ONE-STEP FAST GRADIENT METHOD FOR NANOANTIBODY GENERATION

Abstract

One-step rapid gradient method for rapid generation of nanoantibodies (HHV) against any antigen. Where the method comprises the separation of nanoantibodies against a specific antigen from a nanoantibody expression library through the following steps: (a) binding the antigen of interest to beads made of protein-binding polymers (b) incubating the microorganisms of the expression library with the pearls of stage (a), (c) placing the beads of step (b) in a tube with an inert medium with a density greater than or equal to 1 g/mL and centrifuge for 45 s to 2 minutes at a rate between 150 and 250 g; d) discarding the upper fraction and supernatant with the free microorganisms, and obtain the beads with the linked microorganisms, which correspond to those that express HHV that recognizes the antigen.

Claims

1. Method of separation of nanoantibodies (HHV) against a specific antigen from a nanoantibody expression library CHARACTERIZED in that it comprises the following steps: (a) binding the antigen of interest to beads made of protein-binding polymers (b) incubating the microorganisms of the expression library with the beads of step (a), where the expression library corresponds to microorganisms transformed with the cDNA of fragments corresponding to the HHV domains of HHV-producing animal previously immunized with that antigen of interest, or synthetic libraries; (c) placing the beads of step (b) in a tube with an inert medium with a density greater than or equal to 1 g/mL and centrifuge for 45 s to 2 minutes at a rate between 150 and 250 g; d) discarding the upper fraction and supernatant with the free microorganisms, and obtaining the beads with the linked microorganisms, which correspond to those that express HHV that recognizes the antigen.

2. Method according to claim 1 CHARACTERIZED in that in step (a) the beads are sepharose, cellulose, latex, agarose or nickel and are modified to bind proteins.

3. Method according to claim 2 CHARACTERIZED in that in the beads have a density greater than the medium used in (c).

3. Method according to claim 1 CHARACTERIZED in that in step (c) the inert medium is reactive Ficol, percol or sucrose.

4. Method according to claim 3 CHARACTERIZED in that in step (a) the beads or equivalents bind stably to the antigen of interest in a time of between 2 to 12 hours.

5. Method according to claim 4 CHARACTERIZED in that sites which have not reacted with the antigen are blocked.

6. Method according to claim 1 CHARACTERIZED in that the microorganisms of the expression library are treated with a protein expression inducer for 2 to 4 hours prior to step (b).

7. Method according to claim 6 CHARACTERIZED in that the protein expression inducer is Isopropyl-β-D-1-tiogalactopyranoside (IPTG) at a concentration between 20 to 100 μM.

8. Method according to claim 1 CHARACTERIZED in that in step (b) is incubated for between 20 to 60 minutes at room temperature.

9. Method according to claim 1 CHARACTERIZED in that in step (d) the beads are washed with PBS and seeded on agar plates with culture medium in order to obtain isolated colonies.

10. Method according to claim 9 CHARACTERIZED in that each colony corresponds to a microorganism expressing an HHV capable of binding to the antigen of interest.

Description

DESCRIPTION OF THE FIGURES

[0022] FIG. 1. Schematic representation of the protocol for HHV isolation using density gradient separation: the bacterial presentation library (I) expressing HHV on the surface of bacteria is briefly incubated (II) with conventional sepharosase beads coated with the antigen of interest. Immediately after depositing the mixture in a conical Ficoll density gradient (III) tube and centrifuging at 200 g for 1 min, the beads pass through the sequential selection of the density gradient to the bottom of the tube with the bacteria expressing specific HHV, while the unbound bacteria remain on the gradient surface. Next, the beads are resuspended and (IV) the incubating bacterial clones are isolated in agar plate.

[0023] FIG. 2 Immunodetection of Spike-SARS-CoV2 in a nitrocellulose membrane with a pore size of 0.2 μm with the HHV of the invention as primary antibody, followed by mouse anti-Myc and goat anti-mouse IgG coupled to HRP. It includes photograph of each reaction for different nanoantibody selected with the method of the invention, as well as qualitative assessment (+, ++, +++)

[0024] FIG. 3 Dot Blot against recombinant and synthetic polyubiquitin chains, by the use of Nb No. 34, mouse anti-myc and HRP-coupled anti-mouse. Nb No. 34 was expressed in the plasmid pNae2 (fused with intimin) with labels 6xHis and Myc.

