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US-12618838-B2 - Single domain VHH antibodies against SARS-CoV-2 virus

US12618838B2US 12618838 B2US12618838 B2US 12618838B2US-12618838-B2

Abstract

The invention provides a set of novel Single domain VHH antibodies against SARS-CoV-2 (SEQ ID No 1-6) and their use to detect and neutralize the wild type virus.

Inventors

  • Alejandro Rojas
  • Zaray MIRANDA
  • Benjamín UBERTI
  • Ananda MULLER
  • Guillermo VALENZUELA
  • Ronald JARA
  • Johanna HIMELREICHS
  • Constanza SALINAS
  • Teresa PINTO
  • Natalia LÓPEZ
  • Yorka CHEUQUEMILLA
  • Alexei CUEVAS

Assignees

  • UNIVERSIDAD AUSTRAL DE CHILE

Dates

Publication Date
20260505
Application Date
20221115

Claims (14)

  1. 1 . A VHH against the Spike protein of SARS-COV-2, wherein the VHH comprises a set of three CDRs having aminoacidic sequences selected from the group consisting of: SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 10, 11, and 12; and SEQ ID NOs: 13, 14, and 15.
  2. 2 . The VHH according to claim 1 having an aminoacidic sequence of SEQ ID NO: 2.
  3. 3 . The VHH according to claim 2 , wherein the VHH is encoded by a nucleotidic sequence of SEQ ID NO: 1.
  4. 4 . The VHH according to claim 1 having an aminoacidic sequence of SEQ ID NO: 4.
  5. 5 . The VHH according to claim 4 , wherein the VHH is encoded by a nucleotidic sequence of SEQ ID NO:3.
  6. 6 . The VHH according to claim 1 an aminoacidic sequence of SEQ ID NO: 6.
  7. 7 . The VHH according to claim 6 , wherein the VHH is encoded by a nucleotidic sequence of SEQ ID NO:5.
  8. 8 . A method for detecting a SARS-COV-2 virus, comprising detecting a presence of the virus in a sample by an immunoassay using the VHH according to claim 1 .
  9. 9 . The method according claim 8 , wherein the immunoassay is an assay selected from the group consisting of ELISA, immunoblotting, immunohistochemistry, and immunoprecipitation.
  10. 10 . A method for neutralizing a SARS-COV-2 virus, the method comprising neutralizing the virus using wherein the VHH according to claim 1 .
  11. 11 . The method according to claim 10 , wherein the neutralizing is performed in vivo, and the neutralizing of the virus allows to control COVID-19 in an individual.
  12. 12 . The method according claim 11 , wherein the VHH is humanized.
  13. 13 . The method according claim 10 , wherein the VHH is bound to a carrier molecule selected from the group consisting of an antibody fragment, human Fc fragment, polyethylene glycol, sialic acid polymers, beta carboxyterminal peptides, albumin, and albumin binding peptides.
  14. 14 . The method according claim 13 , wherein the VHH is bound to a human Fc fragment.

Description

FIELD OF THE INVENTION The invention relates to a set of single domain VHH antibodies against SARS-CoV-2 and their use in detection, diagnostic, neutralization and therapies against the wild type SARS-CoV-2 virus and its variants. SEQUENCE LISTING The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence_Listing_20898.0006 USW1.xml, created on Nov. 14, 2023, which is 18.5 bytes (about 19 KB) in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. STATE OF THE ART Coronavirus disease 2019 (COVID-19) is the name given to the illness caused by the SARS-CoV-2 infection, a novel coronavirus identified in China and related to pneumonia, that constitutes an unprecedented threat for health and economic systems. Many months have passed since it was first reported and we are still continuously learning about the virus and the associated clinical manifestations, its clinical implications, and possible treatments. By the end of April 2020, over three million people have been infected and more than two hundred thousand have died worldwide, reinforcing the urgent need for better diagnostics and treatment procedures. Currently, there are no vaccines or drugs that can effectively contain the pandemic, and as a result, non-pharmacologic public health measures such as social distancing, border closures, and lockdowns have been enforced globally to flatten the curve and avoid the collapse of health systems. Genetic studies determined that the pathogen responsible for this outbreak belongs to the genus beta-coronavirus, sub-genus sarbecovirus, and the family coronaviridae. It has high sequence homology with the bat coronavirus RaTG13, providing evidence that the new virus may have originated in bats. There are eight coronaviruses associated with human disease: HKU1, hCoV-0043, hCoV-NL-63, hCoV-229E, and the pandemics SARS-CoV-1, MERS and SARS-CoV-2. The SARS-CoV-2 sequence contains 29903 bp, which includes 14 open reading frames (ORFs) coding for the replicase ORF1ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, and several non-structural (nsps) and accessory proteins. The glycoprotein spike (S) on the surface of the virus is responsible for the attachment and invasion of host cells. The spike is a highly glycosylated trimeric class I fusion protein and contains two subunits, S1 and S2. CryoEM studies showed that it remains in a metastable pre-fusion conformation with a hinge-like movement of the S1 subunit, which binds to the host cell receptor. After attachment, S1 sheds and leaves the S2 subunit in a stable post-fusion conformation. The latter mediates membrane fusion and is responsible for the invasion of the host cell. The angiotensin-converting-enzyme 2 protein (ACE2) appears to be the entryway to the host, as the SARS-CoV-1 spike binds to this receptor, the virus requires priming proteolysis by the TMPRRS2 serine protease, which attacks a furin-like cleavage site on the S protein. The conformational change then stabilizes the complex, allowing the virus to infect the host cell. The presence of ACE2 was confirmed in different tissues, which can be related to some clinical manifestations of COVID-19; some of them already have in-vitro proof of infection by SARS-CoV-2. This mechanism mediated by S glycoprotein highlights the importance and antigenic properties of this protein as a target for the development of therapies such as neutralizing antibodies and vaccines. Isolation of specific antibodies could be used for the development of an effective diagnostic and therapeutic arsenal against the virus. Some naturally occurring antibodies lack light chains, known as single-domain antibodies (HCAb, Heavy Chain only Antibodies). They are derivates of IgG and occur in the entire Camelidae family. The camelid family comprises of camels, dromedaries, llamas, vicuñas, guanaco, and alpacas. The antigen-binding fragment of an HCAb contains a single variable VHH domain consisting of 3 hypervariable regions (CDR). The target-specific VHH derived from camelid HCAbs are obtained after immunization with the target protein, plus adjuvant. Our platform has developed an improved procedure for VHH production using alpacas as a donor species. To isolate the genetic sequences of the target-specific VHH produced after immunization, we must first isolate the peripheral B-lymphocytes to obtain total RNA, followed by cDNA preparation to finally amplify the VHH region. The cDNA fragment encoding the VHH is as short as 360 nt, and up to ˜3×106 single clones can be obtained in a bacterial display library from 120 mL of blood. We used a bacterial display system to clone the full single VHH. Here, we have obtained and selected a set of VHH against the Spike RBD domain of SARS-CoV-2. Single Domain VHH Antibodies The target-specific VHH derived from HCAbs of c