US20130244883A1

US20130244883A1 - Molecular Display Method

Info

Publication number: US20130244883A1
Application number: US13/629,520
Authority: US
Inventors: Sachdev Sidhu; Jason Moffat; Helena Persson; Nish Patel; Saravanan Sundararajan; Amandeep Gakhal; Wei Ye
Original assignee: University of Toronto
Current assignee: University of Toronto
Priority date: 2011-09-27
Filing date: 2012-09-27
Publication date: 2013-09-19
Also published as: US10746743B2; US20170184608A1; EP2761064A2; WO2013080055A3; EP2761064B1; CA2877081A1; US11649561B2; EP2761064A4; WO2013080055A2; CA2877081C; US20210148928A1

Abstract

There is provided herein a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule by screening using molecular display in conjunction with the sequencing of positive and negative selection pools from the screen.

Description

RELATED APPLICATIONS

This application claims the benefit of provisional applications U.S. Ser. No. 61/539,546 filed Sep. 27, 2011, the contents of which are herein incorporated by reference in their entirety.

FIELD OF INVENTION

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.

BACKGROUND

Molecular display technologies are widely used to screen for potential affinity binders to a specific target molecule, however, there is potential for improving thereon. For example, 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. However, many 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]. Indeed, the high specificity of monoclonal antibodies, combined with their ability to engage immune mechanisms, makes this class of biologics of particular interest in the treatment of numerous cancers and infectious diseases [3,4,5]. A reliable selection methodology for targeting exposed epitopes (e.g. cell-surface epitopes), which eliminates the need for highly purified antigens, would significantly expand the range of antigens that could be targeted by therapeutic monoclonal antibodies.
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.

SUMMARY OF THE INVENTION

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. As used herein, the term “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. For example, 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.
The methods provided herein are useful for identifying binding agents, particularly, binding polypeptides including antibodies and antigen-binding fragments thereof, also referred to herein as immunologically active fragments. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′)₂fragment, a scFv, a scab, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds a given antigen, e.g., a cell-surface target, is a mouse, chimeric, humanized or fully human monoclonal antibody.
In some embodiments, the cell-surface target is selected from the group consisting of HER2, CD133, ErbB3, Fzd7, ROR1, ROR2, exon16 deleted ErbB2, and ITGA11. In some embodiments, 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.
These cell-surface targets are expressed on mammalian cells. 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.
The methods provided herein are used in combination with phage-display libraries referred to herein as Libraries F and G, but those of ordinary skill in the art will appreciate that these methods can be used in conjunction with any peptide/polypeptide display system in which cell-surface targets/antigens are expressed. 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.
In an aspect, there is provided a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule, the method 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.
In a further aspect, there is provided an antibody or antibody fragment comprising any one of CDR regions outlined in FIG. 2, FIG. 5 or FIG. 9. Preferably, 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. In one embodiment, 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. In some embodiments, 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.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 242), where X₁, X₂, X₃, and X₄are Y, S, G, A, F, W, H, P or V and X₅is P or L and X₆is I or L; a CDR-H1 comprising the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-136.
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-X₁-X₂-X₃-X₄-X₅(SEQ ID NO: 245), where X₁, X₂, X₃, X₄, and X₅are Y, S or, G; a CDR-L2 comprising the amino acid sequence X₁-A-S-X₂-L-Y (SEQ ID NO: 246), where X₁, and X₃are 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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 247), where X₁is I or L, X₂, X₃, X₄, and X₅are Y, S or G and where X₆is I or M; a CDR-H2 that includes the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y, S or G, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 and 160.
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-X₁-X₂-X₃-X₄-X₅(SEQ ID NO: 245), where X₁, X₂, X₃, X₄, and X₅are Y, S or, G; a CDR-L2 comprising the amino acid sequence X₁-A-S-X₂-L-Y (SEQ ID NO: 246), where X₁, and X₃are 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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 247), where X₁is I or L, X₂, X₃, X₄, and X₅are Y, S or G and where X₆is I or M; a CDR-H2 that includes the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y, S or G, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 164, 166, 168, 170, 172, 174 and 176.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 178, 180 and 182.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 184, 186 and 188.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 190, 192, 194, 196, 198 and 200.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 202, 204 and 206.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 208, 210, 212 and 214.
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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 comprising the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and a CDR-H3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216, 218, 220 and 222.
In a further aspect, there is provided 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.
In a further aspect, there is provided 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.
In a further aspect, there is provided a use of the antibody or antibody fragment described herein 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.
In a further aspect, there is provided a use of the antibody or antibody fragment described herein for the treatment of a disorder that is associated with aberrant expression and/or activity of the cell-surface target against which the antibody has been selected.
In a further aspect, there is provided 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.
In a further aspect, there is provided a use of the antibody or antibody fragment described herein in the preparation of a medicament for the treatment of a disorder that is associated with aberrant expression and/or activity of the cell-surface target against which the antibody has been selected.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the description and drawings, like numerals refer to like structures or processes. In the drawings:

FIG. 1 is a flow chart of the selection strategy used to isolate Fab clones specific for cell-surface displayed Her2. (A) 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. In parallel, 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. (B) Phage clones are rescued from the positive selection output pool using a PCR strategy in which abutting forward and reverse PCR primers (shown in red) anneal to the unique heavy chain CDR3 sequence (represented by the colored portion of the circular DNA molecules). PCR amplification results in synthesis of the complete phagemid vector. Ligation of the PCR product yields closed, circular, double-stranded DNA that can be transformed into bacteria for rescue. Dpn1 digestion of the PCR product degrades undesired phagemid DNA present from the positive selection pool (represented in gray), due to the presence of methylated Dpn1 recognition sites. As a consequence of in vitro synthesis, the PCR product is not methylated and, therefore, is not recognized by Dpn1.

