US20230324403A1 - One-step fast gradient method for nanoantibody generation - Google Patents

One-step fast gradient method for nanoantibody generation Download PDF

Info

Publication number
US20230324403A1
US20230324403A1 US17/998,753 US202117998753A US2023324403A1 US 20230324403 A1 US20230324403 A1 US 20230324403A1 US 202117998753 A US202117998753 A US 202117998753A US 2023324403 A1 US2023324403 A1 US 2023324403A1
Authority
US
United States
Prior art keywords
beads
antigen
nanoantibodies
hhv
microorganisms
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US17/998,753
Other languages
English (en)
Inventor
Alejandro Rojas
Guillermo VALENZUELA
Ronald JARA
Johanna HIMELREICHS
Constanza SALINAS
Teresa PINTO
Natalia LÓPEZ
Yorka CHEUQUEMILLA
Alexei CUEVAS
Zaray MIRANDA
Benjamín UBERTI
Ananda MULLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad Austral de Chile
Original Assignee
Universidad Austral de Chile
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad Austral de Chile filed Critical Universidad Austral de Chile
Priority to US17/998,753 priority Critical patent/US20230324403A1/en
Assigned to UNIVERSIDAD AUSTRAL DE CHILE reassignment UNIVERSIDAD AUSTRAL DE CHILE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PINTO, Teresa, UBERTI, Benjamín, MULLER, Ananda, ROJAS, ALEJANDRO, HIMELREICHS, Johanna, LÓPEZ, Natalia, MIRANDA, Zaray, CHEUQUEMILLA, Yorka, CUEVAS, Alexei, JARA, Ronald, SALINAS, Constanza, VALENZUELA, Guillermo
Publication of US20230324403A1 publication Critical patent/US20230324403A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention relates to a new one-step fast gradient method for the rapid separation of nanoantibodies against any antigen.
  • HCAb single-domain antibodies
  • the Camelidae or camelid family is composed of camels, dromedaries, llamas, vicu ⁇ as, guanacos and alpacas.
  • the antigen-binding fragment of an HCAb contains a single variable HHV domain consisting of 3 hypervariable regions (RDA). When isolated, the HHV domain is also known as a nanoantibody.
  • Target-specific nanoantibodies derived from camelid HCAbs are obtained after immunization with the antigen, plus the adjuvant. Our platform has developed an improved procedure for obtaining nanoantibodies by using alpacas as a donor species.
  • RNA RNA
  • cDNA preparation to finally amplify the nanoantibody region.
  • the cDNA fragment encoding the nanoantibody is as short as 360 nt, and up to ⁇ 3 ⁇ 10 6 individual clones can be obtained from 120 ml of blood from an immunized alpaca.
  • a bacteria or yeast visualization system is used to clone the various complete individual nanoantibodies, generating what is known as a nanoantibody or HHV library.
  • Microorganism presentation technology allows nanoantibodies to be expressed on the surfaces of microorganisms and therefore researchers can separate and enrich bacteria or yeasts expressing the nanoantibody of interest on their surface by means of affinity purification. Final isolated nanoantibodies are expressed recombinantly in bacteria and their binding capabilities can be characterized by ELISA and quantitative biochemical parameters such as ITC. In addition to targeted immunization, it is also possible to use very large libraries of nanoantibodies (1 ⁇ 10 9 ) to find binding nanoantibodies using a stochastic approach. The nanoantibodies are then produced in a renewable and economical manner.
  • single-domain antibodies HCAb
  • HCAb single-domain antibodies
  • the Camilidae or camelid family is composed of camels, dromedaries, llamas, vicu ⁇ as, guanacos and alpacas.
  • the antigen-binding fragment of an HCAb contains a single variable HHV domain consisting of 3 hypervariable regions. When isolated, the HHV domain is also known as a nanoantibody.
  • Target-specific nanoantibodies derived from camelid HCAb are usually obtained rapidly after immunization with the most adjuvant target protein. Analysis of the structure of the nanoantibody reveals how hypervariable regions project in loops outside the structure of the nucleus.
  • nanoantibodies are the best tools available today for affinity-based diagnostics and therapies.
  • nanoantibodies Purification of nanoantibodies is simple compared to any other source of antibodies. They are often expressed attached to an affinity label, such as 6 ⁇ histidine labels, to allow affinity purification. Enrichment often sets in the bacterial periplasm where the oxidizing environment allows for the formation of suitable disulfide bonds. Several milligrams of a liter of culture can be isolated and recombinant isolated nanoantibodies can be further isolated using standard biochemical techniques.
  • affinity label such as 6 ⁇ histidine labels
  • Nanoantibodies are small, compact polypeptides and are often expressed in the periplasm of bacteria. They are very stable at high temperatures, starting at 6° C. compared to human VH, and are also resistant to denaturing chemical agents.
  • molecular engineering of the nanoantibody structure has shown that stability increases when a cysteine is introduced at positions 54 and 78 to form an additional disulfide bond.
  • the resulting superstable nanoantibody is also more resistant to proteases such as pepsin or chymotrypsin.