[0025] FIG. 4 Western Blot against recombinant and synthetic polyubiquitin chains, by the use of Nb No. 34, mouse anti-myc and mouse anti-HRP. Nb No. 34 was expressed in BL21 cells by the use of pNae2 (without intimin) with the Nb coding sequence between 6xHis and Myc.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention allows for obtaining nanoantibodies quickly and easily, against any type of antigen, such as viruses, bacteria or microorganisms usually proteins or molecules, among others. But due to its speed, the method of this invention is very efficient for the generation of nanoantibodies against pathogens of emerging diseases, such as COVID.

[0027] The invention relates to a method of separation of nanoantibodies (HHV) against a specific antigen from a nanoantibody expression library, wherein the method comprises the following steps: [0028] (a) binding the antigen of interest to polymeric beads that allow protein binding [0029] (b) incubating the microorganisms of the expression library with the beads of step (a), where the expression library corresponds to microorganisms transformed with the cDNA of fragments corresponding to the HHV domains of HHV-producing animal previously immunized with that antigen of interest, or synthetic libraries; [0030] (c) placing the beads of step (b) in a tube with an inert medium with a density greater than or equal to 1 g/mL and centrifuging for 45 s to 2 minutes at a rate between 150 and 250 g; [0031] d) discarding the upper fraction and supernatant with the free microorganisms, and obtaining the beads with the linked microorganisms, which correspond to those that express HHV that recognizes the antigen.

[0032] Where the beads used are sepharose, cellulose, latex, agarose or nickel and can be modified to bind proteins, and have a density greater than the medium used in (c). These beads bind to the antigen of interest in a stable manner in a time of between 2 to 12 hours and then the sites that have not reacted with the antigen are blocked, by any means available in the technique.

[0033] The bond between the beads and the antigen can be non-covalent covalent.

[0034] Where this inert medium of stage (c) is preferably chosen between Ficol, percol or sucrose.

[0035] To ensure that the microorganisms in the expression library are expressing HHV they should be treated with a protein expression inducer for 2 to 4 hours prior to step (b). In one embodiment, the protein expression inducer is IPTG at a concentration between 20 to 100 μM. Later in step (b) the microorganisms of the expression library are incubated with the beads attached to the antigen for between 20 to 60 minutes at room temperature.

[0036] Finally, after separation in the density gradient, in step (d) the beads are washed with PBS and seeded on agar plates with culture medium in order to obtain isolated colonies. Where each colony corresponds to a microorganism that expresses an HHV capable of binding to the antigen of interest.

[0037] First, you must have a nanoantibody or HHV library.

[0038] If you want to generate this library yourself, you require a purified antigen, which can be obtained with a system of expression of baculovirus, bacteria, yeast or directly from its natural source. Next, an animal producing nanoantibodies, such as an alpaca or llama, is immunized between one-to four times with 50 to 500 μg of the antigen. The immune response of alpaca serum against the antigen after immunization can be observed rapidly and qualitatively by means of Dot Blot analysis, by immobilization of the epitope to a nitrocellulose membrane and by the use of alpaca serum as a source of primary antibodies; or analytically and comparatively by means of ELISA by the use of the complete antigen that is immobilized in the ELISA plate and the alpaca serum as a source of primary antibodies. Thus, a bacterial presentation library of individual nanoantibody clones is quickly built.

[0039] Once the library is obtained, the method of the invention is applied for the selection of nanoantibodies based on a density gradient, for example the simple use of Ficoll, percol or sucrose. Ficoll is an inexpensive reagent available worldwide commonly used for the fragmentation of blood samples. The inventors developed the method of the invention inspired by the main observation that red blood cells accumulate at the bottom of the Ficoll density gradient, while PBMCs remain in the upper fraction.

