US20130244883A1 - Molecular Display Method - Google Patents

Molecular Display Method Download PDF

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US20130244883A1
US20130244883A1 US13/629,520 US201213629520A US2013244883A1 US 20130244883 A1 US20130244883 A1 US 20130244883A1 US 201213629520 A US201213629520 A US 201213629520A US 2013244883 A1 US2013244883 A1 US 2013244883A1
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cdr
library
seq
cell
sequencing
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Sachdev Sidhu
Jason Moffat
Helena Persson
Nish Patel
Saravanan Sundararajan
Amandeep Gakhal
Wei Ye
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University of Toronto
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University of Toronto
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Assigned to THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO reassignment THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SIDHU, SACHDEV S., SUNDARARAJAN, SARAVANAN, PERSSON, HELENA, GAKHAL, Amandeep, PATEL, Nish, MOFFAT, Jason, YE, WEI
Assigned to THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO reassignment THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MCLAUGHLIN, MEGAN, TOMIC, Jelena
Priority to US15/344,971 priority patent/US10746743B2/en
Priority to US16/994,062 priority patent/US11649561B2/en
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    • 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
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • 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
    • G01N33/6857—Antibody fragments

Definitions

  • This invention relates to the field of screening for affinity reagents to a molecular target, and more specifically to molecular display methods used in conjunction with sequencing.
  • phage display antibody technologies are used for isolating antibody fragments specific to antigens of interest, but selection of libraries against cell-surface antigens remains very challenging.
  • the heterogeneity of the cell-surface and, accordingly, the relatively low concentration of the target antigen give rise to large numbers of background phage clones.
  • These phage clones may be non-specific binding clones, or may be specific for antigens other than the desired cell-surface target. Consequently, poor enrichment for binding phage clones is typically observed in cell selections.
  • proteins require the membrane environment for proper folding and stability and, as such, the ability to select phage-displayed antibody libraries against cell-surface epitopes remains crucial. If a protein is not properly folded, certain epitopes may not be available for binding by, for example, an affinity reagent. Likewise, proteins that are part of large complexes or associated with DNA, histones or other subcellular structures contain epitopes that are not necessarily made available for binding following traditional purification methods. For example, the properties of multi-pass membrane G-protein coupled receptors make their expression and purification very difficult, yet they are particularly relevant drug targets [1,2].
  • Phage display selection strategies to reduce background binding to cells have included negative or competitive pre-absorption steps against multiple cell-lines [6,7,8,9,10] and various strategies to remove unbound from bound phage, including centrifugation through a density gradient [11,12] and the pathfinder approach [13,14]. Although these methods may help to enrich for phage clones specific to the antigen of interest, the number of unique antibody fragments recovered by these methods often remains relatively low, as phage display methodologies typically exhibit an affinity based selection pressure that promotes sequence convergence in later rounds of selection. New strategies are required to identify less prevalent clones that may exhibit desirable binding properties.
  • the methods described herein provide a rapid, efficient method of identifying binding agents, e.g., antibodies and antigen-binding fragments thereof, that specifically bind to cell-surface targets and other cell-surface expressed antigens. These methods include deep sequencing/high-throughput sequencing followed by a recovery method, also referred to herein as a rescue strategy.
  • deep sequencing and variations thereof refers to the number of times a nucleotide is read during the sequencing process. Deep sequencing indicates that the coverage, or depth, of the process is many times larger than the length of the sequence under study. Suitable deep sequencing methods include the methods described herein or any other art-recognized techniques.
  • Suitable rescue strategies include the clonal ELISA assays and PCR rescue strategies described herein or any other art-recognized techniques.
  • the methods provided herein do not require additional purification and/or isolation steps prior to identification and recovery of the binding agent, e.g., antibody or antigen-binding fragment thereof.
  • the methods provided herein are useful in identifying binding agents, e.g., antibodies and antigen-binding fragments thereof, which are not highly expressed in a given display.
  • the methods provided herein are useful in identifying polypeptide sequences that comprise less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5% and/or less than 0.25% of the selection pool.
  • the methods provided herein are useful in differential selection strategies, for example, to identify binding agents that bind a given cell-surface target only when the target exhibits a particular modification, a particular conformation or other identifying characteristic.
  • the methods provided herein are also useful in differential selection strategies, for example, to identify binding agents that bind a given cell-surface target only under certain metabolic or other biological conditions.
  • the methods provided herein are also useful in differential selection strategies, for example, to identify binding agents that bind a given cell-surface target only in the presence of an effector, a target-binding partner or other molecule that must be present to enable binding between the genetically encoded binding agent and the target.
  • binding agents particularly, binding polypeptides including antibodies and antigen-binding fragments thereof, also referred to herein as immunologically active fragments.
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′) 2 fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • such an antibody or immunologically active fragment thereof that binds a given antigen e.g., a cell-surface target
  • the cell-surface target is selected from the group consisting of HER2, CD133, ErbB3, Fzd7, ROR1, ROR2, exon16 deleted ErbB2, and ITGA11.
  • the cell-surface target includes a modification that is required for epitope binding, such as, for example, an O-linked N-acetylglucosamine (O-GlcNAc) modification.
  • O-GlcNAc O-linked N-acetylglucosamine
  • Suitable mammalian cells for use in the methods provided herein include, but are not limited to, cells such as 293, 293T, C2C12, and/or MC7 cells.
  • Library G is an scFv-phage library that was constructed by introducing degenerate codons into positions in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of a single human ScFv framework.
  • Library F is an Fab-phage library that was constructed by introducing degenerate codons into positions in CDR-H1, CDR-H2, CDR-H3 and CDR-L3 of a single human Fab framework. Library F was constructed using an anti-maltose binding protein Fab as a template.
  • a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule comprising: providing a molecular display system which displays a library of potential genetically encoded affinity reagents; screening the library against the target molecule to produce positive and negative selection pools; sequencing genetically encoded affinity reagents in each of the positive and negative selection pools; identifying at least one sequence that is more abundant in the positive selection pool as compared to the negative selection pool; and recovering at least one clone corresponding to the sequence.
  • an antibody or antibody fragment comprising any one of CDR regions outlined in FIG. 2 , FIG. 5 or FIG. 9 .
  • the antibody or antibody fragment is selected from the group consisting of antibodies or antibody fragments comprising CDRL3, CDRH1, CDRH2 and CDRH3 of any one of clones WY574B, WY574E, WY574F, WY677C and WY677D described herein, the CDRH3 regions shown in FIG. 5 or the combinations of CDRL3 and CDRH3 regions shown in FIG. 9 .
  • the antibody or antibody fragment is useful for the treatment of cancer, e.g., Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • the invention provides antibodies and antigen-binding fragments thereof that bind HER2 and include a variable heavy chain complementarity determining region 1 (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 22, 26, 30 and 34; a variable heavy chain complementarity determining region 2 (CDR-H2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 23, 27, 31 and 35; a variable heavy chain complementarity determining region 3 (CDR-H3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 24, 28, 32 and 36.
  • CDR-H1 variable heavy chain complementarity determining region 1
  • CDR-H2 variable heavy chain complementarity determining region 2
  • CDR-H3 variable heavy chain complementarity determining region 3
  • these anti-HER2 antibodies and antigen-binding fragments thereof also include a variable light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence SVSSA (SEQ ID NO: 240); a variable light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence SASSLYS (SEQ ID NO: 241); and a variable light chain complementarity determining region 3 (CDR-L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 29 and 33.
  • CDR-L1 comprising the amino acid sequence SVSSA
  • CDR-L2 variable light chain complementarity determining region 2
  • SASSLYS SEQ ID NO: 241
  • CDR-L3 variable light chain complementarity determining region 3
  • the invention provides antibodies and antigen-binding fragments thereof that bind HER2 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 242), where X 1 , X 2 , X 3 , and X 4 are Y, S, G, A, F, W, H, P or V and X 5 is P or L and X 6 is I or L; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where
  • the invention provides antibodies and antigen-binding fragments thereof that bind CD133 and include a CDR-L1 comprising the amino acid sequence the amino acid sequence Q-X 1 -X 2 -X 3 -X 4 -X 5 (SEQ ID NO: 245), where X 1 , X 2 , X 3 , X 4 , and X 5 are Y, S or, G; a CDR-L2 comprising the amino acid sequence X 1 -A-S-X 2 -L-Y (SEQ ID NO: 246), where X 1 , and X 3 are Y, S or, G; a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 and 159; a CDR-H1 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X
  • the invention provides antibodies and antigen-binding fragments thereof that bind ErbB3 and include a CDR-L1 comprising the amino acid sequence the amino acid sequence Q-X 1 -X 2 -X 3 -X 4 -X 5 (SEQ ID NO: 245), where X 1 , X 2 , X 3 , X 4 , and X 5 are Y, S or, G; a CDR-L2 comprising the amino acid sequence X 1 -A-S-X 2 -L-Y (SEQ ID NO: 246), where X 1 , and X 3 are Y, S or, G; a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 161, 163, 165, 167, 169, 171, 173 and 175; a CDR-H1 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 247), where X
  • the invention provides antibodies and antigen-binding fragments thereof that bind Fzd7 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 177, 179 and 181; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -T-X 9 (SEQ ID NO
  • the invention provides antibodies and antigen-binding fragments thereof that bind ROR1 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 183, 185 and 187; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -T-X 9 (SEQ ID NO:
  • the invention provides antibodies and antigen-binding fragments thereof that bind ROR2 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 189, 191, 193, 195, 197 and 199; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -T-X
  • the invention provides antibodies and antigen-binding fragments thereof that bind an ErbB2 variant known as exon 16 deleted ErbB2 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201, 203 and 205; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X
  • the invention provides antibodies and antigen-binding fragments thereof that bind ITGA11 and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 207, 209, 211 and 213; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -T-X 9 (SEQ
  • the invention provides antibodies and antigen-binding fragments thereof that recognize a modification known as O-GlcNac modification and include a CDR-L1 comprising the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 215, 217, 219 and 221; a CDR-H1 comprising the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y or S and where X 6 is I or M; and a CDR-H2 comprising the amino acid sequence X 1 -I-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8
  • a method of treating cancer e.g., Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer, in a patient comprising administering to the patient a therapeutically effective amount of the antibody or antibody fragment described herein.
  • cancer e.g., Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer
  • a method of treating a disorder that is associated with aberrant expression and/or activity of the cell-surface target against which the antibody has been selected comprising administering to the patient a therapeutically effective amount of the antibody or antibody fragment described herein.
  • cancer e.g., Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • a use of the antibody or antibody fragment described herein in the preparation of a medicament for the treatment of Her-2 positive cancer preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • FIG. 1 is a flow chart of the selection strategy used to isolate Fab clones specific for cell-surface displayed Her2.
  • the positive selection begins a pre-absorption step in which the library phage are incubated with untransfected 293T cells. After incubation, the mixture is pelleted to remove the library clones bound to the cells. These clones are likely specific for cell-surface epitopes that are not of interest, or are non-specific binding clones. The phage of interest for subsequent steps are circled in red.
  • the library phage remaining in the supernatant are incubated with the Her2 transfected 293T cells, non-binding phage are washed away, and the phage bound to the transfected cells are amplified in an E. coli host.
  • the amplified phage are then purified and used in the next round of selection.
  • the negative selection is carried out by incubating library phage with untransfected 293T cells. Phage clones that do not bind to the cells are washed away, and the remaining bound phage (circled in red) are amplified in an E. coli host for the next round of selection.
  • FIG. 2 shows Her2 specific clones rescued from the positive selection pool. Five clones were rescued from the Her2 positive selection output pool by PCR amplification with primers specific to their unique CDR H3 sequence. The total number of times each CDRH3 was observed in the positive and negative selection pools is listed. The abundance of each sequence in the positive pool is also listed, as a percentage of the total number of sequences isolated.
  • the CDR loops are defined by the IMGT nomenclature (Lefranc, Pommie, Ruiz et al (2003) Dev Comp Immunol 27, 55-77).
  • FIG. 3 shows the analysis of Fab binding to cell-surface Her2 by flow cytometry.
  • Fabs WY574B (left panel), WY574E (middle panel), and WY 547F (right panel) were tested for binding to (a) Her2 and EGFR transfected 293T cells.
  • Binding of the anti-Her2 Fab proteins was detected using an Alexa488-conjugated secondary antibody (AF488) against a Flag-epitope on the C terminus of the Fab light-chain.
  • AF488 Alexa488-conjugated secondary antibody
  • the stained anti-Her2 transfected population is shown in green, the stained EGFR transfected population is shown in blue, and the unstained Her2 transfected population is shown in gray.
  • the AF-488 positive cell gate is indicated.
  • Fabs were also testing for binding to Her2 positive (BT474) and negative (T47D) human breast cancer cell-lines, using the same secondary detection as in (a).
  • the stained BT474 population is shown in green in the bottom panel, with the unstained population shown in gray.
  • the stained T47D cell population is indicated in blue.
  • FIG. 4 shows the binding specificity of synthetic anti-HER2 antibodies against live cells.
  • A 293T cells were seeded on coverslips coated with 50 mg/ml poly-D-lysine for 24 h followed by transient transfection of plasmid encoding HER2.
  • B BT474 and T47D breast cancer cells were seeded onto uncoated coverslips. 48 h post-transfection or post-seeding, the cells were fixed with 3.7% formaldehyde without permeabilization and stained with anti-HER2 Fab protein (5 mg/ml) followed Alexa488-conjugated secondary antibody against a Flag-epitope on the C terminus of the Fab light-chain. The nuclei were stained using the Hoechst dye. The images were acquired using the WaveFX spinning disk confocal microscope by Quorom Technologies Inc. Composite images of the ‘xy’ and ‘yz’ planes are represented (scale bar, 10 um).
  • FIG. 5 shows the 100 most frequent CDRH3 sequences obtained from Illumina sequencing of the positive selection pool.
  • the 100 most frequently observed CDR H3 sequences positions 107-117 per IMGT obtained from the round 3 positive selection output are listed, starting with the most frequently observed sequence.
  • the number of counts reflects the number of times each sequence was observed in the positive or negative selection pool, or in the unselected na ⁇ ve library. Sequences highlighted in yellow represent those clones that were rescued from the positive selection pool. Sequence number 13 corresponds to the wild type sequence that was used as the template in the library construction process.
  • FIG. 6 shows rescue strategies that utilize both the unique heavy chain CDR3 (CDRH3) sequence and light chain CDR3 (CDRL3) sequences identified using the methods provided herein.
  • CDRH3 unique heavy chain CDR3
  • CDRL3 light chain CDR3
  • FIG. 7 is a flow chart of the selection strategy used to isolate Fab clones specific for cell surface O-GlcNAc-dependent epitopes.
  • FIG. 8 is an ELISA graph of binders identified from the selection strategy used to identify Fab clones specific for cell surface O-GlcNAc-dependent epitopes.
  • FIG. 9 shows the phage-Fab clones that were rescued from the positive selection pool.
  • FIG. 10 shows deep sequencing strategies to decode variable complementarity determining regions (CDRs) in pools of synthetic antibody fragments.
  • the region of the phagemid encoding the Fab scaffold (solid black line) and its six CDRs (white boxes labeled L1, L2, L3, H1, H2, H3) is shown.
  • PCR primers to generate amplicon sequencing libraries are shown as solid black arrows.
  • Sequencing read orientations are shown as white block arrows.
  • Strategies 1 and 2 are compatible with Illumina platforms and decode two or more CDRs.
  • Strategy 3 is compatible with IonTorrent platforms and decodes only CDR-H3.
  • CellectSeq combines the use of phage-displayed synthetic antibody libraries and high throughput DNA sequencing technology.
  • the antigen binding site contains ‘man-made’ diversity, which is introduced into human framework regions based on existing knowledge of antibody structure and function [15]. Consequently, synthetic libraries can be biased towards antibody clones with favorable properties, such as high stability and expression.
  • the use of high throughput DNA sequencing enables the rapid identification of high affinity clones specific to cells that express the antigen of interest.
  • the methodology we report here allows rare binding clones, which may compose as little as 0.25% of the selection pool, to be identified and successfully rescued.
  • Her2 human epidermal growth factor receptor 2
  • EGFR human epidermal growth factor receptor
  • Her2 is a transmembrane tyrosine kinase receptor involved in signalling pathways that promote cell proliferation and survival [16,17].
  • Her2 is overexpressed in approximately 20 to 25% of invasive breast cancers [18,19], and its overexpression correlates with increased tumor aggressiveness, an increased chance of recurrence, and poor prognosis in breast cancer patients [20,21].
  • a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule comprising: providing a molecular display system which displays a library of potential genetically encoded affinity reagents; screening the library against the target molecule to produce positive and negative selection pools, preferably with multiple rounds of selection; sequencing genetically encoded affinity reagents in each of the positive and negative selection pools; identifying at least one sequence that is more abundant in the positive selection pool as compared to the negative selection pool; and recovering at least one clone corresponding to the sequence.
  • affinity reagent is any molecule that specifically binds to a target molecule, for example, to identify, track, capture or influence the activity of the target molecule.
  • the affinity reagents identified or recovered by the methods described herein are “genetically encoded”, for example an antibody, peptide or nucleic acid, and are thus capable of being sequenced.
  • protein protein
  • polypeptide and “peptide” are used interchangeably to refer to two or more amino acids linked together.
  • “molecular display system” is any system capable of presenting a library of potential affinity reagents to screen for potential binders to a target molecule or ligand, for example, through in vitro protein evolution.
  • Examples of display systems include phage display, bacterial display, yeast display, ribosome display and mRNA display. In one embodiment of the method, phage display is used.
  • the sequencing is deep/high-throughput sequencing.
  • deep/high-throughput sequencing include Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion semiconductor sequencing (Ion Torrent by Life TechnologiesTM), and DNA nanoball sequencing.
  • MSS Lynx Therapeutics' Massively Parallel Signature Sequencing
  • Polony sequencing Polony sequencing
  • 454 pyrosequencing 454 pyrosequencing
  • Illumina (Solexa) sequencing SOLiD sequencing
  • Ion semiconductor sequencing Ion Torrent by Life TechnologiesTM
  • DNA nanoball sequencing a preferable embodiment, Illumina sequencing is used.
  • the rescue strategy is a clonal ELISA assay, a PCR-based rescue strategy, including the clonal ELISA assay and PCR-based rescue strategies described herein.
  • the affinity reagents are selected from the group consisting of nucleic acid molecules and polypeptides.
  • the affinity reagents are antibodies, preferably synthetic antibodies, and further preferably the library is a synthetic Fab or scFv library.
  • each of the affinity reagents in the library contains unique sequence tags and the sequencing identifies the unique sequence tags.
  • the at least one clone is recovered by annealing primers specific for the unique sequence tags.
  • the library is a synthetic Fab library and the unique sequence tag is in the CDR H3 region.
  • the target molecule is a cell surface protein.
  • the screening is performed against the target molecule presented on a cell surface. In some embodiments, the screening is performed against the target molecule presented on a mammalian cell surface.
  • sequences identified are more abundant in the positive selection pool as compared to the negative selection pool by a factor of at least 2, and in increasing preferably at least 3, at least 4 and at least 5.
  • Library G is an scFv-phage library that was constructed by introducing degenerate codons into positions in CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of a single human ScFv framework.
  • the library has a total diversity of 1.08 ⁇ 10 11 unique clones, and the details of the library design are shown in Table 2 below, where the shading in the CDR-L3 and CDR-H3 regions represents positions that were replaced by random loops of all possible varying lengths, as indicated.
  • nucleotide sequence of the vector encoding Library G is shown below:
  • Library F is an Fab-phage library that was constructed by introducing degenerate codons into positions in CDR-H1, CDR-H2, CDR-H3 and CDR-L3 of a single human Fab framework.
  • the loop length of the CDR-L3 and/or CDR-H3 in Library F can vary as shown in the table below.
  • the library has a total diversity of 3 ⁇ 10 10 unique clones, and the details of the library design are shown in Table 2 below, where the shading in the CDR-L3 and CDR-H3 regions represents positions that were replaced by random loops of all possible varying lengths, as indicated.
  • nucleotide sequence of the vector encoding Library F is shown below:
  • an antibody or antibody fragment comprising any one of CDR regions outlined in FIG. 2 , FIG. 5 or FIG. 9 .
  • the antibody or fragment contains a CDR-L1 that includes the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), and one of the combinations of CDR-L3, CDR-H1, CDR-H2 and CDR-H3 shown in FIG. 2 .
  • the antibody or fragment contains a CDR-L1 that includes the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 242), where X 1 , X 2 , X 3 , and X 4 are Y, S, G, A, F, W, H, P or V and X 5 is P or L and X 6 is I or L; a CDR-H1 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 , X 4 , and
  • the antibody or fragment contains a CDR-L1 that includes the amino acid sequence SVSSA (SEQ ID NO: 240), a CDR-L2 that includes the amino acid sequence SASSLYS (SEQ ID NO: 241), a CDR-L3 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 242), where X 1 , X 2 , X 3 , and X 4 are Y, S, G, A, F, W, H, P or V and X 5 is P or L and X 6 is I or L; a CDR-H1 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 243), where X 1 is I or L, X 2 , X 3 ,
  • the antibody or fragment contains a CDR-L1 that includes the amino acid sequence Q-X 1 -X 2 -X 3 -X 4 -X 5 (SEQ ID NO: 245), where X 1 , X 2 , X 3 , X 4 , and X 5 are Y, S or, G; a CDR-L2 that includes the amino acid sequence X 1 -A-S-X 2 -L-Y (SEQ ID NO: 246), where X 1 , and X 3 are Y, S or, G; a CDR-H1 that includes the amino acid sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 (SEQ ID NO: 247), where X 1 is I or L, X 2 , X 3 , X 4 , and X 5 are Y, S or G and where X
  • the antibody or antibody fragment is selected from the group consisting of antibodies or antibody fragments comprising CDRL3, CDRH1, CDRH2 and CDRH3 of any one of clones WY574B, WY574E, WY574F, WY677C and WY677D described herein, the CDRH3 regions shown in FIG. 5 or the combinations of CDRL3 and CDRH3 regions shown in FIG. 9 .
  • the antibody or antibody fragment is for the treatment of cancer, e.g., Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • a method of treating a disorder that is associated with aberrant expression and/or activity of the cell-surface target against which the antibody has been selected comprising administering to the patient a therapeutically effective amount of the antibody or antibody fragment described herein.
  • a method of treating a cancer such as a Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer, in a patient comprising administering to the patient a therapeutically effective amount of the antibody or antibody fragment described herein.
  • a cancer such as a Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • a cancer such as Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
  • 293T cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle medium
  • FBS heat inactivated fetal bovine serum
  • Human breast cancer cell lines T47D and BT474 cells were cultured in DMEM supplemented with 10% FBS and penicillin and streptomycin. All cells were cultured at 37° C. in a humid incubator with 5% CO 2 .
  • Library F was cycled through three rounds of selection, each consisting of a pre-absorption step followed by a positive selection step.
  • 293T cells were trypsinized briefly and re-suspended in a single cell suspension in DMEM with 10% FBS.
  • Ten million cells were pelleted at 1200 rpm for three minutes and cells were mixed with approximately 10 12 cfu of library F phage in DMEM containing 10% FBS, 50 mM HEPES, 2 mM EDTA.
  • the cells and library were incubated for 1.5 to 2 hours at 4° C. with gentle rocking, after which the cells were pelleted and the library supernatant was used in the following positive selection step.
  • 293T cells were harvested and plated at 2 ⁇ 10 6 cells in 150 mm tissue culture dishes (BD Falcon). Twenty-four hours after plating, cells were co-transfected with a Her2 expression plasmid (8 ⁇ g) and a GFP expression plasmid (2 ⁇ g) using Fugene 6 (Roche Applied Sciences), following the manufacturer's instructions. Approximately 48 hours post-transfection, cells were harvested as described above for the pre-absorption step. Five million cells were pelleted and re-suspended in the phage library supernatant from the pre-absorption step. The library and transfected cells were incubated for 2 hours at 4° C. with gentle shaking.
  • Positively selected phage were amplified similarly to previous described methods [30]. Briefly, XL1blue cells were grown to an OD 600 of 0.8 in 2YT media containing 10 ⁇ g/ml tetracycline. Following washing of the positively selected cells, 3 ml of the XL1 blue culture was added directly to the cell pellet. Cells and bacteria were incubated for 30 to 40 minutes at 37° C. with gentle shaking and approximately 10 10 cfu of M13 K07 helper phage was added. The culture was incubated for 45 minutes at 37° C., shaking at 200 rpm, and then transferred to a 40 ml 2YT culture (100 ⁇ g/ml carbenicillin, 25 ⁇ g/ml kanamycin). The culture was grown overnight at 37° C., shaking at 200 rpm. The amplified phage culture was harvested for subsequent selection rounds as previously described [30].
  • an adaptor sequence
  • a reverse primer containing a second Illumina-compatible adaptor region (5′-CAAGCAGAAGACGGCATACGAGCTCTTC-3′) (SEQ ID NO: 6) and an annealing site to the phagemid vector (5′-TCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 7) was used.
  • PCR reactions were performed with the high fidelity polymerase Phusion (Finnzyme) and 400 to 600 ng of template DNA. Reactions were subjected to 15 cycles of annealing and extension, consisting of 30 s at 57° C. and 45 s at 72° C.
  • PCR products were digested with Exol (USB), SAP (USB), and Dpn1 (NEB) and then purified on a PCR purification column (Qiagen). Successful amplification of the correct DNA fragment from each phage pool was verified by agarose gel electrophoresis.
  • the amplified DNA fragments were pooled and subjected to Illumina DNA sequencing on an Illumina GAII, with 72 base pair reads. Each sequencing read was assigned to its correct pool on the basis of its unique barcode sequence. The reads were filtered according to their Phred score [31]. Since a constant aligner region was sequenced, these regions were used to optimize the phred score cutoffs. Briefly, all sequences with phred scores of 20 and higher for every base were kept. A tolerance number (5) of medium quality (phred score higher than 15) was allowed. DNA sequences were translated to decode the sequence of the heavy chain CDR3.
  • primers (described below) were phosphorylated as previously described [30]. The phosphorylated primers were then used in a PCR reaction, in which phage pool DNA was used as a template. The amount of DNA template per reaction was varied between 1 to 100 ng. The amount of DNA template varied with the prevalence of the given clone in the Illumina pool, with greatest amount of DNA template (100 ng) being used in PCR reactions to rescue the least prevalent clones. Reactions were performed with the high fidelity polymerase Phusion (Finnzyme), using the manufacturer recommended conditions. Reactions were subjected to 30 to 35 cycles of annealing and extension, consisting of 30 s at 65 or 68° C.
  • PCR products were confirmed by agarose gel electrophoresis and approximately 50 ng of the PCR product was used directly in ligation reaction (400 U T4 ligase, NEB). Ligations were incubated overnight at room temperature, and then heat inactivated at 65° C. for 10 minutes. Following the PCR, Dpn1 (NEB) was added to digest template DNA present in the reactions and samples were transformed into chemically competent XL1blue cells. Rescued transformations were plated on 2YT agar plates with carbenicillin and incubated overnight at 37° C. Single colonies were inoculated into 96-well culture plates for overnight growth of single phage clones as previously described [30]. The heavy and light chains of individual phage clones were PCR amplified and the PCR products were sequenced to ensure the recovery of clones with the desired CDR H3.
  • WY574B 5′-CCAGTAATGAACAACAGC-3′, 5′-TACGGTTACGTTTCTGGT-3′
  • WY574E 5′-AGCCGGAACCCAACCGCG-3′, 5′-TACCCGTCTTACGGTTTG-3′
  • WY574F 5′-AGCGTAAACAGAAGAACCCCA-3′, 5′-TGGTCTCCGGCTTCTTGGTCT-3′
  • WY677C 5′-ACCCCACCAGTAGTAAGA-3′, 5′-CCGTGGTCTGGTTACTCT-3′
  • WY677D 5′-GTACGGAATGTACGGATGCGG-3′, 5′-TACTCTTACTGGGGTCCGTACTAC-3′.
  • V H Heavy (V H ) and light-chain (V L ) variable regions were amplified for sequencing with the following primers that add M13 forward and reverse binding sites, respectively:
  • Her2 and EGFR were both expressed from pCDNA3 (Invitrogen) [32,33], and GFP was expressed from a previously reported plasmid [34].
  • Fab proteins were expressed in 55244 E. coli from the phage display phagemid engineered with an amber stop codon between the Fab and pill proteins, introduced by a standard Kunkel mutagenesis reaction [30].
  • Single colonies of each clone were grown overnight at 30° C. in 2YT media supplemented with 50 ⁇ g/ml carbenicillin and 25 ⁇ g/ml kanamycin. Overnight cultures were centrifuged at 3000 g for 10 minutes and pellets were re-suspended in 25 ml of complete CRAP media [30].
  • HER2 was immobilized on a GLC chip by standard amine coupling chemistry and serial dilutions of Fab in PBS with 0.05% Tween 20 were injected over the Her2 and blank channels (for reference subtraction) for 60 seconds at a flow rate of 100 ⁇ l/min, followed by ten minutes of buffer to monitor Fab dissociation.
  • the chip surface was regenerated with 0.85% H 3 PO 4 prior to new analyte injection.
  • Kinetic parameters were determined by globally fitting a reference cell-subtracted concentration series to a 1:1 (Langmuir) binding model.
  • transfected 293T cells 3 ⁇ 10 6 cells were plated on 10 cm dishes (BD Falcon). Twenty-four hours after plating, cells were transfected with 10 ⁇ g of a Her2, EGFR, or GFP expression vector using Fugene 6 (Roche Applied Sciences), following the manufacturer's instructions. Approximately 24 hours post-transfection, cells were harvested using a cell scraper into PBS containing 2% FBS (wash buffer). The cells were washed once with wash buffer and re-suspended into a single cell suspension. Approximately 1.0 to 1.5 ⁇ 10 6 cells were placed into 1.5 ml tubes for staining with individual Fab clones.
  • cells were incubated for 45 minutes at room temperature in PBS containing 2% FBS to block non-specific epitopes.
  • cells were incubated with 2 ⁇ g of the Her2 specific Fabs (diluted in wash buffer) for 20 minutes at room temperature and then washed twice with wash buffer.
  • the samples were incubated for 20 minutes at room temperature with a 1:100 dilution of anti-FLAG-Alexa488 secondary antibody (Cell Signaling) in wash buffer.
  • cells were washed twice and re-suspended in 0.5 ml of PBS for analysis on a BD FacsAria I flow cytometer (BD Biosciences).
  • Fab binding to the human cancer cell-lines 5 ⁇ 10 5 BT474 and T47D cells were plated per well of 6-well plates (BD Falcon). Approximately 48 hours after plating, media was aspirated from the 6-well plates and cells were washed twice with cold PBS. Wells were then blocked with wash buffer for 45 minutes at 4° C. The blocking solution was aspirated and 4 ⁇ g of the Fab sample in 0.5 ml of wash buffer was added to the appropriate well. Wells were washed twice with wash buffer, and then incubated with secondary antibody as above for 30 minutes at 4° C. Wells were washed three times; cells were harvested into PBS using a cell scraper, and analyzed as above.
  • Immunofluorescence for cell-surface HER2 was carried out on intact cells seeded on round glass coverslips uncoated or coated with 50 ⁇ g/mL poly-D-lysine (BD Biosciences). 48 hours post-seeding or post-transfection with a plasmid encoding HER2, the cells were washed with ice-cold PBS containing 1 mM MgCl 2 and 1 mM CaCl 2 on ice. The subsequent steps were performed at 4° C., unless otherwise indicated.
  • the cells were fixed for 10 min with 3% paraformaldehyde (Electron Microscopy Sciences) and then stained with anti-HER2 Fab protein (5 mg/ml) in 1% (wt/vol) BSA for 1 h followed by extensive washing and incubation with Alexa488-conjugated secondary antibody against a Flag-epitope on the C terminus of the Fab light-chain.
  • the nuclei were stained using the Hoechst dye (Invitrogen) and then mounted with ProLong antifade reagent (Invitrogen).
  • the images were acquired using the WaveFX spinning disk confocal microscope by Quorom Technologies Inc. Acquisition parameters were adjusted to exclude saturation of the pixels. For assessing binding specificity in HER2+ (BT474) and HER2 ⁇ (T47D) cells, such parameters were kept constant between the two cell lines.
  • FIG. 1 a We subjected the synthetic Fab library F to three rounds of selection on 293T cells transiently transfected to express Her2 ( FIG. 1 a ).
  • the library was incubated with untransfected 293T cells prior to incubation with the Her2 expressing cells. These undesired background phage were removed with the cell pellet and the library phage left in solution were incubated with the Her2 transfected cells. After washing away non-binding phage, the remaining phage, which should include the Her2 specific binders (positive selection pool), were amplified in E. coli .
  • the positive and negative selection pools, and the na ⁇ ve library were next subjected to Illumina sequencing analysis.
  • 20 were also present in the negative selection pool ( FIG. 5 ).
  • a similar number of sequences, 20, from the positive selection pool also overlap with the na ⁇ ve library pool.
  • Sixteen of the sequences observed in the na ⁇ ve library are also present in the negative selection pool.
  • sequences in the na ⁇ ve library exhibited a much greater degree of diversity than the sequences of the two selected pools.
  • Single clones of interest were isolated from the positive selection output pool using a PCR based recovery method in which phosphorylated primers annealed to unique CDR H3 sequences ( FIG. 1 b ).
  • the primers were designed so that the 5′ ends of the forward and reverse primers were abutting, resulting in the amplification of the complete phagemid clone vector.
  • single colonies can be isolated and sequenced to verify recovery of the desired CDR H3. Using this method, we successfully recovered five unique phage clones from the positive selection pool ( FIG. 2 ).
  • the successfully recovered clones vary in their abundance in the positive pool, with the least abundant clone representing only 0.25% of the pool.
  • five of the PCR reactions we attempted failed to generate a PCR product This may be due to their low abundance in the output pool used for the PCR template, as each of these five clones represented less than 0.5% of the pool.
  • a unique aspect of the methodology described here is the use of deep sequencing to identify phage clones specific to the cell-surface antigen of interest.
  • sequences distinctive/exclusive to the positive selection output pool represent clones that have a high probability of being specific for the target antigen.
  • combining cell-surface selections with deep sequencing allows rare clones to be identified.
  • Trastuzumab (Genentech, also known as Herceptin) is a humanized IgG1 specific for the extracellular domain of Her2 [22], which is approved for clinical treatment of Her2 positive breast cancer.
  • Herceptin is a humanized IgG1 specific for the extracellular domain of Her2 [22], which is approved for clinical treatment of Her2 positive breast cancer.
  • Trastuzumab represents a very successful therapeutic option for patients, not all Her2 positive cancers are responsive to Trastuzumab treatment [23]. In addition, resistance to Trastuzumab may also develop during the course of treatment [20,24].
  • the synthetic antibody fragments we have identified using the CellectSeq method exhibit binding characteristics that are highly desirable for potential new therapeutic antibody candidates. Specifically, the synthetic antibody fragments we have isolated bind with both high affinity and specificity to Her2.
  • the five synthetic antibody fragments rescued from our positive selection pool exhibit specific binding to Her2, both by SPR analysis to recombinant Her2 and by flow-cytometry and IF to cell-surface Her2.
  • the methodology we report here may allow for the identification of antibody fragments specific for proteins that are over-expressed as a consequence of the over-expression of Her2 itself.
  • a limiting step to molecular display technologies is the need for correctly folded, purified antigen.
  • multi-domain membrane represent more than 70% of current drug targets due to their role in the progression and tumorigenesis of numerous cancers [1], yet the properties of these proteins makes their production and purification extremely difficult.
  • the instability of membrane proteins also makes them challenging targets to work with during in vitro library selections, as many of these proteins depend on the membrane environment for their correct structure and function.
  • the methodology reported here bypasses the need for purified antigen and allows library selection directly to cell-surface targets. Consequently, the CellectSeq methodology increases the likelihood that the selected antibodies will recognize epitopes on the native, functionally relevant structure of the target antigen.
  • the ability to select for specifically binding phage clones without the need for purified antigen will significantly expand the range of antigens that can be targeted using phage display technology.
  • the described methods could also be tailored to the specific needs of the antigen of interest.
  • the CellectSeq approach can be combined with protocols that involve screening libraries against cells in the presence of ligands, with the goal of targeting active forms of receptors [6].
  • the selection can be performed using cells co-transfected to express all of the relevant complex members, with the intention of isolating antibodies specific to the multimerized protein.
  • Her2 is a member of an oligomeric complex.
  • FIGS. 6A , 6 B Two different PCR based recovery methods are used ( FIGS. 6A , 6 B). As depicted in FIG. 6A , two primer sets specific for both CDR H3 and L3 are used to make the recovery more specific. Primers are designed to anneal to the L3 and H3, and amplify two fragments, in both directions. This results in two fragments that both contain the L3 and H3 regions. The two fragments can be annealed, and then a single round of DNA extension is done. The resulting product can then be ligated and transformed into E. coli to recover the desired phage clone in the original library display vector. As depicted in FIG.
  • three primer sets are used to amplify three fragments, in a strategy that makes use of both the H3 and L3 unique sequences and unique Nsi1 and Nhe1 sites in the library phage vector.
  • the three fragments are annealed, extended by PCR, and subcloned into an IPTG inducible protein expression vector with compatible Nsi1 and Nhe1 restriction enzyme sites.
  • the rescued Fab can be expressed directly from the resulting vector.
  • the phage-Fab clones that were rescued from the positive selection pool are shown in FIG. 9 .
  • cdrL3:cdrH3 PCR the rank of the sequence in the round four positive selection pool based on raw counts which reflects the number of times the sequence was observed in the pool; the CDR L3 and H3 sequences obtained from the round four positive selection output; the raw counts and percentage those counts represent of the entire output pool for the round four and round three positive and negative pools; whether the rescued clones have been validated for cell binding.
  • the conditions used for positive and negative selections are also annotated.
  • GUP is a cocktail of Glucosamine, Uridine and PugNAc.
  • FIG. 7 depicts a flow chart of the selection strategy used to isolate Fab clones specific for cell surface O-GlcNAc-dependent epitopes, demonstrating that the CellectSeq method can be used to isolate Fabs specific to cell growth conditions.
  • the positive selection begins a pre-absorption step in which the library phage are incubated with MCF7 breast cancer cells grown in DMEM (Dulbecco's Modified Eagle Medium) (high glucose version) supplemented with 10% FBS. After incubation, the mixture is pelleted to remove the library clones bound to the cells. These clones are likely specific for cell-surface epitopes that are not of interest, or are non-specific binding clones.
  • the library phage remaining in the supernatant are incubated with the MCF7 cells grown in DMEM (high glucose version) supplemented with 10% FBS plus 30 mM Glucosamine (G), 5 mM Uridine (U) and 50 ⁇ M PugNAc (P) (collectively referred to as GUP), non-binding phage are then washed away, and the phage bound to the GUP treated MCF7 cells are amplified in an E. coli host. The amplified phage are then purified and used in the next round of selection. In parallel, the negative selection is carried out by incubating library phage with MCF7 cells that have been grown in the absence of GUP treatment.
  • Phage clones that do not bind to the cells are washed away, and the remaining bound phage are amplified in an E. coli host for the next round of selection.
  • O-GlcNAc enrichment is achieved by adding GUP (a cocktail of Glucosamine, Uridine and PugNAc).
  • FIG. 8 depicts an ELISA graph of binders chosen from R4O from the selection strategy used to isolated Fabs specific for surface O-GlcNAc-dependent epitopes.
  • FIG. 10 provides diagrams for some deep/high-throughput sequencing strategies used to decode variable regions of affinity reagents in a positive or negative selection pool.
  • one or more complementarity determining regions (CDRs) of synthetic antibodies are decoded by deep sequencing.
  • Positive and negative selection pool phages from rounds three and four were infected into XL1Blue cells and grown overnight in 2YT supplemented with 100 ug/ml carbenicillin. Cultures were miniprepped to obtain phagemid DNA and normalized to 25 ng/ul to use as templates for PCR. PCR primers added barcodes and platform-specific adapters, while amplifying one or more variable regions of the affinity reagent by annealing to adjacent regions of the affinity reagent framework.
  • the forward PCR primer was composed of a paired-end compatible Illumina adaptor sequence (5′AATGATACGGCGACCACCGAGATCT-3′) (SEQ ID NO: 223) and an annealing site upstream of CDR-L3 (5′ GCAGCCGGAAGACTTCGCAACTTATTACTGTCAGC-3′) (SEQ ID NO: 224).
  • the reverse PCR primer was composed of a paired-end compatible Illumina adaptor sequence (5′ CAAGCAGAAGACGGCATACGAGAT-3′) (SEQ ID NO: 225), a five base barcode (5′NNNNN-3) (SEQ ID NO: 226), and an annealing site downstream of CDR-H3 (5′GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 227).
  • PCR reactions were performed with the high fidelity polymerase ExTaq (TaKaRa) and 400 ng of template phagemid DNA. Reactions were subjected to one denaturation step for 30 sec at 95° C., followed by 14 cycles of 30 sec at 94° C. and 60 sec at 72° C., with a final extension for 5 min at 72° C. PCR products were cleaned enzymatically with Exol to remove residual primers, SAP to dephosphorylate dNTPs and Dpn1 to digest methylated phagemid template DNA. PCR products were quantitated using dsDNA-specific fluorescent dye (PicoGreen), normalized, pooled and purified by gel extraction of the correct fragment size (1007 bp).
  • dsDNA-specific fluorescent dye PicoGreen
  • the purified DNA fragments were subjected to Illumina DNA sequencing on GAIIx or HiSeq platforms, using custom read primers and read lengths: Read 1 forward (L3) primer (5′ CAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAA-3′) (SEQ ID NO: 228) for a minimum of 30 bases; Read 2 forward (barcode) primer (5′ GACTACTGGGGTCAAGGAACCCTGGTCACC-3′) (SEQ ID NO: 229) for a minimum of 5 bases; Read 3 reverse (H3) (5′ GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 230) for a minimum of 65 bases. Each sequencing read was assigned to its correct pool of the basis of its unique barcode sequence. The reads were filtered according to their Phred score [31]. Briefly, all sequences with phred scores of 20 or higher for every base were kept. DNA sequences were translated to decode the sequences of CDRs L3 and H3.
  • the forward PCR primer was composed of a paired end Read 1 Illumina adaptor sequence (5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3) (SEQ ID NO: 231), barcode (5′NNNNNN-3′) (SEQ ID NO: 232) and an annealing site downstream of CDR-H3 (5′GGTGACCAGGGTTCCTTGACCCCAGTAGTC-3′) (SEQ ID NO: 233).
  • the reverse PCR primer was composed of a paired end Read 2 Illumina adaptor sequence (5′ CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′) (SEQ ID NO: 234), optional barcode (5′NNNNNN-3′), and an annealing site upstream of CDR-L3 (5′ CAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAA-3′) (SEQ ID NO: 235). PCR reactions were carried out using ExTaq, as described for Strategy 1.
  • the purified DNA fragments were subjected to Illumina DNA sequencing on GAIIx, HiSeq or Miseq platforms, using standard paired end read primers and 2 ⁇ 150 bp read lengths or longer, to span CDR-H2 and CDR-H1 in addition to barcode and CDR-H3 (read 1) or CDR-L3 (read 2).
  • the forward PCR primer was composed of an IonTorrent Adapter A sequence (5′ CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) (SEQ ID NO: 236), barcode (5′ NNNNNNNC-3′) (SEQ ID NO: 236) and an annealing site upstream of CDR-H3 (5′AGGACACTGCCGTCTATTAT-3′) (SEQ ID NO: 237).
  • the reverse PCR primer was composed of IonTorrent adapter P1 sequence (5′ CCTCTCTATGGGCAGTCGGTGAT-3′) (SEQ ID NO: 238) and an annealing site downstream of CDR-H3 (5′AGGACACTGCCGTCTATTAT-3′) (SEQ ID NO: 239).
  • PCR reactions were carried out using Phusion with one denaturation step at 98 C for 5 min, followed by 14 cycles of 5 sec at 98° C., 10 sec at 54° C., 15 sec at 72 vC, with a final extension for 10 min at 72° C. Residual primers and dNTPs were removed using column (Qiagen), and PCR products were quantitated, normalized and pooled, for single end sequencing on an IonTorrent platform.

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