Method of generating an antibody naïve library, said library and application(s) thereof
Inventors
Chatterjee, Sohang • Rodrigues, Kavitha Iyer • Ghosh, Maloy • Maity, Sunit • Unnikrishnan, Divya • Bangalore Muniraju, Yogendra Manjunath • Murugesan, Sathyabalan • Mukunda, Pavithra • Prasad, Bhargav • Kamanagowda, Veeresha • Bhattacharjee, Sanghamitra • Dakshinamurthy, Pravin Kumar • Halan, Vivek • Srinivasan, Sankaranarayanan • Hora, Anuradha • Natarajan, Bairavabalakumar • Nair, Karthika • Thanigaivel, Aswini • Maliwalave, Amol • Shenoy, Bharath Ravindra • Rao, Sahana Bhima • Chakrabarty, Subhra Prakash • Dubey, Ashvini Kumar • Khan, Amir • Tiwari, Anurag • Kumar, Santosh • PATEL, Shivani • M, Nikitha
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11384354-B2
Publication Date
2022-07-12
Expiration Date
Abstract
The present disclosure relates to a method of generating an antibody library, not limiting to a human naïve antibody gene expression library encompassing a pool of nucleic acid sequences derived from a natural antibody repertoire comprising humoral immunity from healthy and genetically diverse human populations. More specifically, the method employs a combination of phage and/or yeast antibody surface display concept which allows to screen large antibody library size and facilitates better folding of antibody structure. The present disclosure also relates to a human naïve antibody library generated by employing the process of the present disclosure, a set of primers employed in the method and application(s) of said antibody library.
Core Innovation
The disclosure describes a method of generating a naive monoclonal antibody gene expression library, or a screened naive monoclonal antibody gene expression library, by processing a biological sample comprising a source antibody gene pool to isolate a plurality of nucleic acids encoding a naturally occurring antibody repertoire, followed by amplification. The method introduces a specific tri-nucleotide sequence marker into a portion of the nucleic acid encoding a heavy chain of a naturally occurring antibody so that the nucleic acid is catalogued to the source antibody gene pool and displayed antibodies are traceable back to the source antibody gene pool.
The tri-nucleotide sequence marker is introduced through amplification by one or more degenerate primers selected from SEQ ID NO:1 to SEQ ID NO:68, where the one or more degenerate primers include the specific tri-nucleotide marker specific to the source antibody gene pool. After amplification, amplified nucleic acids from separate amplifications are pooled, each nucleic acid having the tri-nucleotide sequence marker, and the pooled amplified nucleic acids are cloned into one or more phagemid vectors.
Antibodies are displayed on a surface of at least one phage, optionally followed by screening of the displayed antibodies against a specific antigen to remove non-binders and obtain a screened antibody molecule. The nucleic acid of the displayed antibody or the screened antibody molecule is then transferred into yeast for display of the antibody molecules on a yeast surface, optionally followed by screening of the yeast-displayed antibodies, after which nucleic acids showing desired functional properties are selected or isolated to form a naive monoclonal antibody gene expression library, or nucleic acids showing higher affinity to the target antigen are selected or isolated to form a screened naive monoclonal antibody gene expression library.
Claims Coverage
The independent claim provides three inventive feature clusters centered on tri-nucleotide sequence-marker cataloguing and tracing during amplification, phagemid-based phage display with optional antigen screening, and yeast-display screening followed by selecting or isolating naive monoclonal antibody gene expression library nucleic acids based on desired functional properties or higher affinity.
Tri-nucleotide sequence marker for cataloguing and tracing
introducing a specific tri-nucleotide sequence marker in a portion of the nucleic acid encoding a heavy chain of a naturally occurring antibody so as to catalogue the nucleic acid to the source antibody gene pool, and to enable tracing of displayed antibodies back to the source antibody gene pool, wherein the tri-nucleotide sequence marker is introduced through the amplification of the nucleic acid by one or more degenerate primers selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:68, wherein the one or more degenerate primers include the specific tri-nucleotide marker that is specific to the source antibody gene pool
Phagemid cloning and phage display with optional antigen screening
pooling of the amplified nucleic acids from separate amplifications, each of the nucleic acids having the tri-nucleotide sequence marker, so as to obtain pooled amplified nucleic acids, and cloning of the pooled amplified nucleic acids into one or more phagemid vectors, displaying of the antibodies on a surface of at least one phage so as to obtain the displayed antibodies, wherein the displaying is optionally followed by a screening of the displayed antibodies against a specific antigen so as to remove an antibody non-binder to the specific antigen and to obtain a screened antibody molecule
Yeast transfer for display and optional antigen screening, followed by selecting or isolating nucleic acids
transferring the nucleic acid of the displayed antibody or the screened antibody molecule into a yeast for display of said antibody molecules on a surface of the yeast so as to obtain a yeast-displayed antibody, wherein the transferring is optionally followed by a screening of the yeast-displayed antibodies against a specific antigenic target; selecting or isolating the nucleic acid of the yeast displayed antibodies showing desired functional properties to form a naive monoclonal antibody gene expression library, or selecting or isolating the nucleic acid of the yeast displayed antibodies showing higher affinity to the target antigen to form the screened naive monoclonal antibody gene expression library
The coverage centers on introducing a tri-nucleotide sequence marker via degenerate primers to catalogue nucleic acids and enable tracing, performing phagemid cloning followed by phage display with optional antigen screening, and transferring into yeast for optional antigen screening and selection or isolation to form either a naive monoclonal antibody gene expression library based on desired functional properties or a screened naive library based on higher affinity.
Stated Advantages
Large library sizes are described, including clone ranges and phage particle libraries.
Improved folding and processing is described through yeast display.
A two-stage approach is described: optional screening on phage to remove non-binders and optional screening on yeast to enable affinity-focused selection.
Tracing of displayed antibodies back to the source antibody gene pool is enabled by the tri-nucleotide sequence marker.
A PCR-free transfer approach preserving diversity is described conceptually in the disclosure summary.
Documented Applications
Antibody generation workflows are documented for constructing a human naive antibody gene expression library from PBMCs of genetically diverse donors, including phage display and yeast display screening.
Example antigen screening is documented using an anti-Her2 antigen context, including confirmation by ELISA and SPR/Biacore.
Interested in licensing this patent?