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WO2019099454A2 - Bibliothèques d'anticorps hautement fonctionnelles - Google Patents

Bibliothèques d'anticorps hautement fonctionnelles Download PDF

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WO2019099454A2
WO2019099454A2 PCT/US2018/060933 US2018060933W WO2019099454A2 WO 2019099454 A2 WO2019099454 A2 WO 2019099454A2 US 2018060933 W US2018060933 W US 2018060933W WO 2019099454 A2 WO2019099454 A2 WO 2019099454A2
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antibody
sequence
cdr
seqa
protein
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WO2019099454A3 (fr
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Philippe Valadon
Juan Carlos Almagro
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with the file“PCT_sequences_WorkFile.txf’ created on November 14, 2018, filed on November 14, 2018 and having a size of 43 KB.
  • sequence listing contained in this ASCII formatted document forms part of the specification and is herein incorporated by reference in its entirety.
  • the present invention relates to methods for preparing antibody libraries of improved functionality and relates to the design of antibody libraries of improved functionality and uses thereof.
  • Antibodies are immunoglobulins, or specialized immune proteins, having a heterodimeric structure.
  • the antibody structure consists of two heavy chains and two light chains, folded into constant and variable domains.
  • the variable domains of the heavy chains and light chains form the antigen binding site.
  • Each variable region contains three hypervariable loops known as complementarity determining regions (CDRs) which alternate with less variable regions, called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Monoclonal antibodies are antibodies that are made from identical immune cells that are all clones of one parent cell. Monoclonal antibodies possess unique characteristics such as specificity, affinity, potency, stability, solubility, and clinical tolerability.
  • Hybridoma technology also known as monoclonal antibody technology, is an efficient means to isolate single specificity antibodies and produce them in unlimited amounts (Kohler and Milstein, 1975).
  • the HAMA response prevents the use of multiple administrations of an antibody, for example required for cancer therapy. Further complicating the use of murine monoclonal antibodies in human therapy is their association with the generation of severe allergic reactions. Together, such issues hamper the use of murine antibodies in human therapy (Shawler et al, 1985).
  • V variable domains
  • C constant domains
  • This method is called chimerization and led to the approval of the first cancer-treating therapeutic antibody, Rituximab (Rituxan ® ) (Morrison et al., 1984).
  • Rituximab has been a tremendous medical and commercial success, currently being the fourth best-selling innovative drug of any kind (Strohl, 2017).
  • Phage display technology connects proteins displayed at bacteriophage surface to their genes in the bacteriophage’s genome.
  • phage display opened the possibility of designing and manipulating the repertoire of antibody genes to be used as source of antibodies in phage antibody libraries, hence enabling the selection of fully human antibodies (Hanes and Pluckthun, 1997; Francisco et al., 1993; Beerli et al., 2008; Cherf and Cochran, 2015; Finlay and Almagro, 2012).
  • these technology platforms allow selection for desired pre-defmed epitopes, thus avoiding immunodominant epitopes.
  • these technology platforms can also focus on specific conformations, rare cross-reactive epitopes, or conditions to select antibody variants with enhanced biophysical and biochemical properties.
  • PBMCs peripheral blood mononuclear cells
  • Other strategies are the construction of fully synthetic libraries, which are computationally designed and generated by chemical synthesis (Griffiths et al., 1994; Knappik et al., 2000; Shi et al., 2010), and semisynthetic libraries, which combine natural diversity and synthetic diversity (Hoet et al., 2005).
  • Antibody libraries composed of natural diversity also known in the art as naive libraries, were the first generation of phage displayed antibody libraries (Marks et ah, 1991). Although successful, naive libraries included antibody genes toxic to E. coli and thus with low expression levels or no expression at all on the phage surface. These toxic genes severely compromised the number of functional antibodies displayed in the library. Synthetic antibody libraries followed and partially mitigated this limitation (Knappik et ah, 2000) (US patent 6,828,422). However, synthetic libraries must carefully be designed by making assumptions regarding the number of positions to diversify and type of amino acids to include in the design and proportion of each amino acid per position.
  • CDR-H3 which is by far the most diverse region of the antigen-binding site and key in defining the specificity and affinity of antibodies.
  • the structure of CDR-H1 and CDR-H2 can be predicted with an accuracy of ⁇ 1.0 A. This is a critical step in the successful design of the diversity of a synthetic library.
  • no current method is available in the art to reliably predict the CDR-H3 structure (Almagro et al. 2014).
  • This invention discloses highly functional antibody libraries in addition to methods to make such highly functional antibody libraries.
  • the exemplary antibody libraries of this invention combine highly stable and developable antibody variants with natural diversity in CDR-H3, as well as methods to remove non-productive antibody sequences such as out-of-frame variants and/or poorly folded antibodies due to imprecisions in the design, without compromising the functional size of the library.
  • the present invention includes methods for producing highly functional antibody libraries, comprising the steps of: (1) designing and preparing primary antibody libraries (PLs); (2) applying a selection process (filtration) to enrich the PLs of step 1 with variants of improved functionality and/or developability to prepare intermediate filtered libraries (FLs); and (3) preparing highly functional secondary antibody libraries (SLs) by combining FLs with diverse CDR-H3 fragments.
  • PLs primary antibody libraries
  • filtration selection process
  • FLs intermediate filtered libraries
  • SLs highly functional secondary antibody libraries
  • V L scaffolds Two PLs were designed so that each of the PLs had one V H scaffold binding to a Staphylococcus aureus Protein A. Two distinct V L scaffolds were designed as counterpart of the V H scaffold.
  • One of the V L scaffolds contained a short CDR-L1 loop (PL1).
  • the other V L scaffold contained a long CDR-L1 loop (PL2) ( Figure 1).
  • the libraries of this invention can be used for selection of antibodies against protein or peptide targets. Selection of the two libraries, PL1 and PL2, would potentially generate antibodies that bind diverse types of epitopes on a given target.
  • the scaffolds of the two PLs were diversified in positions that bind both protein and peptide targets and amino acids observed in human germline genes and natural antibodies. Amino acids associated with developability liabilities were avoided. Such amino acids included: (i) asparagine (N) followed by any amino acid but proline (XnoP) followed by serine (S) or threonine (S/T) [NXnoP(S/T)], which generates N-glycosylation sites; (ii) aspartic acid (D) followed by glycine (G) [DG], which tends to isomerize; (iii) asparagine (N) followed by glycine (G) or serine [NG/S], which tends to deamidate, (iv) exposed methionine (M), which tends to oxidize and (v) exposed tryptophan (W), which leads to aggregation spots.