[0040] The inventors created a method using beads made of polymers such as conventional sepharose or agarose which, in a density gradient, such as one obtained by the Ficoll reagent have shown that the density of the chosen beads was suitable for precipitating at the bottom of a 15 ml tube. At the same time, the inventors showed that, when performing the same test with free bacteria, the bacteria remained in the upper fraction. Revealing the possibility of separating free bacteria from those that express HHV and bind beads with the antigen, in a density gradient.

[0041] To obtain specific clones of HHV or nanoantibodies, a bacterial presentation system is usually used, where each bacterium expresses a single type of nanoantibody in its outer bacterial membrane after IPTG induction. Therefore, the inventors obtained sefarose beads coated with the antigen of interest to bind to individual bacteria expressing specific nanoantibodies on their surface against said antigen. Thus, bacteria expressing an HHV or nanoantibody that binds to the antigen sink to the bottom of the density gradient, while unbound bacteria would remain in the upper fraction (FIG. 1).

Examples

Example 1. The Simple Density Gradient Method of the Invention and its use to Obtain Nanoantibodies Against the Spike Protein of SARS-CoV-2

[0042] First, the inventors obtained the lyophilized SARS-CoV-2 Spike protein generated in a baculovirus expression system. Prior to immunization, Spike protein integrity was tested by SDS-Page and Coomassie staining. An alpaca named Buddha was immunized twice with 100 μg of the entire Spike protein. The immune response of alpaca serum prior to immunization revealed a fortunate basal cross-reaction against the Spike protein. Then, after the second immunization, a significant increase in IgG antibodies was observed in alpaca serum qualitatively rapidly by Dot Blot analysis, by immobilization of the epitope in a nitrocellulose membrane and by the use of alpaca serum as a primary antibody source (FIG. 1). In addition, the inventors verified the increase of IgG antibodies (analytically and comparatively) by means of ELISA by the use of the complete Spike protein immobilized in the ELISA plate and by the use of alpaca serum as a source of primary antibodies. Therefore, the inventors quickly built a bacteria presentation library consisting of 2.3×10.sup.6 clones of individual nanoantibodies with 0.7% vector religation.

Building Immunization and HHV Libraries

[0043] The alpaca immunization process followed the protocol “Animal use in research” generated by the Bioethics Committee of the Universidad Austral de Chile. One day before immunization, 5 ml of blood was collected for preimmune serum testing. For immunization (day 1), 100 μg of full-length SARS-CoV2 spike protein (SINOBiological) was used. The cold lyophilized protein was dissolved in 2 ml of adjuvant (veterinary vaccine adjuvant, GERBU FAMA) diluted 1:1 in sterile water and injected subcutaneously into a male alpaca (Vicugna pacos). A total volume of 4 ml was injected into four different sites of the alpaca. A 5 ml blood sample was collected seven days after the first immunization. On day 14, the alpaca was immunized again with 100 μg of Spike and on day 15 a 120 ml sample of blood was collected from the jugular vein in tubes containing 3.8% sodium citrate as an anticoagulant. The noncoagulated blood sample was mixed with the same volume of calcium-free HBSS medium (Gibco), divided into 10 ml aliquots, and 5 ml of Ficoll-Paque Premium (GE Healthcare) was added on top of each aliquot in 15 ml of sterile Falcon tubes. After centrifugation (1,200×rpm, 80 min, room temperature), the PBMC fraction was recovered from the interface, washed twice in HBSS by centrifugation (3,500×rpm, 10 min), resuspended in 4 ml sterile PBS 1X (Gibco). RNA extraction and cDNA production were carried out using the commercial RNeasy Mini (Qiagen) kit and the QuantiTect Reverse Transcription Kit (Qiagen), respectively. Approximately 2 μl of each synthesized cDNA was used as a template in a total PCR reaction volume of 50 μl with the oligonucleotides CALL001 (5′-GTC CTG GCT CTC TTC TAC AAG G-3′) and CALL002 (5′-GGTACGTGCTGTTGAACTGTTCC-3′) (Conrath et al., 2001). The amplified fragments of ˜0.6 kb, corresponding to the VHH-CH2 domains, and ˜0.9 kb, corresponding to the conventional VH-CH1-CH2 domains, were separated into 1.2% low melting point agarose gel (w/v) and a band of ˜0.6 kb was purified (QIAEX II Gel Extraction Kit, Qiagen). This fragment was used as a template in a second PCR reaction with oligonucleotides VHH-Sfi2 (5′-GTC CTC GCA ACT GCG GCC CAG CCGGCC ATG GCT CAG GTG CAG CTG GTG GA-3′) and VHH-Nott (5′-GGA CTA GTG CGG CCG CTG AGG AGA CGG TGA CCT GGG T-3′) to finally obtain the amplified fragments of ˜0.4 kb, corresponding to VHH domains. The amplified HHV fragments were digested with Sfi I and Notl (Thermo Scientific) restriction enzymes and bound at the same sites of the purified pNeae2 vector (Salema et al., 2016). The ligations were subjected to electroporation in cells of E. coli DH10B-T1 R achieving a library size of 2.3×10.sup.6 individual clones, as determined by seeding on LB-Cloramphenicol agar plates with glucose at 2% w/v incubated at 30° C. Less than 0.7% of vectors re-linked from a control ligation carried out in parallel without the DNA insert was estimated. The transformed bacteria were scraped off the plates and stored at −80 degrees in LB broth with 30% glycerol.

[0044] Once the library was obtained, the inventors applied the method of the invention for the selection of nanoantibodies based on a simple density gradient by the use of Ficoll. (FIG. 1).

Coupling Epitopes to Beads

[0045] 1 ml of NHS-activated sepharose 4 Fast Flow beads (General Electric) with 2 ml of cold HCl 1 mM were washed immediately before use, then washed 5 times with PBS (Saline phosphate buffer) 1Xsterile cold. 200 μg of purified protein in PBS 1X were added to the beads and incubated in rotation until the next day. The groups that did not react in the medium were blocked by the addition of ethanolamine at a final concentration of 0.5 M. The beads were washed 5 times with PBS 1X and stored at 4° C.

Density Gradient Separation

[0046] 1 ml of glycerol stock solution from the library was inoculated into a flask containing 20 ml of LB medium with 25 μg/ml chloramphenicol and 2% glucose. The flask was incubated (pre-inoculum) until the next day at 37° C. with 200 rpm stirring. The same procedure was repeated with control bacteria transformed with a kanamycin-resistant plasmid (control). The preinoculum was sedimented and resuspended in LB medium with 25 μg/mL chloramphenicol and then diluted to 0.02 optical density at 600 nm in 100 ml of fresh LB medium with 25 μg/mL chloramphenicol without glucose, incubated at 37° C. with 200 rpm stirring to reach an optical density of 600 nm of 0.45 to 0.6. IPTG was added at a final concentration of 50 μM to induce protein expression for 3 hours at 30° C. and 200 rpm. The OD 600 nm absorbance of the library and control bacteria cultures was measured. 50 mL of both cultures were washed three times with 10 mL of PBS 1X filtered. Centrifugation was always at 3000×g for 5 min. Both cultures were resuspended in a final volume of 10 ml of PBS 1X. 2 ml of library culture and 2 ml of control culture were mixed (if the final 600 nm optical density was the same, if not, the volume of the control bacteria was adjusted according to the OD to ensure an equal number of bacteria) and incubated with 300 μL of beads coupled to the epitope in a 15 ml conical tube on a rotating platform for 30 min at temperature environment. The mixture was added to Ficoll 6 ml (Ficoll-Paque™ PLUS GE Healthcare) in a 15 ml conical tube and centrifuged at 200×g for 1 min. The unbound fraction was discarded (upper fractions).

[0047] The visible bead sediment contains bacteria that express an HHV that binds to antigens, in this case Sars-CoV2 Spike. This sediment was resuspended in 4 ml of PBS 1X and rotated for 5 min at room temperature. This step was repeated six times, in order to eliminate any bacteria not attached to the beads.

[0048] In order to amplify the bacteria attached to the beads, 1 mL of LB medium was added and incubated for 5 min at room temperature, then 50 μL were seeded in LB agar plates with 25 μg/mL chloramphenicol and 2% glucose a, incubated at 37° C. until the next day (>20 hours recommended).

[0049] Where each colony obtained corresponds to a bacterium containing a nanoantibody or HHV that binds specifically to the antigen of interest.

Binding Test of Selected HHV to SARS-CoV-2 Spike

[0050] To verify that the nanoantibodies of the invention bind to the Spike protein, an immunoblot was made for the 22 nanoantibodies, which faced Spike-GFP expressed in natural viral conditions in human cells (FIG. 2).

[0051] It was observed that all nanoantibodies selected by the method of the invention were attached to the Spike antigen of SARS-CoV-2. The results demonstrate that the method of the invention allows to quickly and efficiently select nanoantibodies that bind specifically to an antigen of interest, such as the SARS-CoV2 Spike.

Ubiquitin

[0052] Ubiquitin is the founding member of the UBL family. It participates in many biological processes, but its most studied effect is its covalent conjugation with other proteins, which targets proteins modified for proteasome-mediated degradation. Ubiquitin is one of the most conserved proteins in eukaryotes, however, it is found neither in bacteria nor in archaea. In humans, 14 copies of ubiquitin are expressed from different loci. Ubiquitin translates as an inactive precursor that is activated by means of a proteolytic cleavage immediately behind its amino acid glycine 76 by the same enzymes also responsible for the deubiquitination of ubiquitin substrates. Mature and active ubiquitin is a small globular protein of 76 amino acids (˜8 KDa) [107]. Covalent modification of proteins by ubiquitin (ubiquitination) is defined as the formation of an isopeptide bond between the E-amino group of a lysine in the target protein and the C-terminal glycine (G76) of ubiquitin. Ubiquitination occurs in different ways that regulate biological processes. It changes the specific properties of the target proteins that depend on i) the target itself and ii) the way ubiquitin binds. Ubiquitination of proteins with a single ubiquitin molecule (mono-ubiquitination) has been described as regulating biological processes such as endocytosis, trafficking, and gene expression regulation.

[0053] To exemplify the method of the invention VHH against ubiquitin were developed.

Example 2. The Simple Density Gradient Method of the Invention and its use to Obtain Nanoantibodies Against Ubiquitin

[0054] First, the inventors obtained the various chains of polyubiquitins (K6, K11, K27, K29, K33, K48 and K63). An alpaca named Nick was immunized four times with 100 μg of the K48 polyubiquitin chain. Then, after the third immunization, a significant increase in IgG antibodies was observed in alpaca serum qualitatively rapidly by Dot Blot analysis, by immobilization of the epitope in a nitrocellulose membrane and by the use of alpaca serum as a source of primary antibodies. Therefore, the inventors quickly built a bacterial presentation library consisting of 1×10.sup.6 clones of individual nanoantibodies with 1.2% vector religation.

Building Immunization and HHV Libraries

[0055] The alpaca immunization process followed the protocol “Animal use in research” generated by the Bioethics Committee of the Universidad Austral de Chile. One day before immunization, 5 ml of blood was collected for preimmune serum testing. For immunization (day 1), 100 μg of polyubiquitin k48 chain was used. The cold lyophilized protein was dissolved in 2 ml of adjuvant (veterinary vaccine adjuvant, GERBU FAMA) diluted 1:1 in sterile water and injected subcutaneously into a male alpaca (Vicugna pacos). A total volume of 4 ml was injected into four different sites of the alpaca. A 5 ml blood sample was collected seven days after the first immunization. On day 14, the alpaca was immunized again with 100 μg of the polyubiquitin k48 chain and so on for two more immunizations, until a day after the fourth immunization a 120 ml sample of blood was collected from the jugular vein in tubes containing 3.8% sodium citrate as an anticoagulant. And a library was generated in the same way as indicated for example 1.

[0056] Once the library was obtained, the inventors applied the method of the invention for the selection of nanoantibodies based on a simple density gradient.

Coupling Epitopes to Beads

[0057] 1 ml of NHS-activated sepharose 4 Fast Flow beads (General Electric) with 2 ml of cold HCl 1 mM were washed immediately before use, then washed 5 times with cold sterile PBS. 200 μg of polyubiquitin k48 chains in PBS1X were added to the beads and incubated in rotation until the next day. The groups that did not react in the medium were blocked by the addition of ethanolamine at a final concentration of 0.5 M. The beads were washed 5 times with PBS 1X and stored at 4° C.

Density Gradient Separation

[0058] 1 ml of glycerol stock solution from the library was inoculated into a flask containing 20 ml of LB medium with 25 μg/ml chloramphenicol and 2% glucose. The flask was incubated (pre-inoculum) until the next day at 37° C. with 200 rpm stirring. The same procedure was repeated with control bacteria transformed with a kanamycin-resistant plasmid (control). The preinoculum was sedimented and resuspended in LB medium with 25 μg mL-1 chloramphenicol and then diluted to 0.02 OD 600 nm in 100 ml of fresh LB medium with 25 μg mL-1 chloramphenicol without glucose, incubated at 37° C. with 200 rpm stirring to a OD 600 nm of 0.45 to 0.6. IPTG was added at a final concentration of 50 μM to induce protein expression for 3 hours at 30° C. and 200 rpm. The OD 600 nm absorbance of the library and control bacteria cultures was measured. 50 mL of both cultures were washed three times with 10 mL of filtered PBS 1C. Centrifugation was always at 3000×g for 5 min. Both cultures were resuspended in a final volume of 10 ml of PBS1X. 2 ml of library culture and 2 ml of control culture were mixed (if the final 600 nm optical density was the same, if not, the volume of the control bacteria was adjusted according to the optical density to ensure an equal number of bacteria) and incubated with 300 μL of NHS beads coupled to the epitope protein in a 15 ml conical tube on a rotating platform for 30 min at room temperature. The mixture was added to Ficoll 6 ml (Ficoll-Paque™ PLUS GE Healthcare) in a 15 ml conical tube and centrifuged at 200×g for 1 min. Unbound fraction was discarded (upper fractions)

[0059] The visible bead sediment contains bacteria that express an HHV that binds to antigens, in this case ubiquitin. This sediment was resuspended in 4 ml of PBS1X and rotated for 5 min at room temperature. This step was repeated six times, in order to eliminate any bacteria not attached to the beads.

[0060] In order to amplify the bacteria attached to the beads, 1 mL of LB medium was added and incubated for 5 min at room temperature, then 50 μL were seeded in LB agar plates with 25 μg/mL chloramphenicol and 2% glucose incubated at 37° C. until the next day (>20 hours recommended).

[0061] Where each colony obtained corresponds to a bacterium containing a nanoantibody or HHV that binds specifically to the antigen of interest

Confirmation of Antigen Binding

[0062] The bacterial presentation system expresses nanoantibodies on the surface of bacteria fused with an intein protein and a myc tag. Buffer conditions were optimized to extract nanoantibody-intein fusion from the bacterial membrane and bacterial extract was used directly to confirm binding to polyubiquitin chains by dot blot. After selection of nanoantibodies using our simple density gradient protocol, 100 colonies were used to inoculate liquid LB medium and additionally induced for the expression of intein nanoantibodies. The cells were lysed under optimized conditions and the extract was used as a source of nanoantibodies as primary antibodies for the selection of antibodies that bind effectively by dot blot of the various chains of polyubiquitins immobilized in a nitrocellulose membrane.

[0063] They were revealed by sequential incubation with mouse anti-myc antibody and an antibody conjugated with anti-mouse HRP.

[0064] In this way, it was possible to identify Nb. No. 34 capable of recognizing all the ubiquitin chains tested by Dot Blot (FIG. 3) and also by Western Blot (FIG. 4). This was expressed in the plasmid pNae2 (fused with intimin) with 6xHis and Myc tags and then purified by nickel affinity chromatography. To perform the western blot, the different chains of polyubiquitins were separated electrophoretically in a 12% polyacrylamide gel and then transferred by electrotransfer to a nitrocellulose membrane which was incubated with the extract of Nb. No. 34 and successively with anti-myc antibody and anti-mouse antibody coupled to HRP.

[0065] Thus the invention proved useful for quickly and efficiently selecting nanoantibodies that bind specifically to an antigen of interest.