  • Nanoantibodies can be used as therapeutic bullets against tumors, pathogens and chronic diseases, however, as foreign proteins, they could trigger an immune response on their own. Fortunately, the small size, rapid blood clearance and high homology with the human variable region of the VH heavy chain make them immunogenically small. Only some amino acids differ between nanoantibodies and human OAB, replacing these camelid amino acids with human amino acids has been used to humanize camelid nanoantibodies and make them even safer for therapies.
  • Nanoantibodies are strict monomers, their affinity for substrates depends on the projection of the three hypervariable loops. Consequently, nanoantibodies tend to interact with cavities of the spatial structure of polypeptides, but not efficiently with peptides. For example, several identified nanoantibodies directly block active enzyme sites. The FC5 nanoantibody can even cross the blood-brain barrier via transcytosis and form partially bispecific antibodies used for therapeutic approaches. 52.53. Finally, molecular accessibility impacts access to macromolecular complexes.
  • Nanoantibodies have been used for the purification by affinity of proteins in a specific way and even for the detection of post-translational modifications. However, the number of nanoantibodies available is very limited and many more are needed. Nanoantibodies can be used for various experimental environments such as ELISA, immunoprecipitation, CHIP-seq, immunostaining, to induce degradation of specific proteins by recruiting degradation machinery and blocking and activating cellular pathways.
  • a nanoantibody obtained from alpacas specifically recognizes the green fluorescence protein, known as the GFP trap system, became the first-line tool for interaction and proteomics studies based on its extraordinary affinity and ability to bind to GFP fusion proteins. Today, it can even be used for GFP extraction from endogenously labeled proteins in combination with CRISPR/Cas9. GFP nanoantibody can also be coupled to fluorescent dyes to detect GFP by microscopy approaches when GFP levels are very low.
  • nanoantibodies When a nanoantibody binds to a three-dimensional structure, some properties of the target protein change.
  • One of the common features is that nanoantibodies limit the conformational changes of the target protein by promoting a unique conformation step. Surprisingly, these phenomena positively impact the possibilities of crystallization, therefore, nanoantibodies have been used as helpers to facilitate protein crystallization.
  • Nanoantibodies are a superior tool for diagnosis. Their unlimited in vitro production capacity makes nanoantibodies more reliable than conventional antibodies and independent of batch preparation or animal serum limitations. Nanoantibodies can be produced as a protein fused with informant peptides or proteins for staining or direct visualization, including affinity tags (Flag, HA, V 5 , and cMyc), fluorescent proteins (GFP, RFP, etc.), and enzymes for colorimetric measurements such as horseradish peroxidase (HRP).
  • affinity tags Flag, HA, V 5 , and cMyc
  • fluorescent proteins GFP, RFP, etc.
  • enzymes for colorimetric measurements such as horseradish peroxidase (HRP).
  • ELISA assays can be improved by the use of nanoantibodies for specific immobilization or detection by the use of a specific nanoantibody coupled to horseradish peroxidase (HRP).
  • Nanoantibodies meet most of the requirements of an ideal probe for successful molecular imaging.
  • imaging techniques such as SPECT, PET, optical imaging and ultrasound have been successfully employed for molecular imaging by the use of in vivo nanoantibodies.
  • SPECT specific targeted nanoantibodies are coupled to an irradiation source y administered systemically.
  • the nanoantibodies are then detected in a whole-body scanner.
  • the PET strategy is similar to SPECT, but instead uses positron-emitting radionuclide-labeled nanoantibodies.
  • These imaging technologies are used for various tumor diagnostic techniques in animal models: for example, an anti-EGFR nanoantibody can be used to detect human squamous cell carcinoma and human prostate carcinoma.
  • nanoantibodies have been developed in the context of different experimental therapeutic applications against different viruses: HIV, hepatitis B virus, influenza virus, respiratory syncytial virus, rabies virus, foot-and-mouth disease virus, poliovirus, rotavirus and PERV.
  • nanoantibodies can neutralize HIV infection; cell-to-cell spread has been inhibited by the use of HIV isolated from patients. Due to the low immunoreactivity of nanoantibodies, they can be injected into patients with very few or no side effects.
  • nanoantibodies can be linked to produce bivalent, multivalent, and/or multispecific nanoantibodies, or combined with other nanoantibodies or circulating proteins such as albumin to increase their turnover and therapeutic effectiveness.
  • the rabies virus causes a lethal brain infection in people. Shortly after exposure, rabies prophylaxis with immunoglobulins and plasma-derived vaccines is administered. Often, this occurs directly after the attack of an animal that might be infected. Anti-rabies nanoantibodies can significantly prolong survival or even completely cure the disease in animal models.
