjacent antigen of a nucleophilic function such as NH2, COOH, OH, optionally an electrophilic function. 20 4) Device according to any one of claims 1 to 3, characterized in that the polyvalent support of immunization and diamond selection is responsive to a field, magnetic, electric, so that said fields exert on the multipurpose support d immunization and selection a force capable of moving, attracting, immobilizing, repelling, and in a particular embodiment said polyvalent immunization and selection support will include particles having a magnetic heart, paramagnetic such that a core of iron, iron oxide, nickel, boron, magnetic silica, neodymium, and more generally at least one element sensitive to a magnetic field, electrical, any combination of these elements possibly directly integrated with the crystalline structure SP3 or SP2 of the particles of Odiamant. 5) Device according to any one of claims 1 to 3, characterized in that the polyvalent carrier for immunization and diamond selection is fixed on balls or microparticles sensitive to a field, magnetic, electrical, such that said fields exert on the balls a force likely to move them, to attract them, to immobilize them, to repel them. 6) Device according to any one of claims 1 to 5, for its use to immunize an animal or a human. 7) Device according to any one of claims 1 to 5 for its use to generate an ignition under the action of radiation, a magnetic field, an electric field. 8) In vitro focusing method for the selection, isolation, amplification, stimulation of a clone, cellular, viral, of an antibody or part of antibodies using a device according to any one of the Claims 1 to 5, characterized in that the polyvalent carrier for immunization and selection functionalised with an antigen, makes it possible to select at least one phage clone expressing a type of antibody such as at least one phage fixed on the polyvalent support. Immunization maintains an ability to infect bacteria. 9) Process according to claim 8, characterized in that a screening of a bank expresses all or part of an antibody such as said library, single chain phage, phage display, cell mixture, bacterial, viral or any other type eukaryotic or prokaryotic cell, express all or part of an antibody, and in that it comprises: at least one step of bringing the bank into contact with a polyvalent immunization and selection support, functionalized by a antigen of interest. At least one isolation step, polyvalent immunization and selection media, and library elements expressing an antigen-recognizing antibody or antibody moiety attached to the polyvalent carrier of the invention. immunization and selection - At least one step of culturing, bacterial infection, amplification, library elements expressing an antibody or antibody fraction recognizing the antigen and fixed on the polyvalent carrier of immu At least one deposition step on a preferably flat solid support, elements isolated from the bank so as to obtain a matrix of spots or deposits whose XY coordinates can identify the isolated elements of 301a bank, - At least a step of contacting hybridization complexing of the matrix with a solution of antigens optionally labeled with a fluorescent label, colorimetric, detectable by any physical detection means such as the signal intensity, fluorescent, of any other physical measurement allowing to quantify the antigens, at each spot of the matrix, at least one sequencing step, nucleic acid, quantitative semi-quantitative, isolated element, their culture, the results of bacterial infections by said isolated elements. 3025605 20 10) Process according to any one of claims 8 and 9, characterized in that a polyvalent carrier for immunization and selection will comprise in addition to the antigen of interest, at least one nucleic acid tag sequence or the like such that PNA 5 (nucleic acid peptide) or any other analog for identifying, specifically addressing the polyvalent immunization and selection support on a flat surface, a plate, combs, any object comprising nucleic acids complementary to the tag, antigens with the tag. 11) Method according to any one of claims 8 to 10, characterized in that a polyvalent immunization support comprises particles functionalized with different antigens, such as an antigen of interest and variants of the same antigen altered, in some embodiment each antigen or type of antigen attached to a particle is identifiable by a nucleic acid tag attached to said particle. 12) Method according to any one of claims 8 to 11, 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 which can be independently screened, such that the independent results are combined to make a molecule, antibody-forming antibody subunits, antibody fraction, synergistic so that the first selection of the screening of a first bank is used in the screening of a second bank. 25 13) Method according to any one of claims 8 to 12, characterized in that the phage banks, cells, used comprise elements such that phages or cells express fluorescent proteins. 14) Method according to any one of claims 8 to 13, characterized in that phage libraries are focused from cell extract, optionally fractionated, fixed on a polyvalent support of immunization and selection.