  • amino acids included: (i) asparagine (N) followed by any amino acid but proline (X
  • V H and V L scaffolds were assembled as single chain Fv fragments (scFv) in a V L - linker-V H configuration and synthesized using trinucleotide phosphoramidites.
  • Trinucleotide phosphoramidites synthesis also known as trimer technology, is a type of synthesis that is based on synthetic codons instead of single nucleotides.
  • Trinucleotide phosphoramidites synthesis generates precise combinations of amino acids at specifically targeted positions for diversifications while avoiding stop codons and unwanted amino acids which may disrupt the folding of the scaffolds used to generate the libraries.
  • the quality control of the synthetic fragments was assessed via sequencing of 96 fragments in each library. The results indicated that 50% to 60% sequences were in-frame and matched the design.
  • the percentage of clones binding Protein A after incubation at 70°C for 10 min was 48.8% and 55.8% for PL1 and PL2, respectively, down from 58.5% and 69.8%, respectively in the PL1 and PL2.
  • PL1 and PL2 were incubated for 10 min at 70°C and well- folded and developable antibody fragments were rescued with Protein A, while unstable and non- developable antibody variants, which were either denatured or aggregated, were removed by a simple washing.
  • Other harsh conditions include, but by no means are limited to, other temperatures and incubation times, high or low pH, high salt concentrations, and protease digestion.
  • PL1 and PL2 are built with two human V L scaffolds that belong to two different germline gene families and only share 68.3% identities.
  • the kinetic of unfolding, as showed in the examples of this invention, is significantly different.
  • a consistent average decrease of -12% in Protein A binders represents a loss of 1.2 x 10 9 unique and potentially functional antibody variants in a library containing 1 xlO 10 unique antibody variants. Therefore, improving stability after filtering came with the price of reducing the diversity of the libraries.
  • nucleotide sequences encoding the FLs were amplified by molecular biology techniques known to those skilled in the art and combined with diverse natural CDR-
  • H3 fragments called“natural H3Js” to produce the SLs. It was reasoned that by replacing the neutral H3Js fragments in the FLs with natural H3Js, highly stable and functional libraries can be obtained. This rationale is supported by a substantial body of work indicating that CDR-H3 is key to determine the specificity and affinity of the antibodies and hence antibody libraries with highly diverse CDR-H3 fragments should increase the probability of obtaining diverse and high affinity antibodies.
  • the natural H3 Js were isolated from a pool of 200 donors by molecular biology methods known to those skilled in the art. Amplification of diverse CDR-H3 fragments from other sources including CDR-H3 fragments obtained by synthetic means are clear extensions of this invention.
  • the combination of highly stable scaffolds used to build the libraries led to a collection of highly stable variants suited to accommodate a collection of highly diverse natural H3J fragments, and hence, a highly functional antibody library.
  • NGS next generation sequencing
  • the antigen model is Tumor Necrosis Factor (TNF).
  • TNF Tumor Necrosis Factor
  • HSA human serum albumin
  • Figure 1 provides a ribbon representation of the Y and V L scaffolds.
  • Figure 1A shows a drawing showing the ribbon representation of an Fv with a short Ll loop (PDB ID: 1ILC) [3-20/3-23]
  • Figure IB shows a drawing showing the ribbon representation of an Fv with a long CDR-L1 loop (PBD
  • This dataset of unique antibody: antigen complexes was obtained by starting from all the antibody structures compiled at the PDB and curated by the IMGT as of March l .l 2017. The initial dataset consisted of 2,645 antibody structures from diverse species and specificities. This initial data set was mined to extract the unique and well-solved antigen: antibody listed in the table.
  • Figure 3 provides a table showing a diversification regime at CDR-H1 and CDR-H2 of the V H scaffold.
  • the residues in contact with antigens were determined in the set of structures listed in Figure 2 and mapped onto the structures of the V H scaffold paired with the two V L scaffolds PDB IDs: 1ILC and 1ILD.
  • a total of 10 positions were targeted for diversification, four in the CDR-H1 and six in the CDR- H2.
  • the diversification regime was designed using three sources of information: the dataset of curated antibody structures listed in Figure 2, the V H sequences available at NCBI, and the germline genes of the human IGHV3 family compiled at the IMGT.
  • the estimated number of amino acids per position is listed in the last column of the figure and yields a diversity of 5.4 x lO 5 unique amino acid V H sequences.
  • Figure 4 shows the diversification regime of the 3-20 ⁇ scaffold. To identify positions for diversification and the diversification regime the same procedure than that for the scaffold was followed. The estimated number of amino acids per position is listed in the last column of the figure and yields a diversity of 1.1 x 10 6 unique amino acid V L sequences.
  • Figure 5 shows the diversification regime of the 4-01 ⁇ scaffold. To identify positions for diversification and the diversification regime the same procedure than that for the V scaffold was followed. The estimated number of amino acids per position is listed in the last column of the figure and yields a diversity of 1.3 x 10 6 unique amino acid V L sequences.
  • Figure 6 provides the configuration of the two primary scFv repertoires, diversified regions and cloning sites.
  • V L and V H are linked to form a scFv by the repetitive stretch of amino acids GS19.
  • the V L -linker-V H configuration places the H3J fragment on the C-terminal side.
  • Two Bgll/Sfil sites located on each side of the construct allow for cloning into the acceptor vector.
  • Ncol and Kpnl sites are common to both repertoires, same for the JK1 anchor in the light chain sequence and the human VH conserveed Motif (CM) in the heavy chain sequence.
  • Figure 7 provides a graph showing phage binding to Protein A by ELISA.
  • the y-axis shows the optical density measurements at 490 nm and the x-axis shows dilutions in virions/ml for the 3-20/3-23 control scFv, the 4-1/3-23 control scFv, the 3-20/3-23 library, and the 4-01/3-23 library.
  • COSTAR plates 3369 (Coming) were coated with Protein A (Sigma Aldrich, cat# P6031) at 4 pg/ml in TBS overnight at 4°C.
  • virions ( ⁇ 2.6 x 10 12 virions/ml) in TBST with 5% w/v nonfat dry milk were added to the wells, 2- fold serially diluted and incubated for 2 h at 37°C.
  • virions derived from the parent scaffolds with the CDR-H3 of CNTO 888 cloned in the same vector were added and similarly diluted on the plate.
  • Bound phage was detected with A4G1.6 monoclonal antibody (Antibody Design Labs, San Diego, CA) conjugated to HRP. Binding of the secondary antibody, a murine IgGl, to Protein A was blocked by polyclonal human IgG at 100 pg/ml added to the incubation buffer.
  • Figure 8 provides a graph showing, on the y-axis, optical density measurements at 490 nm taken at different temperature points during heat shock showing the thermal unfolding of the 4-01/3-23 and 3- 20/3-23 control scFvs.
  • the 3-20/3-23 and 4-01/3-23 control scFvs displayed as fusion proteins to pill on the phage surface were incubated for 10 min over a range of temperatures, starting at 40°C and increasing the temperature up to 80°C in steps of 5°C.
  • the unfolding process is monitored with a direct Protein A ELISA shown in Figure 7.
  • Figure 9 provides a graph showing, on the y-axis, optical density measurements at 490 nm taken at different time points during heat shock showing the thermal unfolding of the 4-01/3-23 and 3-20/3-23 control scFvs at 60°C and 72°C.
  • the 3-20/3-23 scFv and 4-01/3-23 scFv were displayed as fusion proteins to pill on the phage surface and the unfolding process is monitored with a direct Protein A ELISA shown in Figure 7.
  • Figure 10 provides a bar graph showing different Protein A binding relative to control scFvs at 37°C. The binding shown is measuring PL1 single clone scFv-phages binding to Protein A relative to control scFv. A sample of 44 ampicillin-resistant colonies was chosen at random from PL1. The phage displaying scFvs were prepared in 96 deep-well plates and, after pelleting the bacteria by centrifugation,
  • Figure 13 provides a bar graph showing residual Protein A binding of PL2 scFv-phage single clones following 10 min at 70°C. The binding shown is measuring PL2 single clone scFv-phages binding to Protein A relative to control scFv. The 44 clones selected from PL2 were incubated for 10 min at
  • Figure 15 provides a bar graph showing Protein A binding relative to control scFvs after incubation at 70°C for 10 min and Protein A filtering. The binding shown is measuring FL2 single clone scFv-phage binding to Protein A relative to control scFv. A sample of 44 ampicillin-resistant colonies was selected at random from FL2. The phages were prepared and assayed on Protein A as described in
  • Figure 16 provides a drawing depicting a strategy for assembling seamlessly semisynthetic secondary repertoires.
  • the top line shows a strategy for the filtrated primary library scaffold amplification
  • the middle line shows a strategy for an assembly with natural H3J fragments
  • the bottom line of Figure 16 shows the final product.
  • the diversified scaffold fragments from the filtrated primary libraries were amplified by a pair of primer located in the pelB leader and 5’ of the human VH Consensus Motif (CM).
  • CM human VH Consensus Motif
  • Two Bsal sites added at the end of the C-terminal of the Fv and at the beginning of the natural H3J fragments are used to assemble the complete scFv in a single digestion step plus ligation reaction.
  • the assembled product is amplified as a whole scFv prior to ligation and cloning.
  • Figure 17 shows a series of photographs of DNA gel electrophoresis, comparing between SL1 (on the left) and SL2 (on the right) library assembly by seamless amplification.
  • Figure 17A shows an electrophoresis gel of the amplified filtrated primary library scaffolds.
  • Figure 17B shows an electrophoresis gel before (left lane) and after (right lane) the fragment seamless assembly with natural
  • Figure 17C shows an electrophoresis gel of the amplification of the full scFv fragments.
  • Figure 18 provides a bar graph showing Protein A binding of SL1 single clones relative to control scFvs after incubation at 37°C for 10 min. A sample of 44 ampicillin-resistant colonies was selected at random from the SL1. The phage were prepared and assayed on Protein A as described in
  • Figure 19 provides a bar graph showing Protein A binding of SL2 single clones relative to control scFvs after incubation at 37°C for 10 min. A sample of 44 ampicillin-resistant colonies was selected at random from the SL2. The phages were prepared and assayed on Protein A as described in
  • Figure 20 provides a bar graph showing Protein A binding of SL1 single clones relative to control scFvs after incubation at 70°C for 10 min.
  • the phages were prepared and assayed on Protein A as described in Figure 7.
  • Figure 21 provides a bar graph showing Protein A binding of SL2 single clones relative to control scFvs after incubation at 70°C for 10 min.
  • the phages were prepared and assayed on Protein A as described in Figure 7.
  • Figure 22 provides a bar graph showing a comparison of Protein A survival between PLs and SLs after incubation at 70°C for 10 min. Survival is defined >10% with respect to the control scaffold in the Protein A binding ELISA after incubation for 10 min at 70°C for phage clones having 25% or more binding to Protein A relative to the control scaffolds.
  • Figure 23 provides a graph showing, on the y-axis, optical density measurements at 450 nm versus 570 nm showing the binding of the purified anti-TNFalpha scFv TNF-E12 taken at different concentrations on the x-axis to either TNFalpha or BSA as a negative control.
  • “X and/or Y” can mean“X” or‘ ⁇ ” or“X and Y”.
  • the use of“comprise,”“comprises,”“comprising,”“include,”“includes,” and“including” are interchangeable and not intended to be limiting.
  • the description of one or more embodiments uses the term“comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language“consisting essentially of’ and/or“consisting of’.
  • the term“and/or” means one or all of the listed elements or a combination of any two or more of the listed elements.
  • the practice of the present invention may employ conventional techniques and descriptions of bacteriology, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art.
  • Such conventional techniques include PCR, extension reaction, oligonucleotide synthesis and oligonucleotide annealing, ELISA.
  • Specific illustrations of suitable techniques can be added by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols.
  • antibody is used in the broadest sense and refers to monoclonal antibodies and one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., lysozyme). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • antigen-binding fragments encompassed a Fv fragment consisting of the Y and V H domains of a single arm of an antibody, a Fab fragment, a F(ab)2, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region fragment a monovalent fragment consisting of the Y V H , CF and Cj 11 domains, a Fd fragment consisting of the V H and C H l domains.
  • the two domains of the Fv fragment, V L and V H domains are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules, known as single chain Fv (scFv).
  • scFv single chain Fv
  • Such scFvs are also intended to be encompassed within the term "antibody”.
  • antibody fragments are obtained using conventional techniques known to those of skill in the art and the fragments are screened using, but not limited to, phage, yeast and mammalian display for utility in the same manner as are intact antibodies.
  • “antibody variable domain” refers to the portions of the light and heavy chains of antibody molecules that interact specifically with an antigen (e.g., lysozyme).
  • ‘(jV or“V H domain” refers to a variable domain of an antibody heavy chain.
  • “V L ” or“V L domain” refers to a variable domain of an antibody light chain.
  • the Y domain is produced by the recombination of the IGVF and IGVJ germline genes, whereas the V H domain is encoded by repertoires of IGVH, IGVD and IGJH germline genes.
  • V H and V L contain the antigen-binding site and hence define the capacity of antibodies to bind virtually any antigen with extraordinar specificity and high affinity.
  • the term“antigen-binding site”, as used herein, contains the portion of the variable domains that interact with the antigens. Definitions of the antigen-binding site include the complementarity determining regions (CDRs) as defined by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Bethesda, Md.: National Center for Biotechnology Information, National Fibrary of Medicine, 1991). There are three CDRs in V L : CDR-F1, CDR-F2, and CDR-F3, and three in V H CDR- Hl, CDR-H2, and CDR-H3.
  • CDRs complementarity determining regions
  • the CDRs alternate with conserved regions called Framework Regions (FRs), four in V L : FR-F1, FR-F2, FR-F3 and FR-F4, and four in V H : FR-H1, FR-H2, FR-H3 and FR-H4.
  • FRs Framework Regions
  • the antigen-binding site can also be defined as specificity-determining regions as defined and the SDR usage regions (SDRUs). There are six SDRs or SDRUs, which approximately correspond with three CDRs - a comparison of the different definitions of antigen-binding site can be found at Gilliland et al, Methods Mol Biol. 841:321, 2012.
  • amino acid position refers to a position of an amino acid located in V H or V L amino acid sequence.
  • A“diversified position” refers herein to an amino acid position with different amino acids represented at said position.
  • positions to diversify are determined by identifying amino acids in the antigen-binding site contact with antigens in the structure of antibody:antigen complexes determined by x-ray crystallography.
  • Antibody amino acids in contact with the antigen are defined by the distance between an atom of said amino acid in the antibody and an atom of an amino acid in the antigen. Typically, two atoms are in contact when distance between said atoms is ⁇
  • the positions to diversify are defined by the exposure of the amino acids to the solvent, defined as accessible surface area (ASA).
  • The“diversification regime” as used in this invention refers to amino acids used to diversify the amino acid position.
  • the diversification regime is derived from amino acid sequences of known and/or naturally occurring antibodies or antigen binding fragments. Diversified positions are typically found in the CDRs in known and/or naturally occurring antibodies and their discovery are facilitated by the antibody sequences and structures compiled at Internet-based databases. Databases compiling amino acid structures include the Protein Data Bank (PDB; http://www.rcsb.org/pdb ).
  • Antibody sequence databases include V-base (http://www2.mrc-lmb.cam.ac.uk/vbase/), The National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/), the IMGT, and Abysis (http://www.bioinf.org.uk/abs/). These electronic resources provide extensive collections and alignments of human light and heavy chain sequences and facilitate the determination of highly diverse positions in these sequences.
  • “repertoire” or“library” refers to a plurality of antibodies, antibody fragment sequences, antibody variable domains, diversified scaffolds, or the nucleic acids that encode these sequences, the sequences being different in the combination of variant amino acids that are introduced into these sequences according to the methods of the invention.
  • A“scaffold”, as used herein, refers to a polypeptide or portion thereof that maintains a stable structure or structural element when a heterologous polypeptide or amino acid is inserted into the polypeptide.
  • the scaffold provides for maintenance of a structural and/or functional feature of the polypeptide after the heterologous polypeptide has been inserted.
  • a scaffold comprises an antibody variable domain, and maintains a stable structure when a heterologous CDR or amino acids are inserted into the scaffold.
  • library size refers to the number of phages that comprise the library.
  • the term“functional space” or“effective size” of a library as used herein refers to the number of unique antibody sequences in a library that produce functional antibody fragments fused to a phage particle. For instance, nucleotide sequences with stop codons like UAA (“ochre") or UGA (“opal”) lead to truncated sequences and do not produce functional antibody fragments fused to a virion particle. Insertions or deletions of one or two nucleotides change the reading frame of the gene sequence leading to stretches of amino acids that may impair folding or lead to non-functional clones.
  • the functional space of a library can correspond with the library size if all the sequences are functional or is a fraction of the library size.
  • the functional space can also be called 'shape space' or ‘sequence space’.
  • the term“stability” as used herein refers to the ability of a molecule to maintain a folded state such that it retains at least one of its normal functional activities, for example, binding to an antigen or to a molecule like Protein A.
  • the stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melt (“Tm”) temperature. The Tm is the temperature in degrees Celsius at which 1 ⁇ 2 of the molecules become unfolded. Typically, the higher the Tm, the more stable the molecule.
  • “natural” or“naturally occurring” polypeptides or polynucleotides refers to a polypeptide or a polynucleotide having a sequence of a polypeptide or a polynucleotide identified from a no synthetic source.
  • the no synthetic source can be a differentiated antigen-specific B cell obtained ex vivo, or its corresponding hybridoma cell line, or from the serum of an animal.
  • Such antibodies can include antibodies generated in any type of immune response, either natural or otherwise induced.
  • Natural antibodies include the amino acid sequences and the nucleotide sequences that constitute or encode these antibodies, for example, as identified in the Kabat database.
  • natural antibodies are different than“synthetic antibodies”, synthetic antibodies referring to antibody sequences that have been changed, for example, by the replacement, deletion, or addition, of an amino acid, or more than one amino acid, at a certain position with a different amino acid, the different amino acid providing an antibody sequence different from the source antibody sequence.
  • A“plurality” or“population” of a substance generally refers to a collection of two or more types or kinds of the substance.
  • a substance there are two or more types or kinds of a substance if two or more of the substances differ from each other with respect to a particular characteristic, such as the variant amino acid found at a particular amino acid position.
  • a particular characteristic such as the variant amino acid found at a particular amino acid position.
  • there is a plurality or population of polynucleotides of the invention if there are two or more polynucleotides of the invention that are substantially the same, preferably identical, in sequence except for one or more variant amino acids at particular CDR amino acid positions.
  • developerability refers to a set of desirable antibody properties which, as a whole, facilitates clinical development and manufacturing of a therapeutic antibody.
  • Properties that have been associated with developability include, but are not limited to, good expression (>30 mg/L in transient CHO cell expression), thermal stability (>70°C), low or no aggregation ( ⁇ 1% high molecular weight aggregates; HMWA), and solubility of > 40 mg/ml for IV indications and > 100 mg/ml for subcutaneous indications.
  • “Phage display” is a technique by which variant polypeptides are displayed as fusion proteins to at least a portion of a coat protein on the surface of phage, e.g., filamentous phage, particles.
  • a utility of phage display lies in the fact that large libraries of randomized protein variants can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used for screening millions of polypeptides for ones with specific binding properties. Polyvalent phage display methods have been used for displaying small random peptides and small proteins through fusions to either gene III, VIII or IX of filamentous bacteriophage, see references cited therein.
  • a protein or peptide library is fused to a gene III or a portion thereof, and expressed at low levels in the presence of wild type gene III protein so that phage particles display one copy or none of the fusion protein.
  • Avidity effects are reduced relative to polyvalent phage so that sorting is on the basis of intrinsic ligand affinity, and phagemid vectors are used, which simplify DNA manipulations.
  • A“phagemid” is a plasmid vector having a bacterial origin of replication, e.g., ColEl, and a copy of an intergenic region of a bacteriophage.
  • the phagemid may be used on any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage.
  • the plasmid will also generally contain a selectable marker for antibiotic resistance. Segments of DNA cloned into these vectors can be propagated as plasmids. When cells harboring these vectors are provided with all genes necessary for the production of phage particles, the mode of replication of the plasmid changes to rolling circle replication to generate copies of one strand of the plasmid DNA and package phage particles.
  • the phagemid may form infectious or non-infectious phage particles.
  • This term includes phagemids, which contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene as a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle.
  • amplify refers to the process of enzymatically increasing the amount of a specific nucleotide sequence. This amplification is not limited to but is generally accomplished by PCR.
  • Denaturation refers to the separation of two complementary nucleotide strands from an annealed state. Denaturation can be induced factors such as, for example, ionic strength of the buffer, temperature, or chemicals that disrupt base pairing interactions.
  • amplification cycle and“PCR cycle” are used interchangeably herein and as used herein refers to the denaturing of a double -stranded polynucleotide sequence followed by annealing of a primer sequence to its complementary sequence and extension of the primer sequence.
  • polymerase and“nucleic acid polymerase” are used interchangeably and as used herein refer to any polypeptide that catalyzes the synthesis or sequencing of a polynucleotide using an existing polynucleotide as a template.
  • DNA polymerase refers to a nucleic acid polymerase that catalyzes the synthesis or sequencing of DNA using an existing polynucleotide as a template.
  • This invention includes methods to generate highly functional antibody libraries.
  • the methods comprise the steps of: (i) generation of a primary library (PL) of antibody fragments; (ii) a process to select for a well-folded antibody fragment library (FL) from the PL; and (iii) combining the antibody fragments selected in step 2 with diverse CDR-H3 fragments to generate a highly functional secondary antibody library (SL).
  • PL primary library
  • FL well-folded antibody fragment library
  • SL highly functional secondary antibody library
  • the V L scaffold with a short Ll loop was built by assembling the human IGKV3-20*0l germline gene with the human IGJV4*0l germline gene (SEQ ID NO: 1).
  • the Y scaffold with the long Ll was built by assembling the human IGKV4-0l*0l germline gene combined with the same human IGJV4*0l germline gene (SEQ ID NO: 2).
  • IGKV3-20*0l and IGJV4*0l belong to different IGKV families and share only 68% identities.
  • the scaffolds built with these two genes represent two distantly related human germline genes and thus offer distinct exemplary proofs of this invention.
  • V H scaffold built with the human IGHV3-23*0l and the human IGJH3*0l germline genes (SEQ ID NO: 3) was used.
  • Protein A binds the framework region 3 (FR-3) of the V H domain encoded by germline gene IGHV3-23*0l.
  • the FR-3 is formed by discontinuous amino acid stretches distant in the primary sequence but brought together by folding. Therefore, Protein A has been used in prior art for its ability to bind only to well- folded V H domains (Jespers et al., 2004).
  • Protein-F binds the V L domain of antibodies encoded by the human IGKV-l, IGKV-2 and IGKV-4 gene families (Nilson et al., 1992). More specifically, the IGKV4-0l*0l germline gene, which belongs to the human IGKV4 family, binds Protein-F.
  • the use of Protein-F as a ligand to select well-folded antibodies alone and/or in conjunction with Protein A is a clear extension of this invention.
  • human V H domains of antibodies encoded by the gene families IGHV-l, IGHV-2, IGHV-4, IGHV-5, IGHV-6 and IGHV-7, which do not bind Protein A can be paired with libraries of V L domains encoded by scaffolds built with members of the human IGKV-l, IGKV-2 and IGKV-4 gene families. These libraries and antibody pairs can then be submitted to diverse destabilizing conditions to select for well-folded antibodies with Protein-F.
  • the germline genes used to build the V H and V L scaffolds of this invention have frequently been observed in human antibodies elicited against a vast array of diverse antigens (Nilson et al., 1992). These genes have also been used as foundation to build numerous scFv and Fab libraries for antibody discovery in the art (US patent 9,062,305) (Shi et al., 2010), as well as in humanization of therapeutic antibodies (US patent 8,777,044). Therefore, it is expected that antibodies discovered from libraries built with these scaffolds will perform well both in vitro and in vivo settings and will be amenable to further therapeutic development.
  • the Tm of the H Vand V L scaffolds in Fab format has been measured by Differential Scanning Calorimetry (DSC) yielding similar values of 75°C (Teplyakov et al., 2016).
  • This Tm is almost l0°C above the Tm of the C H 2 domain, which is estimated at 68°C (Gilliland et al., 2012).
  • the C H 2 is the least stable domain of the human IgGl molecule and hence the first domain that unfolds. Therefore, the antibodies isolated from the libraries herein described are expected to be highly stable.
  • antibodies encoded by the exemplary V H and V L scaffolds herein disclosed have been solved by x-ray crystallography (Teplyakov et al, 2016) in association the CDR-H3 loop of a known therapeutic antibody, CNTO 888 (Obmolova et al., 2012). This knowledge facilitated the design of diversity in all the CDRs that form the antigen-biding site except in the CDR-H3.
  • the diversity of the V H scaffold was focused on the CDR-H1 and CDR-H2 and was designed at positions and amino acids commonly found in contact with protein and peptide antigens, positions accessible to antigens in the antigen-binding site and/or in contact with the CDR-H3.
  • Example 2 to determine the positions in contact with protein and peptide targets, all the antibody structures compiled at the PDB and curated by the IMGT as of March 17 th , 2017 were analyzed.
  • the initial dataset consisted of 2,645 antibody structures from diverse species and specificities. From this initial dataset, only anti-protein antibodies with different names and those solved at ⁇ 3 ⁇ resolution were considered for identifying positions to diversify.
  • Positions 30 and 55 are in the periphery of the antigen-binding site and are highly exposed to the solvent (> 65% ASA). Hence, although these positions are in contact antigens in less than 50% of the complexes, they were considered for diversification to supplement diversity for binding targets of bigger size than average proteins.
  • V H sequences available at Abysis amounts 76,000 V H sequences, thus complementing the information obtained from the two other sources.
  • the comparison with the frequency of amino acids encoded in an alignment of the human IGHV3 germline family ensured that the amino acids used for diversifying the scaffolds mimicked the human germline diversity.
  • the estimated number of amino acids per position is listed in the last column of the figure and yielded a diversity of 5.4 x lOmique amino acid ⁇ ( sequences. Some positions are relatively conserved such as position 30 at the CDR-H1 with 2 amino acids allowed, whereas other positions are heavily diversified with up to 9 residues, e.g., position 50 of CDR-H2.
  • the neutral H3Js provide enough diversity to avoid biases in amino acids in contact with, or nearby, the neutral H3J sequences while contributing to select for developable antibodies.
  • the set of neutral H3Js fragments comprises ninety (90) fragments (SEQ ID Nos: 4-93).
  • the Ncol restriction site and the Kpnl restriction site are common to both repertoires, allowing for easy replacement of V L -
  • the JK anchor sequence in V L and the human VH Consensus Motif (CM) in V H are used for exchange and cloning of natural H3J fragments in the third step of the invention or for exchanging the light chains. These two short sequence stretches are common to PL1 and PL2.
  • the synthetic fragments of this invention were cloned as fusions to pill by digestion with Bgll restriction enzyme and ligated into pADLTM-23c phagemid vector (Antibody Design Labs, San Diego, cat# PD0T11 ) as described in Example 4.
  • the pADLTM-23c phagemid vector is a classical type 3+3 phage display vector with a cloning site for display on the N-terminal side of the full-length gene III protein. Secretion in the periplasm of the fusion protein is driven by the PelB leader peptide. Display of the scFvs on the phage is obtained with the help of an amber-suppressive bacterial strain and M13K07 helper phage.
  • IMAC immobilized metal affinity chromatography
  • a PL can be obtained from natural sources such as PBMCs and lymphoid organs (lymph nodes, spleen), either from human or animals that have not been immunized against any specific antigen, hyper-immunized animals or individuals suffering or not from a debilitating condition or circumstance of immunological interest such as a recent vaccination or an infectious episode.
  • natural sources such as PBMCs and lymphoid organs (lymph nodes, spleen)
  • the PLs are submitted to diverse conditions to select for highly stable and developable antibody variants.
  • heat was used to eliminate unstable variants from the pool of primary antibodies. It has been shown that transient heat treatment of antibodies displayed on phage can lead to denaturation and aggregation of the less thermostable antibody variants in a library (Jespers et al., 2004). Therefore, following heat-induced denaturation and aggregation of variants with lower stability profiles, rescue by Protein A through direct binding yielded filtered libraries of improved functionality such as in-frame and thermostable clones.
  • the main purpose of this invention is to provide a method to improve the diversity, hence functionality, of antibody library filtrated for improved developability without impacting the quality of the antibody variants collected along the filtration process.
  • Conditions for selecting developable and stable antibody variants from PLs include, but are not limited to heat. Conditions capable to induce unfolding, or combination of thereof, are obvious extensions of this invention. For example, although without clear literature examples, but obvious from the skilled in the art, high or low pH, high salt concentrations or any chaotropic conditions such as urea may be used to interrogate antibody stability. In another application, partial unfolding of the antibody structure may lead to exposure of a stretch of the polypeptide backbone and protease sensitivity. This approach has been used to engineer antibody with high-protease resistance capable to survive the stress conditions found in the gastrointestinal track and is a clear extension of this invention (Hussack et al.,
  • Non-specific, multi-reactive B-cell clones are eliminated during the maturation of the B-cell repertoire.
  • well-folded antibody variants are rescued with a ligand that selectively binds said well-folded antibodies.
  • a ligand that selectively binds said well-folded antibodies.
  • one such a binder is Protein A.
  • Example 6.1 shows the incubation for 10 min of the 3-20/3-23 and 4-
  • the scFvs displayed as fusions to the minor phage coat protein pill on the phage surface were submitted for a range of temperatures, starting at 40°C and increasing the temperature up to 80°C in steps of 5°C.
  • the unfolding process was monitored with a direct Protein A
  • the unfolding process which leads to aggregation of the scFv-phage and hence a drop in the ELISA signal depends on two parameters (Jespers et al., 2004): (i) thermal stability of the scFvs, and (ii) number of copies of the scFvs displayed on the phage surface, which varies from one to five copies.
  • a cooperative aggregation process takes place. ScFv-phages with a higher number of copies aggregate first, serving of aggregation seed for phages with a fewer number of scFv copies.
  • Example 6.1 indicated that the 3-20/3-23 scFv started to unfold at 65°C, whereas, the 4-01/3-23 scFv started the unfolding process at 55°C.
  • the Tm of the 3-20/3-23 scFv is 75°C, at which 50% was unfolded.
  • the Tm of 4-01/3-23 scFv is 65°C.
  • the unfolding kinetic at 60°C and 72°C of the 3-20/3-23 scFv and 4-01/3-23 scFv demonstrated that the 3-20/3-23 scFv remained folded at 60°C for up to one hour (Figure 9). At 72°C it unfolded slowly with approximately a 20% drop in the ELISA signal during the first 30 min.
  • the exemplary PLs of this invention produced unique scFvs with a significant number, on average -52.3% (Table 3), of well-folded antibodies after incubation at 72°C for 10 min. This number was down from on average -64% in both PLs prior to the heat treatment, representing a combined number of more than 2 billion folded but unstable antibodies for PLs of 1 x 10 10 clones or more each.
  • the present invention also includes methods to restore diversity to an antibody library that has lost some of its diversity due to enrichment and/or selection.
  • the highly stable variants selected in the previous step with a collection of natural CDR-H3 fragments the diversity of the libraries can be restored.
  • Two SL libraries, SL1 and SL2 were generated starting from plasmid DNA of FL1 and FL2 as substrate for amplification of well-folded scaffolds by PCR as exemplified in Example 8.
  • the PCR-generated fragments included the pelB leader peptide-encoding nucleotide sequence and a stretch of nucleotides immediately before the CM ( Figure 16).
  • the sequence of the CM allowed the amplification of more than 95% of all antibody sequences found in circulating PBMCs as described in Example 8.
  • a Bsal restriction site was added immediately after the end of the V H fragment.
  • a repertoire of natural H3Js fragments was obtained from 200 healthy donors as described in Example 8.1. It was generated with a pair of primers matching the sequence of the CM and the pADLTM-23c phagemid vector sequence downstream to the second Sfil site. In doing so, a Bsal site was added 5’ to the CM. Simultaneous digestion by Bsal of the two fragments and ligation led to the joining of the FLs with the natural H3J fragments ( Figure 17).
  • the natural CDR-H3 fragments of this invention were isolated from PBMCs of a pool of 200 donors and amplified with primers to generate around 95% of all the CDR- H3 in those individuals.
  • the CDR-H3 is highly variable, with length variation between 3 and more than 20 amino acids, recombined with over 40 IGHV and 6 IGHJ and paired with ⁇ 40 IGKV genes. This enormous diversity when cloned in the highly stable exemplified scaffolds of the invention, only impacted -10% of the overall library diversity while improving stability.
  • NGS analysis of the SL confirmed the presence of very high levels of diversity at both CDR-H3 level, with around 60% unique sequences, and full antibody sequence level with above 99% diversity at the depth of around one million sequences in each SL (Table 12). Analysis of the most prevalent CDR-H3 indicated a limited copy number for each clone, to the contrary of what one would expect from re-amplified libraries.
  • TNF and HSA were performed. In both examples specific antibodies were isolated, demonstrating the potential of the SLs to produce specific antibodies
  • EXAMPLE 1 DESIGN OF TWO HUMAN SCAFFOLDS FOR RECOGNITION OF DIVERSE EPITOPES
  • EXAMPLE 2 DESIGN OF DIVERSIFICATION REGIMES FOR TWO HUMAN
  • V H scaffold The diversity of the V H scaffold was focused on the CDR-H1 and CDR-H2 and was designed at positions and amino acids commonly found in contact with protein and peptide antigens, positions accessible to antigens in the binding site and/or in contact with CDR-H3. To determine the positions in contact with protein and peptide targets, 117 antigen: antibody complexes listed in Figure 2 were used.
  • the positions in contact with antigens were identified by downloading the contact tables of the 117 structures from the IMGT and aligning the contact residues. Then, the residues in contact with antigens were then mapped onto the structures of the V H scaffold paired with the two V L scaffolds (PDB IDs: 1ILC and 1ILD) to determine their AS As and structural environment.
  • the diversification regime at CDR-H1 and CDR-H2 was designed using three sources of information: The dataset of curated antibody structures listed in Figure 2, the V H sequences available at NCBI and curated by Abysis, and the germline genes of the human IGHV3 family compiled at the IMGT. The number of V H sequences available at Abysis amounted for 76,000 V H sequences, thus complementing the information obtained from the two other sources.
  • the final diversification regime at CDR-H1 and CDR-H2 of the V H scaffold is summarized in Figure 3. The estimated number of amino acids per position is listed in the last column of Figure 3 and yields a diversity of 5.4 x 10 6 unique amino acid V H sequences.
  • the IGDH germline genes compiled at the IMGT were translated in the three reading frames into amino acid sequences. Only productive sequences, i.e. without stop codons, were considered. Those IGDH genes with developability liabilities including methionine (M) and tryptophan (W) residues, as well as those encoding three or more hydrophobic residues were removed from the set of neutral H3Js. After this in silico selection process, 18 sequences from the IGDH germline genes were combined with 5 IGJH regions to produce ninety (90) fragments (Table I) (SEQ ID Nos: 4- 93). The length of the neutral H3Js varies between 18 and 27 amino acids.
  • the ligation reactions were electroporated into electro-competent TG1 cells (Lucigen) and transformants were rescued on 2xYT medium supplemented with ampicillin at 37°C in the presence of glucose 1% w/v.
  • the initial diversity was 1.7 x 10 9 and 2.3 x 10 9 primary transformants for PL1 and PL2, respectively.
  • this example demonstrates that: (i) the 3-20/3-23 and 4-01/3-23 control scFvs are stable at 60°C for at least one hour; and (ii) 3-20/3-23 and 4-01/3-23 scFvs can be incubated at 72°C for
  • Double Transformants 6.8% 3/44 2.1% 1/44 Vector Background 0.0% 0/44 0.0% 0/44 In-Frame Clones 61.0% 25/41 83.7% 36/43
  • Protein A binding of single phage results from multiple factors such as: (1) as proper folding of the Fv fragment, (2) concentration of displayed scFv and (3) scFv valency, up to 5 per M13 phage. It should be noted that the last two properties are linked to better expression levels, e.g. the higher the expression, the higher the valency. Therefore, Protein A binding is linked to more developable antibodies (better folded or better expressed or both). On average 24 out of 41 single clones in PL1 bound Protein A (58.5%) and 30 out of 43 single clones in PL2 (69.8%) (Table 3). Therefore, the vast majority of the assayed clones had a signal between 20% and 90% with respect to the control scaffolds, showing a good dynamic range to assess changes in Protein A binding after heat treatment.
  • COSTAR plates 3369 (Coming) were coated with Protein A (Sigma Aldrich, cat# P6031) at 4 pg/ml in TBS overnight at 4°C. After blocking with TBST and 5% w/v nonfat dry milk for one hour, virions ( ⁇ 2.6 x 10 12 virions/ml) in TBST with 5% w/v nonfat dry milk were added to the wells and incubated for 2 h at 37°C. As a reference, virions derived from the parent scaffolds with the CDR-H3 of CNTO 888 cloned in the same vector were added and similarly incubated on the plate.
  • Protein A Sigma Aldrich, cat# P6031
  • Bound phage was detected with A4G1.6 monoclonal antibody (Antibody Design Labs, San Diego, CA) conjugated to HRP. Binding of the secondary antibody, a murine IgGl, to Protein A was blocked by polyclonal human IgG at 100 pg/ml added into the incubation buffer as described in
  • EXAMPLE 8 SECONDARY REPERTOIRES [157]
  • the natural H3J fragments were obtained from the PBMCs of 200 healthy donors, 100 females and 100 males under the age of 40 years. Each donor provided 5 x 10 6 cells and thus potentially yielded 1 x 10 6 unique H3J sequences. Therefore, the pool of 200 donors contained 2 x 10 8 potentially unique H3J sequences.
  • total RNA (tRNA) was individually isolated using Trizol (Invitrogen; Cat# 15596026 and 15596018). Pools of tRNAs from 10 donors were generated after determining the concentration by UV spectrophotometry and mixing the donor tRNAs in equal amounts to generate 20 tRNA pools.
  • Each of the 20 pools were processed to isolate Messenger RNA (mRNA) using polyA SpinTM mRNA Isolation Kit (NEB, Cat #: S1560S) following the manufacturer instructions.
  • the mRNA was used as template to generate cDNA by reverse transcription using OneTaq® RT-PCR Kit (NEB, Cat #:
  • This particular set of antibodies has a highly conserved nucleotide sequence GACACGGCYGTGTATTACTGTGC (SEQ ID NO: 94) located at the FR-3 - CDR-H3 junction. There is polymorphism with a C or a T for the third nucleotide of the alanine codon at position 88, hence the Y (UIPAC code for C or T) symbol in this sequence. This motif is present in more than 95% of the VH sequences found in circulating PBMCs.
  • CM human VH conserved motif
  • Double-stranded DNA containing the repertoire of natural H3J fragments was obtained by PCR using a universal forward primer annealing to the CM and three reverse primers designed to amplify 95% of the human CDR-H3 fragments in circulating PBMCs (SEQ ID Nos: 95-97).
  • H3J fragments were assessed by cloning an aliquot of final pool into a TOPO vector (Life Technologies). Sanger sequencing of 30 clones indicated that all H3J fragments were different, with length variation resembling the human CDR-H3 repertoire. The region introduced by the amplification primers for assembling the full scFvs and cloning into the vector matched 100% the expected nucleotide sequence.
  • EXAMPLE 8.2 CLONING OF A SECONDARY REPERTOIRE BY SEAMLESS ASSEMBLY
  • the second step of the scFv assembly involved a simultaneous digestion by Bsal and ligation by T4 DNA ligase at 37°C which joined the primary filtered fragments and the natural H3J fragments.
  • the resulting full length scFv DNA was further amplified by PCR before cloning ( Figure 16).
  • 60 ng of the joined products were further amplified by the nested primers padllib s (SEQ ID NO: 101) and ALT_huH3J_r (SEQ ID NO: 102) in a final volume of 300 pi using Phusion, 20 cycles and an annealing temperature of 60°C ( Figure 17, Panel C).
  • a total of 5 pg of ligated products was electroporated into electro-competent TG1 cells (800 ng/50 pi cells) as described in Example 1 for each secondary library.
  • the number of primary transformants for the secondary libraries was 1.4 x 10 10 cfii for the SL1 library and 1.1 x 10 10 cfii for the SL2 library.
  • EXAMPLE 9 ASSESSING DIVERSITY OF THE LIBRARIES BY NEXT GENERATION SEQUENCING [169] To study the diversity along the filtration process and the construction of the secondary libraries, two amplicons were prepared from the phagemid DNA. Plasmid DNA were isolated using QIAGEN Plasmid Midi Kit (Cat No.: 12143) from the PL and SL bacterial cultures prior to helper phage superinfection and induction, and after overnight bacterial culture for the FL libraries. The DNA was used as a template to generate amplicons of approximately 300 bps. Amplicon 1 covered the V L scaffolds (3-20 and 4-01) and was amplified with one forward primer for PL1 (SEQ ID 126) and PL2 (SEQ ID
  • PCR fragments were gel-purified using QIAquick PCR Purification Kit (Cat No.: 28104) and used as template to prepare the samples for NGS following the manufacturer instructions.
  • the sequencing was performed on a Miseq platform from Illumina. FASTQ files were processed with the software AptaAnalyzerTM (AptalT; Germany) using the BCR (B-cell receptors) functionality.
  • the frequency was within 1.3% of expected value of 6% of an even distribution (18 neutral fragments) and was similar in both PLs, suggesting no major bias due to the PLs preparation.
  • EXAMPLE 10 ASSES SING FUNCTIONALITY OF THE SECONDARY LIBRARIES USING TWO TARGET MODELS [181] To assess the potential of the secondary libraries to produce specific and developable antibodies, selections were performed with two non-related target models: TNF and HSA.
  • SL1 and SL2 libraries (3 x ll phage) were mixed and diluted to 1 x ltf virion/ml in MPBS and 4 ml were added to the TNF-coated Immunotube.
  • the Immunotube was incubated one hour at RT with slow shaking and one additional hour standing at RT.
  • the unbound phages were washed away 10 times with TPBS (PBS + 0.1% Tween ® 20) washes and 10 washes with PBS.
  • soluble scFvs of 45 clones chosen at random were assayed in ELISA for binding to TNF and BSA.
  • reporter reagent Protein A/HRP was used. 12 positive clones for TNF but not for BSA were sent for Sanger sequencing to determine unique clones. Two unique clones were expressed as soluble scFvs and re-tested for binding to the target and BSA. The confirmed clones were expressed in lOO-mL culture and purified using HisTrap TM (Sigma; GE Catalogue # GE 17-5255-01 ).
  • the purified scFvs exhibited specific binding to the target in a direct ELISA as shown on Figure 23 or clone E12.
  • EXAMPLE 10.2 SELECTION WITH HS A
  • the selections with HSA were conducted following a similar procedure as for TNF with some modifications. That is, only SL2 was used for the selections and a COSTAR plate 3369 (Coming) instead of Immunotubes was used as solid phase.
  • Ten wells were coated with 100 pl per well of HSA 4 pg/ml in PBS overnight at 4°C. After washing 3 times with PBS and blocking with 200 pl TPBS with BSA 3% w/v for one hour at 37°C, 100 m ⁇ of SL2 phage, 6.1 x 10 11 cfu/ml, per well in TPBS with BSA 3% w/v were incubated at 37°C for 2 h.
  • Muromonab-CD3 Orthoclone OKT3: the first monoclonal antibody approved for therapeutic use. Iowa Med 77(2), 78-82.
  • OKT3 antibody response study (OARS): a multicenter comparative study. Transplant Proc 25(1 Pt 1), 558-560.
  • Phage antibodies filamentous phage displaying antibody variable domains. Nature 348(6301), 552-554.
  • Pruzina S., Williams, G.T., Kaneva, G., Davies, S.L., Martin-Lopez, A., Bruggemann, M., et al.
  • EmailAddress pvaladon@abdesignlabs.com
  • EmailAddress juan.c.almagro@gmail.com
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Abstract

La présente invention concerne un procédé de génération de bibliothèques d'anticorps présentant une fonctionnalité améliorée et des utilisations de celles-ci par combinaison d'une bibliothèque d'anticorps présélectionné pour des propriétés fonctionnelles pertinentes pour l'aptitude au développement, comme par exemple une stabilité thermique améliorée, avec une bibliothèque de fragments CDR-H3.
PCT/US2018/060933 2017-11-15 2018-11-14 Bibliothèques d'anticorps hautement fonctionnelles Ceased WO2019099454A2 (fr)

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