  • the respiratory syncytial virus, RSV is one of the leading causes of hospitalization in children, each year more than 1.9 million children under 1 year of age are infected and there are more than 0.3 million children under 5 years of age hospitalized. Current RSV therapy is not available. However, trivalent nanoantibody-based therapy is in phase II clinical trials.
  • the absolute novelty of the RSV therapy developed by Ablynx, ALX-0171, is the direct neutralization of the virus in the lung of infected experimental animals.
  • the nanoantibody is administered by nebulization and reduces the virus titer by 10,000 times. Nanoantibodies are also used for cancer immunotherapies.
  • 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.
  • FIG. 2 Immunodetection of Spike-SARS-CoV2 in a nitrocellulose membrane with a pore size of 0.2 ⁇ m 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 (+, ++, +++)
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • the bond between the beads and the antigen can be non-covalent covalent.
  • this inert medium of stage (c) is preferably chosen between Ficol, percol or sucrose.
  • the protein expression inducer is IPTG at a concentration between 20 to 100 ⁇ M.
  • the microorganisms of the expression library are incubated with the beads attached to the antigen for between 20 to 60 minutes at room temperature.
  • 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.
  • a purified antigen which can be obtained with a system of expression of baculovirus, bacteria, yeast or directly from its natural source.
  • an animal producing nanoantibodies such as an alpaca or llama, is immunized between one-to four times with 50 to 500 ⁇ g 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.
  • a bacterial presentation library of individual nanoantibody clones is quickly built.
  • 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.
  • 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 ).
  • Example 1 The Simple Density Gradient Method of the Invention and its use to Obtain Nanoantibodies against the Spike Protein of SARS-CoV-2
  • 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.
  • 100 ⁇ g 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.
  • adjuvant veterinary vaccine adjuvant
  • RNA extraction and cDNA production were carried out using the commercial RNeasy Mini (Qiagen) kit and the QuantiTect Reverse Transcription Kit (Qiagen), respectively.
  • 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.
  • VHH-Sfi2 5′-GTC CTC GCA ACT GCG GCC CAG CCGGCC ATG GCT CAG GTG CAG CTG GTG GA-3′
  • VHH-Nott 5′-GGA CTA GTG CGG CCG CTG AGG AGA CGG TGA CCT GGG T-3′
  • 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 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.
  • 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.
  • 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 ).
  • Mature and active ubiquitin is a small globular protein of 76 amino acids ( ⁇ 8 KDa) [107].
  • Covalent modification of proteins by ubiquitin 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 (G 7 6) 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 has been described as regulating biological processes such as endocytosis, trafficking, and gene expression regulation.
  • VHH against ubiquitin were developed.
  • Example 2 The Simple Density Gradient Method of the Invention and its use to Obtain Nanoantibodies Against Ubiquitin
  • 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 ⁇ g of the K48 polyubiquitin chain.
  • 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 6 clones of individual nanoantibodies with 1.2% vector religation.
  • 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.
  • 100 ⁇ g of polyubiquitin k 48 chain was used for immunization (day 1 ).
  • 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.
  • the alpaca was immunized again with 100 ⁇ g 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.
  • the inventors applied the method of the invention for the selection of nanoantibodies based on a simple density gradient.
  • the preinoculum was sedimented and resuspended in LB medium with 25 ⁇ g mL-1 chloramphenicol and then diluted to 0.02 OD 600 nm in 100 ml of fresh LB medium with 25 ⁇ g 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 ⁇ M 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.
  • 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.
  • each colony obtained corresponds to a bacterium containing a nanoantibody or HHV that binds specifically to the antigen of interest
  • 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.
  • 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.
  • the invention proved useful for quickly and efficiently selecting nanoantibodies that bind specifically to an antigen of interest.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Pulmonology (AREA)
  • Plant Pathology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
US17/998,753 2020-05-15 2021-05-14 One-step fast gradient method for nanoantibody generation Pending US20230324403A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/998,753 US20230324403A1 (en) 2020-05-15 2021-05-14 One-step fast gradient method for nanoantibody generation