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. 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. (b) The 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 naïve 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. (A) Two primer sets specific for both CDRH3 and CDRL3 are used to make recovery more specific. (B) Three primer sets are used to amplify three fragments in a strategy that makes use of both the CDRH3 and CDRL3 sequences, as well as unique Nsi1 and Nhe1 restriction sites in the library phage vector.

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.

DETAILED DESCRIPTION

There is described herein the development of a new method for selecting for affinity reagents against a target molecule.
In a specific example, there is described a new method for selecting phage display libraries against cell-surface expressed antigens. This methodology, termed CellectSeq, combines the use of phage-displayed synthetic antibody libraries and high throughput DNA sequencing technology. In the synthetic library approach, 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. Moreover, 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.
As an initial model system, we selected synthetic antibody libraries against cells transiently transfected to express the human epidermal growth factor receptor 2 (Her2, also known as ErbB2). A member of the human epidermal growth factor receptor (EGFR) family, 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]. We selected phage-displayed synthetic antibody libraries against 293T cells transiently transfected to express Her2 and, in parallel, untransfected 293T cells. After three rounds of selection, each output pool was subjected to Illumina deep sequencing. We found that comparing the deep sequencing results of the positive and negative selection pools could identify Her2 specific clones. We were able to rescue clones unique to the positive selection pool using primers specific to the third hypervariable loop of the antibody heavy chain (CDR H3), and demonstrated that the rescued clones bind specifically and with high affinity to our target antigen, Her2. Our results suggest that the use of deep sequencing enables efficient identification of antibody fragments specific to target antigens presented on cell-surfaces.
While the initial model system used synthetic antibody libraries screened against cells expressing HER2, it is understood that the methods described herein are useful to identify binding agents that recognize any number of targets that are expressed on a cell-surface.
In an aspect, there is provided a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule, the method 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.
As used herein, “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. As used herein, the terms “protein”, “polypeptide” and “peptide” are used interchangeably to refer to two or more amino acids linked together.
As used herein, “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.
In some embodiments, the sequencing is deep/high-throughput sequencing. Examples of 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 Technologies™), and DNA nanoball sequencing. In a preferable embodiment, Illumina sequencing is used.
In some embodiments, 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.
In some embodiments, the affinity reagents are selected from the group consisting of nucleic acid molecules and polypeptides. In one embodiment, the affinity reagents are antibodies, preferably synthetic antibodies, and further preferably the library is a synthetic Fab or scFv library.
In some embodiments, each of the affinity reagents in the library contains unique sequence tags and the sequencing identifies the unique sequence tags. Preferably, the at least one clone is recovered by annealing primers specific for the unique sequence tags. For example, in a preferred embodiment, the library is a synthetic Fab library and the unique sequence tag is in the CDR H3 region.
In some embodiments, the target molecule is a cell surface protein. In further embodiments, 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.
In some embodiments, the 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.
The methods provided herein are used in combination with phage-display libraries referred to herein as Libraries F and G, but those of ordinary skill in the art will appreciate that these methods can be used in conjunction with any peptide/polypeptide display system in which cell-surface targets/antigens are expressed.
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 1.08×10¹¹unique 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.

TABLE 1

CDR Sequences of Library G clones

The nucleotide sequence of the vector encoding Library G is shown below:


FEATURES	Location/Qualifiers

rep_origin	3764 . . . 4235
	/note = “f1 origin”
sig_peptide	1534 . . . 1602
	/note = “ST2 secr signal”
promoter	21 . . . 52
	/note = “LacIq promoter”
promoter	1412 . . . 1439
	/note = “pTac promoter”
ORF	87 . . . 1169
	/note = “LacIq”
ORF	complement(5461 . . . 6321)
	/note = “AmpR”
ORF	2416 . . . 2880
	/note = “III gene (truncated)”
misc_feature	2008 . . . 2385
	/note = “VH”
misc_feature	1639 . . . 1959
	/note = “VL”
misc_feature	1960 . . . 2007
	/note = “linker C3”
misc_feature	2302 . . . 2346
	/note = “CDRH3”
misc_feature	2155 . . . 2184
	/note = “CDRH2”
misc_feature	1606 . . . 1629
	/note = “FLAG tag”
misc_feature	1786 . . . 1806
	/note = “CDRL2”
misc_feature	2092 . . . 2109
	/note = “CDRH1”
misc_feature	1909 . . . 1926
	/note = “CDRL3”
misc_feature	2389 . . . 2403
	/note = “hinge”
misc_feature	1720 . . . 1734
	/note = “CDRL1”
misc_feature	2404 . . . 2415
	/note = “dimerization domain”

1 gaattcccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga tagcgcccgg

61 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt

121 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg

181 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg

241 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg

301 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg

361 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc

421 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg

481 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg

541 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg

601 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg

661 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc

721 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa

781 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg

841 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg

901 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg

961 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg

1021 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc

1081 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac

1141 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact

1201 cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt

1261 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat

1321 aattcgtgtc gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata

1381 acggttctgg caaatattct gaaatgagct gttgacaatt aatcatcggc tcgtataatg

1441 tgtggaattg tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt

1501 tcacgagcac ttcaccaaca aggaccatag attatgaaaa agaatatcgc atttcttctt

1561 gcatctatgt tcgttttttc tattgctaca aatgcctatg catccgatta caaagatgac

1621 gatgacaaag gcggtggcga tatccagatg acccagtccc cgagctccct gtccgcctct

1681 gtgggcgata gggtcaccat cacctgccgt gccagtcagt ccgtgtccag cgctgtagcc

1741 tggtatcaac agaaaccagg aaaagctccg aagcttctga tttactcggc atccagcctc

1801 tactctggag tcccttctcg cttctctggt agccgttccg ggacggattt cactctgacc

1861 atcagcagtc tgcagccgga agacttcgca acttattact gtcagcaatc ttcttattct

1921 ctgatcacgt tcggacaggg taccaaggtg gagatcaaag gtactactgc cgctagtggt

1981 agtagtggtg gcagtagcag tggtgccgag gttcagctgg tggagtctgg cggtggcctg

2041 gtgcagccag ggggctcact ccgtttgtcc tgtgcagctt ctggcttcaa cttttcttct

2101 tcttctatac actgggtgcg tcaggccccg ggtaagggcc tggaatgggt tgcatctatt

2161 tcttcttctt atggctatac ttattatgcc gatagcgtca agggccgttt cactataagc

2221 gcagacacat ccaaaaacac agcctaccta caaatgaaca gcttaagagc tgaggacact

2281 gccgtctatt attgtgctcg cactgttcgt ggatccaaaa aaccgtactt ctctggttgg

2341 gctatggact actggggtca aggaaccctg gtcaccgtct cctcggccga caaaactcac

2401 acatgcggcc ggccctctgg ttccggtgat tttgattatg aaaagatggc aaacgctaat

2461 aagggggcta tgaccgaaaa tgccgatgaa aacgcgctac agtctgacgc taaaggcaaa

2521 cttgattctg tcgctactga ttacggtgct gctatcgatg gtttcattgg tgacgtttcc

2581 ggccttgcta atggtaatgg tgctactggt gattttgctg gctctaattc ccaaatggct

2641 caagtcggtg acggtgataa ttcaccttta atgaataatt tccgtcaata tttaccttcc

2701 ctccctcaat cggttgaatg tcgccctttt gtctttagcg ctggtaaacc atatgaattt

2761 tctattgatt gtgacaaaat aaacttattc cgtggtgtct ttgcgtttct tttatatgtt

2821 gccaccttta tgtatgtatt ttctacgttt gctaacatac tgcgtaataa ggagtcttaa

2881 tcatgccagt tcttttggct agcgccgccc tataccttgt ctgcctcccc gcgttgcgtc

2941 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg

3001 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac

3061 caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat

3121 ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac

3181 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg

3241 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt

3301 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt

3361 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac

3421 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag

3481 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt

3541 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatt cccccttaca

3601 cggaggcatc aagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga

3661 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac

3721 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcaggatccg gaaattgtaa

3781 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc

3841 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga

3901 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag

3961 ggcgaaaaac cgtctatcag ggctatggcc cactacgtga accatcaccc taatcaagtt

4021 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta

4081 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag

4141 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg

4201 cgcttaatgc gccgctacag ggcgcgtccg gatcctgcct cgcgcgtttc ggtgatgacg

4261 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg

4321 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag

4381 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga

4441 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag

4501 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt

4561 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc

4621 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa

4681 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa

4741 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc

4801 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc

4861 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag

4921 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga

4981 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc

5041 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac

5101 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg

5161 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca

5221 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa

5281 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa

5341 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt

5401 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag

5461 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat

5521 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc

5581 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa

5641 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca

5701 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa

5761 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt

5821 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc

5881 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact

5941 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc

6001 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg

6061 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct

6121 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc

6181 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag

6241 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac

6301 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg

6361 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt

6421 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac

6481 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaa (SEQ ID NO: 249)

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 3×10¹⁰unique 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.

TABLE 2

CDR Sequences of Library F clones

The nucleotide sequence of the vector encoding Library F is shown below:


FEATURES	Location/Qualifiers

promoter	536 . . . 752
	/note = “Pho A”
ORF	complement (4052 . . . 4913)
	/note = “AmpR”
ORF	2461 . . . 2925
	/note = “III gene (G2-CT)”
sig_peptide	804 . . . 872
	/note = “ST2 secr signal”
sig_peptide	1669 . . . 1737
	/note = “ST2 secr signal”
misc_feature	1747 . . . 2124
	/note = “VH”
misc_feature	1197 . . . 1526
	/note = “CL”
misc_feature	876 . . . 1196
	/note = “VL”
misc_feature	2125 . . . 2433
	/note = “CH1”
misc_feature	2041 . . . 2085
	/note = “CDRH3”
misc_feature	1894 . . . 1923
	/note = “CDRH2”
misc_feature	1527 . . . 1550
	/note = “FLAG tag”
misc_feature	1023 . . . 1043
	/note = “CDRL2”
misc_feature	1146 . . . 1163
	/note = “CDRL3”
misc_feature	1831 . . . 1848
	/note = “CDRH1”
misc_feature	2434 . . . 2448
	/note = “Hinge”
misc_feature	957 . . . 971
	/note = “CDRL1”
misc_feature	2449 . . . 2460
	/note = “dimerization domain”

1 ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc

61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga

121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc

181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc

241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag

301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa

361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac

421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct

481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcg catgcgacca

541 acagcggttg attgatcagg tagagggggc gctgtacgag gtaaagcccg atgccagcat

601 tcctgacgac gatacggagc tgctgcgcga ttacgtaaag aagttattga agcatcctcg

661 tcagtaaaaa gttaatcttt tcaacagctg tcataaagtt gtcacggccg agacttatag

721 tcgctttgtt tttatttttt aatgtatttg taactagtac gcaagttcac gtaaaaaggg

781 tatgtagagg ttgaggtgat tttatgaaaa agaatatcgc atttcttctt gcatctatgt

841 tcgttttttc tattgctaca aatgcctatg catccgatat ccagatgacc cagtccccga

901 gctccctgtc cgcctctgtg ggcgataggg tcaccatcac ctgccgtgcc agtcagtccg

961 tgtccagcgc tgtagcctgg tatcaacaga aaccaggaaa agctccgaag cttctgattt

1021 actcggcatc cagcctctac tctggagtcc cttctcgctt ctctggtagc cgttccggga

1081 cggatttcac tctgaccatc agcagtctgc agccggaaga cttcgcaact tattactgtc

1141 agcaatcttc ttattctctg atcacgttcg gacagggtac caaggtggag atcaaacgaa

1201 ctgtggctgc accatctgtc ttcatcttcc cgccatctga ttcacagttg aaatctggaa

1261 ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga

1321 aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca

1381 aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgaaaaac

1441 ataaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct

1501 tcaacagggg agagtgtggt ggttctgatt acaaagatga cgatgacaaa taattaactc

1561 gaggctgagc aaagcagact actaataaca taaagtctac gccggacgca tcgtggccct

1621 agtacgcaag ttcacgtaaa aagggtaact agaggttgag gtgattttat gaaaaagaat

1681 atcgcatttc ttcttgcatc tatgttcgtt ttttctattg ctacaaacgc gtacgctgag

1741 atctccgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc

1801 cgtttgtcct gtgcagcttc tggcttcaac ttttcttctt cttctataca ctgggtgcgt

1861 caggccccgg gtaagggcct ggaatgggtt gcatctattt cttcttctta tggctatact

1921 tattatgccg atagcgtcaa gggccgtttc actataagcg cagacacatc caaaaacaca

1981 gcctacctac aaatgaacag cttaagagct gaggacactg ccgtctatta ttgtgctcgc

2041 actgttcgtg gatccaaaaa accgtacttc tctggttggg ctatggacta ctggggtcaa

2101 ggaaccctgg tcaccgtctc ctcggcctcc accaagggtc catcggtctt ccccctggca

2161 ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac

2221 ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc

2281 ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc

2341 tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc

2401 aaggtcgaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cggccggccc

2461 tctggttccg gtgattttga ttatgaaaag atggcaaacg ctaataaggg ggctatgacc

2521 gaaaatgccg atgaaaacgc gctacagtct gacgctaaag gcaaacttga ttctgtcgct

2581 actgattacg gtgctgctat cgatggtttc attggtgacg tttccggcct tgctaatggt

2641 aatggtgcta ctggtgattt tgctggctct aattcccaaa tggctcaagt cggtgacggt

2701 gataattcac ctttaatgaa taatttccgt caatatttac cttccctccc tcaatcggtt

2761 gaatgtcgcc cttttgtctt tagcgctggt aaaccatatg aattttctat tgattgtgac

2821 aaaataaact tattccgtgg tgtctttgcg tttcttttat atgttgccac ctttatgtat

2881 gtattttcta cgtttgctaa catactgcgt aataaggagt cttaaagctc caattcgccc

2941 tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac

3001 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctgcattaat

3061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc

3121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg

3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag

3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc

3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag

3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga

3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc

3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg

3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt

3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca

3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca

3721 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag

3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca

3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg

3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa

3961 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta

4021 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag

4081 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga

4141 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac

4201 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc

4261 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta

4321 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac

4381 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat

4441 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa

4501 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg

4561 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag

4621 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc

4681 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct

4741 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat

4801 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg

4861 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc

4921 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta

4981 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg (SEQ

ID NO: 248)

In a further aspect, there is provided an antibody or antibody fragment comprising any one of CDR regions outlined in FIG. 2, FIG. 5 or FIG. 9. For antibodies or antigen-binding fragments thereof shown in FIG. 2 or derived from those 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), and one of the combinations of CDR-L3, CDR-H1, CDR-H2 and CDR-H3 shown in FIG. 2.
For antibodies or antigen-binding fragments thereof shown in FIG. 5 or derived from those shown in FIG. 5, 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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 242), where X₁, X₂, X₃, and X₄are Y, S, G, A, F, W, H, P or V and X₅is P or L and X₆is I or L; a CDR-H1 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 that includes the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and one of the CDR-H3 sequences shown in FIG. 5.
For antibodies or antigen-binding fragments thereof shown in FIG. 9 or derived from those shown in FIG. 9 and were identified from Library F, 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 X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 242), where X₁, X₂, X₃, and X₄are Y, S, G, A, F, W, H, P or V and X₅is P or L and X₆is I or L; a CDR-H1 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 243), where X₁is I or L, X₂, X₃, X₄, and X₅are Y or S and where X₆is I or M; and a CDR-H2 that includes the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y or S, X₄is P or S, and where X₇is G or S; and one of the combinations of CDR-L3 and CDR-H3 sequences shown in FIG. 9 (where the Library column indicates F).
For antibodies or antigen-binding fragments thereof shown in FIG. 9 or derived from those shown in FIG. 9 and were identified from Library G, the antibody or fragment contains a CDR-L1 that includes the amino acid sequence Q-X₁-X₂-X₃-X₄-X₅(SEQ ID NO: 245), where X₁, X₂, X₃, X₄, and X₅are Y, S or, G; a CDR-L2 that includes the amino acid sequence X₁-A-S-X₂-L-Y (SEQ ID NO: 246), where X₁, and X₃are Y, S or, G; a CDR-H1 that includes the amino acid sequence X₁-X₂-X₃-X₄-X₅-X₆(SEQ ID NO: 247), where X₁is I or L, X₂, X₃, X₄, and X₅are Y, S or G and where X₆is I or M; a CDR-H2 that includes the amino acid sequence X₁-I-X₂-X₃-X₄-X₅-X₆-X₇-X₈-T-X₉(SEQ ID NO: 244), where X₁, X₃, X₅, X₈, and X₁₀is Y, S or G, X₄is P or S, and where X₇is G or S; and one of the combinations of CDR-L3 and CDR-H3 sequences shown in FIG. 9 (where the Library column indicates G).
Preferably, 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. In one embodiment, 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.
In a further aspect, there is provided 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.
In a further aspect, there is provided 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.
In a further aspect, there is provided a use of the antibody or antibody fragment described herein for the treatment of 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 further aspect, there is provided a use of the antibody or antibody fragment described herein in the preparation of a medicament for the treatment of a cancer, such as Her-2 positive cancer, preferably selected from the group consisting of breast cancer, ovarian cancer, uterine cancer and stomach cancer.
The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.

EXAMPLES

Example 1

Rapid Isolation of Antibody Fragments to Cell-Surface Targets

The considerable heterogeneity of cell-surfaces makes selection of phage-displayed antibody libraries against cell-surface antigens challenging. We report the development of a unique methodology for rapidly isolating phage-displayed antibody fragments to cell-surface targets, using the oncogenic human epidermal growth factor receptor 2 (Her2) as a model. Synthetic phage-displayed libraries were selected in parallel on Her2-positive and negative cells. Following three rounds of selection, the output phage pools were analyzed by Illumina deep sequencing. Comparisons of the sequences from the positive and negative selection pool allowed sequences specific to the antigen-expressing cell-line to be readily identified from background phage clones. A PCR amplification strategy that used primers specific to the unique heavy chain third hypervariable loop enabled the recovery of clones from the positive selection pool, which represented 2.95% to 0.25% of the phage pool. Binding kinetics measured by surface plasmon resonance showed that all of the recovered antibody fragments bind to Her2 specifically and with high affinity. Three of the isolated antibody fragments were assayed for specific binding to Her2 expressed on the surface of transiently transfected cells and a Her2+ breast cancer cell-line by flow cytometry and immunofluorescence. These antibody fragments displayed specific binding to cell-surface Her2, demonstrating that our methodology, termed CellectSeq, is amenable to the rapid identification of high affinity antibody fragments specific to cell-surface epitopes. Together, these results suggest that the CellectSeq approach can increase the efficiency of library selections to cell-surface targets and eliminates the need for purified antigen.

Materials and Methods

Cell Culture

293T cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS). 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% CO₂.

Phage-Displayed Fab Library and Screening

Selections were performed using Library F, a single framework human Fab library constructed similarly to previously described libraries [28,29]. Briefly, a phagemid vector was engineered to bivalent display a human Fab on the pill protein of M13 bacteriophage. All three heavy chain CDRs and the light chain CDR3 were mutagenized using Kunkel mutagenesis and tailored oligonucleotide mixtures. Solvent assessable residues of CDRs H1 and H2 were restricted to tyrosine and serine residues, whereas CDRs H3 and L3 were allowed a much more complex chemical diversity of the following composition: 25% Tyr, 20% Ser, 20% Gly, 10% Ala, and 5% each of Phe, Trp, His, Pro and Val. The CDR H3 and L3 lengths were varied between 5 to 22 and 8 to 12 residues, respectively.
Library F was cycled through three rounds of selection, each consisting of a pre-absorption step followed by a positive selection step. For the pre-absorption 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 10¹²cfu 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.
For positive selection, 293T cells were harvested and plated at 2×10⁶cells in 150 mm tissue culture dishes (BD Falcon). Twenty-four hours after plating, cells were co-transfected with a Her2 expression plasmid (8 μg) and a GFP expression plasmid (2 μg) 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. Following incubation, cells were pelleted as before, the supernatant was discarded, and cells were re-suspended in cold phosphate-buffered saline (PBS). This process was repeated for a total of two washes for round one and three washes for rounds two and three. To obtain the negative selection pool for Illumina sequencing, Library F was also selected for three rounds against 5 million untransfected 293T cells, using the same methods described for the positive selection step.
Positively selected phage were amplified similarly to previous described methods [30]. Briefly, XL1blue cells were grown to an OD₆₀₀of 0.8 in 2YT media containing 10 μg/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 10¹⁰cfu 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 μg/ml carbenicillin, 25 μg/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].

Illumina Sequencing and PCR Amplification of Phagemid Clones

The round three positive and negative selection pool phage, along with the naïve library, were infected into XL1blue cells and grown overnight in 2YT supplement with 100 μg/ml carbenicillin. Cultures were miniprepped (Qiagen) to obtain phagemid DNA to use as the templates for a PCR with individual forward primers comprised of an adaptor sequence (5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) (SEQ ID NO: 1), a five base pair barcode sequence (positive pool: 5′-GAGTA-′3 (SEQ ID NO: 2); negative pool: 5′-CCAAA-′3 (SEQ ID NO: 3); naïve library: 5′-TTGTT-3′ (SEQ ID NO: 4)) and an annealing site to the third antibody framework region of the heavy chain (5′-GTCTATTATTGTGCTCGC-3′) (SEQ ID NO: 5). For all phage pools, 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.
To rescue individual clones from the positive selection pool, 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. and 180 s at 72° 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.

Vectors and Primers

For PCR recovery the following phosphorylated forward and reverse primers were used to recover the phage-Fab clones:

	(SEQ ID NO: 8)
	WY574B: 5′-CCAGTAATGAACAACAGC-3′,

	5′-TACGGTTACGTTTCTGGT-3′;

	(SEQ ID NO: 9)
	WY574E: 5′-AGCCGGAACCCAACCGCG-3′,

	5′-TACCCGTCTTACGGTTTG-3′;

	(SEQ ID NO: 10)
	WY574F: 5′-AGCGTAAACAGAAGAACCCCA-3′,

	5′-TGGTCTCCGGCTTCTTGGTCT-3′;

	(SEQ ID NO: 11)
	WY677C: 5′-ACCCCACCAGTAGTAAGA-3′,

	5′-CCGTGGTCTGGTTACTCT-3′;

	(SEQ ID NO: 12)
	WY677D: 5′-GTACGGAATGTACGGATGCGG-3′,

	5′-TACTCTTACTGGGGTCCGTACTAC-3′.

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:

(SEQ ID NO: 13)

V_H: 5′-TGTAAAACGACGGCCAGTGGACGCATCGTGGCCCTA-3′,

(SEQ ID NO: 14)

5′-CAGGAAACAGCTATGACCCCTTGGTGGAGGCCGAG-3′;

(SEQ ID NO: 15)

VL: 5′-TGTAAAACGACGGCCAGTCTGTCATAAAGTTGTCACGG-3′,

(SEQ ID NO: 16)

5'-CAGGAAACAGCTATGACCCCTTGGTACCCTGTCCG-3′

Her2 and EGFR were both expressed from pCDNA3 (Invitrogen) [32,33], and GFP was expressed from a previously reported plasmid [34].

Protein Expression and Purification

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 μg/ml carbenicillin and 25 μg/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]. Ten milliliters of the re-suspended culture was used to inoculate 1 L of CRAP media, which was subsequently grown for 24 to 27 hours at 30° C., pelleted, re-suspended in 25 ml of PBS, and frozen. After thawing, 15 mg of lysozyme (Bioshop) and 30 μl of DNase I (deoxyribonuclease I, Fermentus) was added to 30 ml of cell suspension and cells were lysed by sonication. Following centrifugation to pellet cell debris, Fab supernatants were loaded onto fast-flow rProtein A-Sepharose (GE Healthcare) pre-equilibrated in PBS. Columns were washed with PBS, eluted with 50 mM NaH₂PO₄, 100 mM H₃PO₄, 140 mM NaCl, pH 2.5. Eluates were neutralized with 1 M Na₂HPO₄, 140 mM NaCl. Recovered Fab proteins were analyzed by SDS-PAGE and quantified using a Bradford assay (Bio-Rad).

Surface Plasmon Resonance

The binding affinities and kinetic parameters for interactions between Her2 specific Fabs and recombinant Her2 (R&D Systems) were measured by surface plasmon resonance using a ProteOn XPR36 instrument (Bio-Rad). 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 μl/min, followed by ten minutes of buffer to monitor Fab dissociation. The chip surface was regenerated with 0.85% H₃PO₄prior to new analyte injection. Kinetic parameters were determined by globally fitting a reference cell-subtracted concentration series to a 1:1 (Langmuir) binding model.

Flow Cytometry and Immunofluorescence Staining

For flow cytometric analysis of transfected 293T cells, 3×10⁶cells were plated on 10 cm dishes (BD Falcon). Twenty-four hours after plating, cells were transfected with 10 μg 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 1.5×10⁶cells were placed into 1.5 ml tubes for staining with individual Fab clones. First, cells were incubated for 45 minutes at room temperature in PBS containing 2% FBS to block non-specific epitopes. Next, cells were incubated with 2 μg 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. Finally, cells were washed twice and re-suspended in 0.5 ml of PBS for analysis on a BD FacsAria I flow cytometer (BD Biosciences).
For flow cytometric analysis of Fab binding to the human cancer cell-lines, 5×10⁵BT474 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 μg 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 μg/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 MgCl₂and 1 mM CaCl₂on 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.

Results and Discussion

Library Screening Against Her2-Transfected Cells and Illumina Sequence Analysis

We subjected the synthetic Fab library F to three rounds of selection on 293T cells transiently transfected to express Her2 (FIG. 1 a). To help reduce background from phage binding to undesired cell-surface epitopes and non-specific binding phage clones, 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. We also carried out three rounds of selection against untransfected 293T cells. This negative selection was carried out with the rational that the sequences obtained from this pool represent undesired background clones that are unlikely to be Her2 specific binders. As such, comparing the sequences from the positive and negative selections should help readily identify sequences in the positive pool that arise from phage clones binding to undesired epitopes.
The positive and negative selection pools, and the naïve library, were next subjected to Illumina sequencing analysis. Of the 100 most frequently observed CDR H3 sequences in the positive pool, which represent anywhere from 0.06 to 14.94% of the total number of sequences obtained, 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 naïve library pool. Sixteen of the sequences observed in the naïve library are also present in the negative selection pool. As expected, sequences in the naïve library exhibited a much greater degree of diversity than the sequences of the two selected pools.
PCR Recovery of Her2 Specific Clones from the Positive Selection Pool
Single clones of interest, identified from the Illumina sequencing results, 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. Following a blunt-end ligation and transformation into E. coli, 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. Of note, 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.

Binding Kinetics of Recovered Anti-Her2 Clones

Kinetic analysis of the purified Fabs by SPR shows that the five recovered Fab clones bind to recombinant Her2 with high affinities (Table 3), with K_Dvalues ranging from 4 nM to 75 nM. These data suggest that the cell-surface selection methodology presented here can be used to rapidly recover multiple Fab clones that bind with high affinity to the target of interest. We chose three of the five Fab clones, WY547B, WY574E, and WY574F, for further analysis based on the observation that they exhibit a range of affinities encompassing the highest (WY574F), lowest (WY574E), and an intermediate (WY574B) affinity value.

TABLE 3

Binding Kinetics of anti-Her2 Fab clones

	Fab	K_a(M⁻¹s⁻¹)	K_d(s⁻¹)	K_D(nM)

WY574B	1.0 × 10⁵	1.4 × 10⁻³	14
WY574E	1.9 × 10⁵	6.8 × 10⁻⁴	4
WY574F	9.7 × 10³	7.3 × 10⁻⁴	75
WY677C	4.37 × 10⁴	1.62 × 10⁻³	37
WY677D	4.01 × 10⁵	1.08 × 10⁻²	27

Cell-Surface Specificity of Anti-Her2 Fabs

Next, the specificity of Fab clones WY574B, WY574E, and WY574E, was examined by flow-cytometry using 293T cells transiently transfected with Her2 or EGFR, which is also a member of the EGFR receptor family. A fluorescence shift was observed in the Her2-transfected cell population for all three Fab clones (FIG. 3 a). A similar shift in fluorescence staining was not observed in the EGFR-transfected cell population or the unstained Her2-transfected 293T cell population. This data suggests that the recovered Fabs are binding specifically to the Her2 transfected cell population. We also evaluated the specificity of the Fab clones by flow-cytometric analysis with the Her2 positive breast cancer cell-line BT474 and the Her2 negative breast cancer cell-line T47D (FIG. 3 b). As expected, a drastic shift in the fluorescence signal of the BT474 cell population was observed in the presence of the anti-Her2 Fabs. In contrast, little or no binding of the three Fabs was detected in the Her2 negative T47D cell population.
Finally, we sought to confirm the specificity of the three Fabs for Her2 presented on the cell-surface by immunofluorescent staining (IF) of Her2-transfected 293T cells and a Her2 expressing cancer cell-line. Fluorescent staining of each Fab clone was observed around the cell periphery in the Her2-transfected 293T cells (FIG. 4 a). Consistent with staining pattern observed in the Her2-transfected cells, fluorescent staining of the Her2 positive BT474 cancer-cell line was also evident by IF (FIG. 4 b). In contrast, no specific staining was observed for the Her2-negative T47D cancer cell-line. Collectively, these data strongly demonstrate that the recovered Fab clones bind specifically to Her2 presented in the context of the cell-surface.
Selection of phage-displayed antibody libraries against cell-surface antigens is often challenging, as the vast array of epitopes presented on the cell-surface gives rise to a high degree of background binding and poor enrichment of clones specific to the target of interest. 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. Here, sequences distinctive/exclusive to the positive selection output pool represent clones that have a high probability of being specific for the target antigen. In addition, combining cell-surface selections with deep sequencing allows rare clones to be identified. It is unlikely that the degree of clonal diversity we observe by deep sequencing could be resolved using traditional phage display methodologies, in part because of the practical limitations of manually screening sufficient numbers of single phage clones to retrieve a similar level of sequence diversity. Although a variety of factors can influence clonal diversity during the selection process, such as the growth advantage of certain clones, selection methodologies also tend to preferentially enrich for higher affinity binders. As a consequence, the diversity of the sequences recovered in later rounds may be diminished. However, high throughput DNA sequencing is becoming an increasingly accessible technology, as evidenced by recent reports that made use of deep sequencing approaches to characterize human antibody libraries and V-gene repertoires of immunized mice [25,26].
We reasoned that Her2 would be an ideal model given the existence of a well-characterized therapeutic monoclonal antibody specific for Her2, which is reflective of our goal of applying the CellectSeq methodology to isolating stable, high affinity, antibody fragments specific to therapeutically relevant cell-surface proteins. 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. Although 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. However, it is also important to note that 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.
Of the ten unique CDR H3 clones we attempted to rescue, five failed to generate a PCR product. This may be due to factors that include the sequence and length of the CDR H3, the abundance of the template in the positive selection pool, or the design of the PCR primers. It is important to consider, however, that these factors were not optimized in this study. In light of this observation, the number of phagemid clones we successfully rescued is considerably high. Another important consideration is whether the methodology presented here introduces bias into the final sequence analysis. For example, many of the sequences in the naïve and negative selection pools that overlap with the positive selection pool are of very short length. However, this type of potential bias can be identified by comparing the abundance of given sequences in the positive pool to the negative pool. It is possible that shorter sequences were preferentially amplified during the PCR reaction used to recover the DNA that was subsequently submitted for Illumina sequencing. In addition, a previous analysis of the naïve Fab library did demonstrate that there was a bias towards shorter CDR H3 loop lengths, which was likely attributable to differences in the efficiency of the library mutagenesis reaction with oligos of different lengths. This issue may be addressed by comparison of the length distribution of the hypervariable regions sequenced by traditional Sanger methods to those sequenced in the deep sequencing analysis.
A limiting step to molecular display technologies is the need for correctly folded, purified antigen. For example, 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. For instance, 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]. In cases in which the target of interest may be a member of an oligomeric complex, 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. One example of relevance here is Her2, as it known to homo- and hetero-dimerize with the other members of the EGFR family [27].

Example 2

PCR-Based Recovery of Antibody Fragments to Additional Cell-Surface Targets

The rescue strategies described herein make use of both the unique H3 and L3 CDR sequences.
As an alternative to identifying positive Fabs by clonal cell ELISAs, two different PCR based recovery methods are used (FIGS. 6A, 6B). 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. 6B, 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. Listed are the phage-Fab clones targeting various cell surface receptors that were successfully rescued from the positive selection pool. The following is displayed: the cell line used to express the antigen of interest; the phage-display library used for selection (F: Fab, G:scFv); the rescue strategy used to recover the clones (1. Clonal ELISA, 2. cdrH3 PCR, 3. 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. Note that GUP is a cocktail of Glucosamine, Uridine and PugNAc.

Example 3

Rapid Isolation of Antibody Fragments Specific to Cell Growth Conditions

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 μM 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. B4 binder ranked #1 with ratio of 1.8, A4 binder ranked #2 with ratio of 1.6, C4 binder ranked #4 with ratio of 1.5 and C3 binder ranked #7 with ratio 1.5.

Example 4

Deep Sequencing to Decode Variable Regions of Affinity Reagents

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. In the examples shown here, one or more complementarity determining regions (CDRs) of synthetic antibodies are decoded by deep sequencing.

Materials and Methods

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.

Strategy 1: Illumina Sequencing of CDRs L3 and H3

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).
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.
Strategy 2: Illumina Sequencing of CDRs L3 and H3, with Optional Sequencing of H2 and H1
The forward PCR primer was composed of a paired end Read 1 Illumina adaptor sequence (5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3) (SEQ ID NO: 231), barcode (5′NNNNNNNN-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).

Strategy 3: IonTorrent Sequencing of CDR H3

The forward PCR primer was composed of an IonTorrent Adapter A sequence (5′ CCATCTCATCCCTGCGTGTCTCCGACTCAG-3′) (SEQ ID NO: 236), barcode (5′ NNNNNNNNNC-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.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references cited herein, including those in the attached reference list, are incorporated by reference.

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Claims

1. A method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule, the method comprising:

(a) providing a molecular display system which displays a library of potential genetically encoded affinity reagents;

(b) screening the library against the target molecule to produce positive and negative selection pools, preferably with multiple rounds of selection;

(c) sequencing genetically encoded affinity reagents in each of the positive and negative selection pools;

(d) identifying at least one sequence that is more abundant in the positive selection pool as compared to the negative selection pool; and

(e) recovering at least one clone corresponding to the sequence.

2. The method of claim 1, wherein the display system is selected from the group consisting of phage display, bacterial display, yeast display, ribosome display and mRNA display.

3. The method of claim 2, wherein the display system is phage display.

4. The method of claim 1, wherein the sequencing comprises deep/high-throughput sequencing.

5. The method of claim 4, wherein the deep sequencing comprises Illumina sequencing.

6. The method of claim 4, wherein the deep sequencing comprises Ion semiconductor sequencing.

7. The method of claim 1, wherein each of the affinity reagents in the library contain unique sequence tags and the sequencing identifies the unique sequence tags.

8. The method of claim 7, wherein the at least one clone is recovered by annealing primers specific for the unique sequence tags.

9. The method of claim 1, wherein the affinity reagents are selected from the group consisting of nucleic acid molecules and polypeptides.

10. The method of claim 9, wherein the affinity reagents are antibodies or antigen-binding fragments thereof.

11. The method of claim 10, wherein the affinity reagents are synthetic antibodies.

12. The method of claim 11, wherein the library is a synthetic Fab library.

13. The method of claim 7, wherein the library is a synthetic Fab library and the unique sequence tag is in the CDR H3 region.

14. The method of claim 1, wherein the target molecule is an epitope of a protein.

15. The method of claim 1, wherein the protein is a cell surface protein.

16. The method of claim 1, wherein the screening is performed against the target molecule presented on a cell surface.

17. The method of claim 16, wherein target molecule is presented on the cell surface of a mammalian cell.

18. The method of claim 1, wherein the 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.