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US202063025534P 2020-05-15 2020-05-15
US17/998,753 US20230324403A1 (en) 2020-05-15 2021-05-14 One-step fast gradient method for nanoantibody generation
PCT/CL2021/050039 WO2021226728A1 (fr) 2020-05-15 2021-05-14 Méthode de gradient rapide à un seul passage pour la production d'anticorps

Publications (1)

Publication Number Publication Date
US20230324403A1 true US20230324403A1 (en) 2023-10-12

Family

ID=78525891

Family Applications (2)

Application Number Title Priority Date Filing Date
US17/998,753 Pending US20230324403A1 (en) 2020-05-15 2021-05-14 One-step fast gradient method for nanoantibody generation
US18/055,589 Pending US20240067705A1 (en) 2020-05-15 2022-11-15 Single domain vhh antibodies against sars-cov-2 virus

Family Applications After (1)

Application Number Title Priority Date Filing Date
US18/055,589 Pending US20240067705A1 (en) 2020-05-15 2022-11-15 Single domain vhh antibodies against sars-cov-2 virus

Country Status (9)

Country Link
US (2) US20230324403A1 (fr)
EP (2) EP4151784A4 (fr)
KR (1) KR20230065931A (fr)
CN (2) CN115867350A (fr)
AR (1) AR122104A1 (fr)
BR (1) BR112022023168A2 (fr)
CA (1) CA3178663A1 (fr)
CL (2) CL2022003165A1 (fr)
WO (3) WO2021229540A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115043934B (zh) * 2022-03-31 2023-06-27 深圳市人民医院 靶向新冠病毒的纳米抗体及其制备方法和应用
CN114478758B (zh) * 2022-04-01 2022-06-21 深圳市人民医院 靶向SARS-CoV-2的纳米抗体及制备方法和应用
CN116375852B (zh) * 2023-03-10 2024-02-02 华南农业大学 重组荧光纳米抗体及其在制备狂犬病病毒检测试剂中的应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1558645B1 (fr) * 2002-11-08 2011-07-27 Ablynx N.V. Anticorps a domaine unique stabilises dans une composition pharmaceutique pour inhalation
US20130332133A1 (en) * 2006-05-11 2013-12-12 Ramot At Tel Aviv University Ltd. Classification of Protein Sequences and Uses of Classified Proteins
CA2677383A1 (fr) * 2007-02-09 2008-08-21 Medimmune, Llc Banque d'anticorps presentes sur la membrane plasmique de cellules de levure
WO2010033913A1 (fr) * 2008-09-22 2010-03-25 Icb International, Inc. Anticorps, analogues et leurs utilisations
US20110214205A1 (en) * 2010-02-26 2011-09-01 Monsanto Technology Llc. Isolated Novel Nucleic Acid and Protein Molecules from Foxtail Millet and Methods of Using Those Molecules to Generate Transgenic Plants with Enhanced Agronomic Traits

Also Published As

Publication number Publication date
BR112022023168A2 (pt) 2023-02-07
EP4149626A2 (fr) 2023-03-22
AR122104A1 (es) 2022-08-10
WO2021229540A2 (fr) 2021-11-18
CN115867350A (zh) 2023-03-28
US20240067705A1 (en) 2024-02-29
EP4149626A4 (fr) 2024-07-24
EP4151784A1 (fr) 2023-03-22
WO2021226729A1 (fr) 2021-11-18
WO2021226728A1 (fr) 2021-11-18
CN115917061A (zh) 2023-04-04
WO2021229540A3 (fr) 2021-12-23
CL2022003165A1 (es) 2023-03-24
CA3178663A1 (fr) 2021-11-18
KR20230065931A (ko) 2023-05-12
CL2022003173A1 (es) 2023-07-07
EP4151784A4 (fr) 2024-08-07

Similar Documents

Publication Publication Date Title
US20230324403A1 (en) One-step fast gradient method for nanoantibody generation
US11866785B2 (en) Tumor specific antibodies and T-cell receptors and methods of identifying the same
JP5941615B2 (ja) ヒトｃｘｃｌ１タンパク質の免疫学的測定方法
KR102671155B1 (ko) Ns1 단백질의 결합 단백질
US20210301000A1 (en) NS1-Binding Protein and Uses Thereof
CN106866820B (zh) 一种用于捕获肿瘤细胞的抗人角蛋白18的单克隆抗体及其应用
CN106854244B (zh) 一种针对her3的纳米抗体及其临床应用
JP2022512630A (ja) 抗ヒト心筋型トロポニンｉ抗体及びその応用
JPWO2016143226A1 (ja) 反応場を制限したプロテアーゼ分解反応による抗体からペプチド断片を得る方法
WO2013015367A1 (fr) Anticorps pour la détection d'un marqueur du cancer épithélial de l'ovaire et méthode pour le diagnostic du cancer épithélial de l'ovaire
JP6744670B2 (ja) タグ付抗体
JP5770092B2 (ja) ヒトｈｉｇ１ポリペプチドに対するモノクローナル抗体
EP2187216B1 (fr) Nouveau marqueur de cancer du foie
CN117683128B (zh) 一种全人源针对鼠疫菌的保护性单克隆抗体及其应用
WO2022071581A1 (fr) Peptide de liaison au sars-cov-2
JP5849275B2 (ja) 抗ヒスチジンタグ抗体
WO2015137441A1 (fr) Anticorps anti-protéine myristoylée ou fragment de liaison à l'antigène de celui-ci, trousse de détection d'une protéine myristoylée, médicament, gène et vecteur
JP5849254B2 (ja) 上皮性卵巣癌マーカー検出用抗体及び上皮性卵巣癌判定方法
US20240168026A1 (en) Method and kit for assisting in determination of malignant pancreatic cystic tumor
WO2019230823A1 (fr) Procédé d'obtention d'anticorps, procédé d'identification d'anticorps, procédé de production d'anticorps et anticorps
WO2024089627A1 (fr) Anticorps à domaine unique -sdab/vhh dirigé contre la nucléoprotéine du virus sars-cov-2
CN116554321A (zh) 一种znf165特异性抗体
CN118667000A (zh) 抗hsa单克隆抗体及其应用
KR100477898B1 (ko) 인간 ＣａｃｙＢＰ／ＳＩＰ에 대한 단일클론 항체 및 이를생산하는 융합세포주
JP2012070648A (ja) マウスｈｉｇ１ポリペプチドに対するモノクローナル抗体

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNIVERSIDAD AUSTRAL DE CHILE, CHILE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ROJAS, ALEJANDRO;VALENZUELA, GUILLERMO;JARA, RONALD;AND OTHERS;SIGNING DATES FROM 20221110 TO 20221114;REEL/FRAME:061780/0153

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION