US20030190598A1 - Single-domain antigen-binding antibody fragments derived from llama antibodies - Google Patents

Single-domain antigen-binding antibody fragments derived from llama antibodies Download PDF

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US20030190598A1
US20030190598A1 US10/031,874 US3187402A US2003190598A1 US 20030190598 A1 US20030190598 A1 US 20030190598A1 US 3187402 A US3187402 A US 3187402A US 2003190598 A1 US2003190598 A1 US 2003190598A1
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Jasmid Tanha
Ginette Dubuc
Saran Narang
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Sanofi Aventis Deutschland GmbH
Halliburton Energy Services Inc
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the invention relates to antigen-binding proteins, in particular to antigen-binding fragments of antibodies derived from a na ⁇ ve library of llama antibodies and to a phage display library of such fragments. More particularly, the present invention relates to antigen-binding fragments of llama antibodies comprising at least a part of the variable heavy domain (VH or V H H) of antibodies derived from a na ⁇ ve library of llama antibodies and to a phage display library of such fragments.
  • VH or V H H variable heavy domain
  • the immune system in vertebrates provides a defense mechanism against foreign intruders, such as foreign macromolecules or infecting microorganisms.
  • foreign invaders both macromolecules (proteins, polysaccharides, or nucleic acids) and microbes (viruses or bacteria)
  • antigenic determinants are recognized through specific binding of the proteins of the host immune system to specific sites on the antigen surface, known as antigenic determinants.
  • B-cells of vertebrate organisms synthesize antigen-recognizing proteins known as antibodies or immunoglobulins (Ig).
  • an antigen activates those B-cells of the host organism that have on their surface immunoglobulins that can recognize and bind the antigen.
  • the binding triggers production of a clone of identical B-cells that secrete soluble antigen-binding immunoglobulins into the bloodstream.
  • Antibodies secreted by B-cells bind to foreign material (antigen) to serve as tags or identifiers for such material.
  • Antibody-tagged antigens are then recognized and disposed of by macrophages and other effector cells of the immune system or are directly lysed by a set of nonspecific serum proteins collectively called complement. In this way a small amount of antigen can elicit an amplified and specific immune response that helps to clear the host organism of the source of antigen.
  • human B-cells Through a complex process of gene splicing combined with additional mutation mechanisms, human B-cells have been estimated to produce a “library” (repertoire) of more than a billion (10 9 ) different antibodies that differ in the composition of their binding sites.
  • immunoglobulins of the IgG class are the major type in normal serum of humans and many other species and have the four-chain structure shown schematically in FIG. 1.
  • Each chain of an IgG molecule is divided into domains of about 110 amino acid residues, with the light chains having two such domains and the heavy chains having four.
  • Comparison of amino acid sequences between different IgGs shows that the amino-terminal domain of each chain (both light and heavy) is highly variable, whereas the remaining domains have substantially constant sequences.
  • the light (L) chains of an IgG molecule are built up from one amino-terminal variable domain (VL) and one carboxy-terminal constant domain (CL), and the heavy (H) chains from one amino-terminal variable domain (VH) followed by three constant domains (CH1, CH2, and CH3).
  • variable domains are not uniformly variable throughout their length.
  • Three small regions of a variable domain known as hypervariable regions (loops) or complementarity determining regions (CDR1, CDR2, and CDR3) show much more variability than the rest of the domain.
  • These regions which vary in size and sequence among various immunoglobulins, determine the specificity of the antigen-antibody interaction.
  • the specificity of an antibody of the type shown in FIG. 1 is determined by the sequence and size of six hypervariable loops (regions), three in the VL domain and three in the VH domain.
  • the IgG molecule By partial digestion with papain, which cleaves the heavy chains in the hinge region, the IgG molecule can be broken down into two identical Fab fragments (Fragment, antigen binding) and one Fc fragment (Fragment, crystallizes easily).
  • Each Fab fragment comprises one complete light chain (consisting of VL and CL domains) linked by a disulfide bridge and noncovalent interactions to a fragment of the heavy chain consisting of VH and CH1 domains.
  • the Fc fragment comprises CH2 and CH3 domains from both heavy chains, also linked by disulfide bridges and noncovalent interactions.
  • the part of the Fab fragment consisting of variable domains of the light and the heavy chain (VL and VH) is known as Fv fragment (Fragment, variable).
  • variable domains VL and VH are not covalently bound.
  • VL and VH domains are covalently linked by a short peptide linker (spacer), usually 15 to 20 amino acids long, introduced at the genetic level (see FIG. 2).
  • scFv fragments are recombinant fusion proteins and are produced by techniques of genetic engineering, by expressing in a suitable host, usually in bacteria, a chimeric gene coding for the fragment.
  • Various other recombinant antibody fragments have been designed to substitute for large intact immunoglobulin molecules (see FIG. 2).
  • these options include Fab or Fv fragments that are stabilized or covalently linked using various strategies (see, for example, Bird et al., Science, 242, 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci.
  • the genetic engineering has also made possible to screen in vitro for antibodies having a predetermined binding specificity. This may be achieved by constructing first a gene library of antibodies or antibody fragments, for example by polymerase chain reaction (PCR)-amplification of cDNA derived from B-lymphocytes using suitable primers, or by in vitro gene synthesis.
  • the gene library may contain sequences corresponding to certain fragments of natural antibodies, or randomized antigen-binding regions, or new combinations of heavy/light chains, thus creating the potential for generating antibodies which could never be obtained from natural sources, for example, antibodies to highly toxic substances or antigens tolerated by the human immune system.
  • the affinity or specificity of the antigen binding can be manipulated, for example, to reach affinities never observed with natural antibodies.
  • a selection system comparable to that of the immune system is required.
  • Such a selection system can be achieved by inserting the library genes into the genome of microorganisms capable of displaying on their surface the antibody corresponding to the inserted gene, in analogy to the expression of an immunoglobulin antigen receptor on the surface of a B-cell.
  • Microorganisms most frequently used for providing such a display are filamentous bacteriophages, such as fd or M13 phages (phage display).
  • the collection of phage particles having inserted genes of a library of proteins, such as antibodies, and displaying these proteins on the particles' surface is known as a phage display library.
  • the display of the library of antibodies on the surface of phage particles provides a physical link between the antigen-binding function of an antibody and the antibody gene.
  • the whole organism (phage) displaying this affinity can be identified and separated out of billions of non-specific clones, usually through binding to the antigen immobilized on a support, technique usually referred to as panning (see, for example, Scott et al., Science, 249, 386-390 (1990); Winter et al., Annual Rev. Immunology, 12, 433-455 (1994)).
  • Phage clones binding to the antigen can be then amplified and used to produce the specific antibody or antibody fragment in E. coli or in other suitable organism.
  • VH domains were isolated from expression libraries derived from immunized mice (Ward et al., Nature, 341, 544-546 (1989)).
  • antigen-binding VH domains were rescued from an antibody phage library that was made from a vaccinated patient (Cai et al., Proc Natl. Acad. Sci. USA, 93, 6280-6285 (1996)).
  • Antigen-binding antibody fragments consisting of a single VH domain known as dAbs or sdAbs (single-domain antibodies), are becoming an attractive alternative to single chain Fv (scFv) fragments.
  • sdAbs are amenable to detailed NMR structural studies (Davies et al., FEBS Letters, 339, 285-290 (1994)). Additionally, due to their simpler structure, sdAbs are more stable and have simpler folding properties.
  • HCA heavy chain antibodies
  • camelids HCA-chain antibodies
  • camelids Hamers-Casterman et al., Nature, 363, 446-448 (1993); see also U.S. Pat. No. 5,759,808; U.S. Pat. No. 5,800,988; U.S. Pat. No. 5,840,526; and U.S. Pat. No. 5,874,541.
  • these antibodies Compared with conventional four-chain immunoglobulins of IgG-type, which are also produced by camelids, these antibodies lack the light chains and CH1 domains of conventional immunoglobulins.
  • V H H H variable domain
  • Glu, Arg and Gly at VL interface positions 44, 45 and 47 Kabat numbering
  • cysteine pairs mediate the formation of a disulfide bridge and are therefore involved in modulating the surface topology of the antibody combining site.
  • a rigid loop protruding from the sdAb and partly stabilized by a CDR disulfide linkage extends out of the combining site and penetrates deeply into the lysozyme active site (Desmyter et al., Nature Struct. Biol., 3, 803-811 (1996)).
  • camelid sdAbs phage display libraries have been generated from the V H H repertoire of camelids immunized with various antigens (Arbabi et al., FEBS Letters, 414, 521-526 (1997); Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decanniere et al., Structure, 7, 361-370 (1999)).
  • camelid sdAbs phage display libraries have been generated from the V H H repertoire of camelids immunized with various antigens.
  • the present invention has overcome the above-discussed prior art limitations by generating a large size (in the order of 10 9 ) phage display library of antibody fragments of a non-immunized llama, which fragments comprise at least a part of the variable heavy domain (VH or V H H domain) of llama antibodies.
  • the fragments consist essentially of the variable heavy domain (VH or V H H of llama antibodies (sdAb fragments).
  • This library possesses a number of unique features which distinguish it from similar libraries generated from other camelids.
  • the large size of the library considerably increases the probability of isolating therefrom antigen-binding fragments having high affinity to almost any predetermined target (antigen) of interest. This has been demonstrated by isolating from the library fragments binding specifically to several preselected antigens as targets.
  • the invention provides a phage display library of antigen-binding fragments of llama antibodies, said fragments comprising at least a part of the variable heavy domain (VH or V H H) of the antibodies.
  • the antigen-binding fragments consist of a complete variable heavy domain (VH or V H H) of the antibodies (sdAb fragments)
  • the invention provides an antigen-binding fragment of a llama antibody, said fragment comprising at least a part of the variable heavy domain (VH or V H H) of the antibody.
  • the antigen-binding fragment consists of a complete variable heavy domain (VH or V H H) of the antibody (sdAb fragment).
  • the invention provides a cDNA library comprising nucleotide sequences coding for antigen-binding fragments of llama antibodies, said library obtained by isolating lymphocytes from a biological sample obtained from a non-immunized llama; isolating total RNA from the lymphocytes; reverse-transcribing and amplifying RNA sequences coding for the antigen-binding fragments; cloning the amplified cDNA in a vector; and recovering the obtained clones.
  • the antigen-binding fragments consist of a complete variable domain (VH or V H H) of the antibodies (sdAb fragment) and the cloning vector is a filamentous bacteriophage.
  • the invention provides a process for the preparation of an antigen-binding fragment of a llama antibody, said fragment binding to a predetermined antigen, said process comprising the steps of isolating lymphocytes from a biological sample obtained from a non-immunized llama; isolating total RNA from the lymphocytes; reverse-transcribing and amplifying RNA sequences coding for antigen-binding fragments; cloning the cDNA sequences so obtained into a first vector, said first vector capable of a surface display of the corresponding antigen-binding fragments; subjecting the clones to antigen affinity selection and recovering clones having the desired affinity; for the recovered clones, amplifying DNA sequences coding for antigen-binding fragments; cloning the amplified DNA sequences into a second vector; transforming prokaryotic cells with the second vector under conditions allowing expression of DNA coding for antigen-binding fragments; and recovering the antibody fragments
  • FIG. 1 is a schematic representation of a typical four-chain IgG-type immunoglobulin (antibody) showing (a) the structure and arrangement of heavy and light chains and the approximate positioning of interchain disulfide bonds, and (b) the organization of the antibody molecule into paired domains.
  • FIG. 2 is a schematic representation of various modifications and fragments of IgG-type antibodies, and antigen-binding fusion proteins derived from such fragments.
  • FIG. 3 is a schematic representation of steps involved in construction of the phage display library of llama sdAb antibody fragments according to the present invention. For simplicity, only the coding sequences of the mRNA transcripts are shown.
  • A, a heavy chain mRNA of conventional four-chain (A) and two-chain heavy chain (a) antibodies; B, b: RT-PCR product derived from A and a, respectively; c: V H H derived from heavy chain antibodies.
  • VH variable
  • CH constant domains are marked with dark and light shading, respectively.
  • FIG. 4 is a bar graph showing fractional occurrence of the CDR3 lengths. Gray bars represent data according to the present invention, whereas the white bars represent the published data for llama V H H (Vu et al., Mol. Immunol., 34, 1121-1131 (1997)).
  • FIG. 5 is a graph showing global fitting to 1:1 interaction model of the binding of Yst9.1 scFv to immobilized Bruc.C6 sdAb fragment at 20, 100, 200, 300, 400, and 600 nM. Open circle lines represent experimental data points, whereas solid lines represent the fit.
  • FIG. 6 is a graph showing overlays of sensograms (A) and the Scatchard plot derived therefrom (B) for the binding of TNG.p1779 sdAb fragment (2.5 (f), 7.5 (e), 10 (d), 15 (c), 20 (b) and 30 (a) ⁇ M) to captured biotinylated p1779 peptide.
  • FIG. 7 is a graph showing the Scatchard plot derived from sensograms for the binding of TNG.PTH50 sdAb fragment to captured biotinylated PTH2 peptide.
  • the present invention provides a large size (in the order of 10 9 ) phage display library of single-domain fragments of variable heavy domains (VH and V H H) of llama antibodies.
  • the library which has been generated using lymphocytes of a non-immunized animal (na ⁇ ve library), can be used for in vitro selection against any antigen of interest as a target.
  • the size of the library makes it highly probable that an antibody specific to the target will be identified among the library's sdAb fragments. This utility of the library has been demonstrated by isolating therefrom sdAbs binding specifically to various preselected antigens as targets.
  • Another advantage of choosing a na ⁇ ve library as the source of llama antibodies concerns anti-idiotypic antibodies.
  • An anti-idiotypic antibody (a second antibody) recognizes the idiotope of another antibody (a first antibody) as an antigen, meaning that the first antibody recognizes in turn the second (anti-idiotypic) antibody as its antigen.
  • Anti-idiotypic antibodies have gained a widespread clinical use, e.g., in vaccine development for cancer and cholera (Grant et al., Clin. Cancer Res., 1319-1323 (1999); Herlyn et al., Ann. Med., 66-78 (1999); Maxwell-Armstrong et al., Br. J.
  • llama is the smallest animal which can survive in a severe, cold climate. Lymphocytes of a llama from a farm located in Osgoode (Canada) have been used to generate the phage display library of variable heavy domains of llama antibodies. From this library, sdAbs binding specifically to several preselected antigens have been subsequently isolated and characterized.
  • FIG. 3 depicts a schematic representation of steps involved in the construction of the V H H-derived sdAb phage display library.
  • lymphocytes from the fresh blood of llama (from a farm located at Osgoode, Ontario, Canada) were prepared and their RNA was isolated using techniques well known to those skilled in the art.
  • RT-PCRs reverse transcriptase-polymerase chain reactions
  • the amplified products were separated and fragments of the expected size derived from conventional IgG ( ⁇ 900 bp) and heavy chain IgG ( ⁇ 600 bp) were observed on the agarose gel.
  • the smaller fragment was gel purified and used in a second PCR to amplify the V H H genes.
  • the amplification products were cloned into fd-tet (GIIID) vector, between the leader signal and gene III, to produce fusion proteins, which were displayed on the filamentous phage particles using a modified procedure.
  • GIIID fd-tet
  • phagemid vectors Two different types of vectors are used for generating phage display libraries: phagemid vectors and phage vectors. Libraries having size in the order of 10 8 can be constructed with relative ease using phagemid vectors. However, a phagemid-based libraries suffers from some serious drawbacks. First, phagemid vectors provide typically a monovalent display and therefore may not select for lower binding (of lower affinity), but potentially important antibody fragments. Second, a phagemid-based library allows for the enrichment of phage particles displaying deleted versions of the antibody fragments.
  • Such particles are preferably selected during the panning process over those displaying the full-length fragments and therefore obscure the process of selection of the full-length binders.
  • constructing a phagemid-based library requires a helper phage and therefore library construction, panning and downstream phage binding assays become a far more complicated and tedious task. For these reasons the use a phage vector for the library construction is preferred.
  • fd-tet Zacher III et al., Gene, 9, 127-140 (1980)
  • Tn10 contains a tetracycline resistance gene, tetA, and thus confers tetracycline resistance to the host cells carrying the fd-tet vector.
  • tetA tetracycline resistance gene
  • the library was propagated as plaques in the absence of tetracycline, resulting in a llama V H H library of size of approximately 8.8 ⁇ 10 8 . This is the largest size library ever obtained using fd-tet vector. Due to its size, the library has an enhanced probability of selecting therefrom proteins (antibody fragments) binding to almost any given target (antigen).
  • the display library of the invention could be generated using vectors other than phages, such as bacteria (e.g., E coli ) (Daugherty et al., Protein Eng., 613-621 (1999); Georgiou et al., Nat. Biotechnol., 29-34 (1997)) or yeast (e.g., Saccharomyces cerevisiae ) (Kieke et al., Proc. Natl. Acad. Sci. USA., 5651-5656 (1999); Kieke et al., Protein Eng., 1303-1310 (1997); Cho et al., J. Immunol.
  • bacteria e.g., E coli
  • yeast e.g., Saccharomyces cerevisiae
  • Previously generated camelid sdAb libraries were characterized by typical presence of Glu, Arg and Gly in positions 44, 45 and 47, respectively, of the VL interface of V H H domain.
  • the occurrence of cysteine at position 45 was also frequent in V H H, as opposed to VH domain of four-chain IgGs.
  • the present library as shown by sequence analysis (Table 1), lacks these characteristics, as only one sdAb (C35) has Glu44, Arg45 and Gly47.
  • the majority of sdAbs of the present library have Arg in position 45 of the VL interface.
  • V H Hs variable heavy chain domains
  • VHs typical conventional variable heavy domains
  • sdAbs C1, C29, C43, C44 and C48 of Table 1, some sdAbs of Table 2 This contamination is most likely the results of PCR crossovers between the VHs and V H Hs during the step of RT-PCR (FIG. 3) (Tomlinson et al., J. Mol. Biol., 227, 776-798 (1992); Muyldermans et al., Protein Eng., 7, 1129-1135(1994)).
  • VHs are genuine antigen binding fragments, as shown in Table 2, produced in high yield in Escherchia coli . They are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies (Tanha et al., manuscript in preparation; Vranken et al., submitted). The very close similarity of these molecules to human VHs makes them potentially very useful as therapeutic sdAbs.
  • amino acids of the VL interface are most frequently:
  • CDRs can be selected from the following sequences: CDR1/H1: GFTFSSYAMS (SEQ ID NO: 85) GFTFSSYYMS (SEQ ID NO: 86) GFTFDEHAIG (SEQ ID NO: 87) GFTVSSNHMT (SEQ ID NO: 88) GFTFSSYHMA (SEQ ID NO: 89) GFTFSRHQMS (SEQ ID NO: 91) GFTFRTYYMN (SEQ ID NO: 92) GFIFSSYAMS (SEQ ID NO: 93) GFTFSTYAMT (SEQ ID NO: 95) GFTFSGYAMS (SEQ ID NO: 99) GFAFSNYRMT (SEQ ID NO: 100) GFTFSRYAMS (SEQ ID NO: 101) CDR2: GIEGGGGITRYADSVKG (SEQ ID NO: 102) TIKPGGGSTYYADSVKG (SEQ ID NO: 103) TIDIGGGRTYADSVKG
  • Antigen-antigen reactions are those in which the antigen (Ag) is itself an antibody (Ab), as discussed above.
  • Single domain anti-idioptypic (anti-id) antibody fragments have been isolated from the library of the present invention using phage display technology and an antibody serving as antigen. Such anti-Id antibody fragments have great potential in both evoking the immune system responses to pathological antigens and in vaccine development.
  • Bruc.C7.2, Bruc.D10 and Bruc.E6 have the same CDR3 in addition to the first two, which share the same CDR2.
  • These common sequences were encoded by identical nucleotides raising the possibility that divergent sdAbs may have arisen as a result of PCR cross-over in vitro.
  • the interface amino acids are generally Gly44, Leu45 and Trp47, typical of human/murine VH domain.
  • none of the isolated sdAbs have any cysteine in CDR1, 2, or 3.
  • Table 2 also shows the identity of amino acids at positions 37, 44, 45 and 47 of the VL interface of V H H domain. Interestingly, all sdAbs shown in the table have VL TABLE 2 CDR/H1 sequences of dAbs which were isolated by panning the llama library against Yst9.1 scFv. The V L interface residues at positions 37, 44, 45 and 47 are also included.
  • interface residues which are typical of murine or human VHs.
  • half of the sdAbs have Val37, Gly44, Leu45 and Trp47, which are highly conserved in murine and human VH.
  • all sdAbs have Val37 and Gly44, and majority has Leu45 and Trp47.
  • Six, three and one sdAbs are characterized by the presence of Phe45 or Pro45, Tyr45 and Ser45, respectively. It is interesting to note that the presence of the same VL interface residues in the conventional antibodies would render the isolated VH highly hydrophobic, resulting in their aggregation, which is not observed for llama antibodies.
  • Proteins of granulin/epithelin family are thought to play a role in inflammation, wound repair, tissue modeling and regulating enzyme activity (Vranken et al., J. Pept. Res., 590-597 (1999); Hrabal et al., Nat. Struct Biol., - 752 (1996)). They are implicated as potential co-factors for HIV Tat protein and in modulating the growth of human epidermal carcinoma cells, and inhibition of their expression is known to inhibit the tumorigenecity of certain cells.
  • the granulin motif has been found throughout the animal kingdom, in fish and insects, and encoded in the genome of a nematode worm.
  • the motif consists of a parallel stacks of beta-hairpins pinned together by disulfide bonds.
  • the structural sub-domain of granulin containing the first two beta-hairpin and spanning the first N-terminal 30 amino acids is also shared by growth factor proteins such as epidermal growth factors, transforming growth factor (TGF)-alpha, as well as the epithelial cell-specific TGF (TGF-e) which modulates the growth of human epidermal carcinoma cells.
  • TGF transforming growth factor
  • TGF-e epithelial cell-specific TGF
  • antibodies can be used to probe the structural changes caused by amino acid substitution.
  • the changes in the stability of a sub-domains brought about by amino acid substitutions may be manifested as changes in its affinity for an antibody probe compared to the wild type.
  • peptides p1779, p1780 and p1781 shown in Table 6 as a model system it was demonstrated that a sdAb isolated from the llama sdAbs phage display library by panning against p1779 may serve as a structural probe.
  • the sdAb binds to the p1779 peptide with a K d of 10 ⁇ M, but shows no binding to the substituted versions of the peptide (peptides p1780 and p1781), which are known to have structures different from p1779. Other than serving as structural probes, such sdAbs can be used, for example, to interfere with granulin binding in pathways leading to cancer cell growth or HIV progression.
  • TABLE 6 Sequences of the human granulinA-derived peptide p1779 and its substituted versions p1780 and p1781. For panning experiments the peptides were labelled at the N-terminal through a (Gly) 4 linker.
  • TNG.P1779 Sequencing of twenty-one p1779-specific sdAb genes identified one fragment, namely, TNG.P1779, which was further expressed for detailed binding studies by BIACORE. In agreement with the phage ELISA results, TNG.P1779 was shown to be active by BIACORE analysis in which biotinylated p1779 was captured on a SA-coated CM5 sensor chip (FIG. 6, part A). No binding was detected to the reference surfaces on which a similar amount of p1780 or p1781 had been captured (data not shown). A Scatchard plot of the binding data gave a K d of 1.1 ⁇ 10 ⁇ 5 M (Table 5). These results demonstrate that the TNG.P1779 behaves like a structural probe, sensing the structural changes, which occur in p1780 or p1781 as a result of amino acid substitutions.
  • Parathyroid hormone is the major regulator of serum calcium levels and its use for the treatment of bone loss due to osteoporosis has been postulated. Osteoporosis, which is characterized by bone loss, strikes at any age, affects both men and women, although women with higher frequency, and can results in hospitalization, disability and death (Morley et al., Current Medicinal Chemistry, 6, 1095-1106 (1999); Whitfield et al., Drugs & Aging, 15(2), 117-129 (1999)). Most of the available drugs slow down or stop further bone loss, but have no bone growth-stimulating effects, hence are not capable of replacing the lost bones.
  • PTH parathyroid hormone
  • TNG.PTH50 shows the binding profile for TNG.PTH50 which was isolated by panning against PTH2.
  • the calculated K d for TNG.PTH50 is 4.3 ⁇ 10 ⁇ 6 which is shown in Table 8.
  • TABLE 8 Equilibrium constants for the binding of TNG.PTH22, TNG.PTH23 and TNG.PTH50 to biotinylated PTH2. The values were determined from the respective sensograms and Scatchard plots, as shown in FIG. 7 for TNG.PTH50.
  • sdAb K d (M) TNG.PTH22 1.4 ⁇ 10 ⁇ 5 TNG.PTH23 5.7 ⁇ 10 ⁇ 5 TNG.PTH50 4.3 ⁇ 10 ⁇ 6
  • the mixture was autoclaved and stored solid at room temperature.
  • the oligonucleotides were synthesized using the Applied Biosystems 394 DNA/RNA synthesizer. DNA sequencing was performed by the dideoxy method (Sanger et al., Biotechnology, 104-108 (1992)) using the AmpliTaq DNA Polymerase FS kit and 373A DNA Sequencer Stretch (PE Applied Biosystems, Mississauga, ON, Canada).
  • the host bacteria used for cloning was TG1: supE hsd5 thi .(lac-proAB) F′ [traD36 proAB + lacI q lacZM15]. All the cloning steps were performed as described (Sambrook et al., supra).
  • the vector fd-tet was purchased from American Type Culture Collection (Manassas, Va.) and engineered such that it contained ApaI and NotI restriction sites immediately following the gIIIp leader sequence codons (Simon J. Foote, personal communications).
  • RT-PCR Reverse transcription-polymerase chain reaction
  • the primers used included a CH2-specific primer, LlamaFOR, 5′(CGCCATCAAGGTACCAGTTGA)3′ [SEQ ID No: 207] and LlamaBACK primer, 5′(GATGTGCAGCTGCAGGCGTCTGGRGGAGG)3′ [SEQ ID No: 208], which anneals to the 5′ flanking region of VH genes.
  • Amplified product of approximately 600 base pair was purified from the agarose gel using QIAquick Gel ExtractionTM kit (QIAGEN) and subjected to a second round of PCR using the primers LlamaApaII, 5′(CATGACCACAGTGCACAGGAKGTSCAGCT)3′ [SEQ ID No: 209] and LlamaNotI, 5′(CGATTCTGCGGCCGCTGAGGAGACGGTGACCTG)3′ [SEQ ID No: 210].
  • the PCR mixture contained 10 pmol/ ⁇ l each of the two primers, 1 ⁇ buffer (Perkin Elmer), 200 ⁇ M each of the four dNTPs and 0.05 unit/ ⁇ l AmpliTaqTM DNA polymerase (Perkin Elmer).
  • PCR protocol consisted of an initial denaturation step at 95° C. for 15 min followed by 35 cycles of 94° C. for 30 sec, 45° C. for 30 sec, and 72° C. for 1 min, and a final extension step at 72° C. for 10 min.
  • the primers were complimentary to the 5′ and 3′ ends of the amplified product and incorporated ApaII and NotI restriction sites (underlined) at the end of VH genes.
  • the amplified products were purified using QIAquick PCR Purification kitTM (QIAGEN), cut sequentially with ApaII and NotI restriction endonucleases, purified again, ligated to the ApaII/NotI-treated fd-tet phage vector and desalted using the above kit.
  • Electrocompetent TG1 cells were prepared (Tung et al., Trends Genet., 128-129 (1995)) and 1.5 ⁇ g of the ligated product was mixed with 40 ⁇ l of competent E. coli strain TG1 and the cells were transformed by electroporation using the BIO-RAD Gene PulserTM according to the manufacturer's instructions.
  • the transformed cells were immediately transferred into 1 ml of SOC medium and split into 3 sterile tubes containing 3 ml of 50° C. agarose top, vortexed immediately, poured onto pre-warmed 2 ⁇ YT petri dishes, and incubated at 37° C. overnight.
  • the phage particles were eluted by adding five ml of sterile PBS to the plates gently shaked at 4° C. for 3 hr.
  • the phage-containing PBS was collected, the plates were rinsed with an additional 5 ml PBS and the two supernatants were combined in a centrifuge bottle.
  • the contents were centrifuged at 6000 g for 15 min at 4° C., the supernatant was decanted into a sterile centrifuge bottle and the phage was purified as described (Harrison et al., supra). At the end of the purification, the phage pellet was dissolved in 20 ml of sterile PBS and stored in liquid nitrogen in 100 ⁇ l aliquots.
  • Panning was performed using the Nunc-Immuno MaxiSorpTM 8-well strips (Nunc). Briefly, the wells were coated overnight by adding 150 ⁇ l of 100 ⁇ g/ml antigen in PBS. In the morning, the wells were rinsed three times with PBS and subsequently blocked with 400 ⁇ l PBS-2% (w/v) skim milk (2% MPBS) at 37° C. for 2 hr. The wells were rinsed as above and 1012 transducing units phage in 2% MPBS were added. The mixture was incubated at room temperature for 1.5 hr after which the unbound phage in the supematant was removed.
  • the wells were rinsed 10 times with PBS-0.1% (v/v) Tween 20 and then 10 times with PBS to remove the detergent.
  • the bound phage was eluted by adding freshly prepared 200 ⁇ l 100 mM triethylamine, pipetting the content of the well up and down several times and incubating the mixture at room temperature for 10 min.
  • the eluted phage was transferred to a tube containing 100 ⁇ l 1 M Tris-HCl, pH 7.4 and vortexed to neutralize the triethylamine.
  • 10 ml of exponentially growing TG1 culture was infected with 150 ⁇ l eluted phage by incubating the mixture at 37° C. for 30 min.
  • SA-PMP (1 mg/ml) obtained from Promega (Madison, Wis.). To maintain SA-PMP in solution during the panning process, the reaction tubes were flicked frequently during the incubation period. Briefly, for each target antigen 2 ⁇ 100 ⁇ l SA-PMPs was first dispersed by gently flicking the bottom of the tubes, and then captured at the side of the tube in a magnetic stand (approximately 30 sec.) followed by careful removal of the supematant. SA-PMPs were re-suspended in 100 ⁇ l 1 ⁇ PBS, re-captured and the supernatant was removed. This washing process was repeated three times.
  • the phage particles were pre-incubated with SA-PMP in 2% MPBS for 1 hr at room temperature and the magnetic beads were captured.
  • 10 12 t.u. phage (10 11 t.u. for further rounds) in the supernatant was incubated in 2% MPBS containing 20 mg/ml BSA, 0.05% Tn20 and 1 ⁇ g/ml biotinylated antigen in a total volume of 150 ⁇ l for 1 hr at room temperature.
  • 100 ⁇ l of the washed SA-PMP was blocked in 400 ⁇ l 2% MPBS at 37° C. for 2 hr.
  • the supematant was discarded and the phage-biotinylated antigen complex solution from the first tube was added to the blocked SA-PMP at room temperature for 30 min.
  • the supematant was removed and the complex-bound SA-PMPs were washed twice with 100 ⁇ l PBS and then once with 100 ⁇ l 2% MPBS containing 0.05% Tn 20; this sequence of washes was repeated another three times and then finally SA-PMPs were washed twice with PBS.
  • the bound phage was eluted by adding 200 ⁇ l of 100 mM freshly prepared triethylamine and standing at room temperature for 10 min. Phage elution, propagation, titering and purification were performed as described for solid phase panning.
  • the procedure preceding the elution step was modified as described below.
  • 100 ⁇ l SA-PMPs were blocked followed by removal of supernatant and subsequent incubation of SA-PMPs with 100 ill of 5 ⁇ g/ml biotinylated antigens in 2% MPBS at room temperature for 30 min.
  • the antigen-bound SA-PMPs were washed 5 times with 0.5% MPBS and then incubated with phage in 2% MPBS at room temperature for 1.5 hr in a total volume of 100 ⁇ l.
  • the supernatant was removed and the phage bound SA-PMPs were washed eight times with 0.5% MPBS and two times with PBS before proceeding with the elution step.
  • Phage Enzyme-Linked Immunosorbent Assay (Phage ELISA)
  • phage-infected TG1 colonies were used to inoculate 200 ⁇ l of LB in sterile 96-well plates. The cells were grown overnight at 100 rpm and 37° C. In the morning, the plates were spun down in a bench top centrifuge, and the sdAb phage-containing supematant was used for phage ELISA as described below. Briefly, Nunc-Immuno MaxiSorpTM plates (Nunc) were coated overnight at 4° C. with 150 ⁇ l of 10 ⁇ g/ml of target antigen or control proteins in PBS. The contents were removed and the plates were tapped on a paper towel to remove any liquid remaining in the wells.
  • the wells were blocked by adding 300 ⁇ l of PBS-2% (w/v) skim milk (2% MPBS) and incubating for 2 hr at 37° C. The contents of the wells were emptied as before, 100 ⁇ l of sdAb phage supematant in 2% MPBS was added, and the wells were incubated at room temperature for 1.5 hr. For biotinylated antigen, the plates were pre-coated with 5 ⁇ g/ml streptavidin overnight followed by blocking. The wells were then coated with the target antigen by incubating plates with 150 ⁇ l of 1 ⁇ g/ml biotinylated antigen at room temperature for 30 min.
  • the wells were washed six times as before and the binding of sdAb to the antigen was detected colorimetrically by adding 100 ⁇ l of equal mixtures of TMB Peroxidase Substrate and H2O2 (KPL, Maryland, USA) at room temperature for several min. The reaction was stopped by adding 100 ⁇ l of 1 M H3PO4 and the A450 was measured by DYNATECH MR5000 ELISA reader (DYNATECH).
  • sdAb genes were amplified out of the phage vector by PCR using the primers, VH.Bbs, 5′(TATGAAGACACCAGGCCGATGTGCAGCTGCAGGCG)3′ [SEQ ID No: 211], and VH.Bam, 5′(TATGGATCCTGAGGAGACGGTGACCTG)3′ [SEQ ID No: 212] which also introduced BbsI and BamHI sites at the ends of the amplified fragments.
  • sdAb genes were subsequently purified, cut sequentially with BbsI and BamHI restriction endonucleases, purified again with QIAquick Gel ExtractionTM kit (QIAGEN), and ligated to the BbsI/BamHI-treated pSJF-2 vector. An aliquot of the ligated product was used to transform E. coli strain TG1. Transformants were selected on ampicillin plates and the clones harbouring the sdAb genes were identified by PCR and sequencing. For expression, single positive clones were used to inoculate 25 ml of LB containing 100 ⁇ g/ml ampicillin and the culture was shaken at 240 rpm at 37° C. overnight.
  • the entire overnight culture was used to inoculate 1 liter of M9 medium supplemented with 5 ⁇ g/ml vitamin B1, 0.4% casamino acid and 100 ⁇ g/ml ampicillin.
  • the culture was shaken at room temperature for 30 hr at 180 rpm and subsequently supplemented with 100 ml of 10 ⁇ induction medium and 100 ⁇ l of 1 M isopropylthio-D-galactoside.
  • the culture was shaken for another 60 hr, the periplasmic fraction was extracted by osmotic shock (Anand et al., Gene, 39-44 (1991) and the presence of sdAb in the extract was detected by Western blotting (MacKenzie et al., Biotechnology N.Y., 390-395 (1994)).
  • the periplasmic fraction was dialyzed extensively in 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) buffer pH 7.0, 500 mM NaCl.
  • the presence of the sdAb C-terminal His5 tag allowed a one step protein purification by immobilized metal affinity chromatography using HiTrap ChelatingTM column (Phamacia).
  • the 5-ml column was charged with Ni 2+ by applying 30 ml of a 5 mg/ml NiCl2.6H2O solution and subsequently washed with 15 ml deionized water.
  • Purification was carried out as described (MacKenzie, supra) except that the starting buffer was 10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the bound protein was eluted with a 10-500 mM imidazole gradient.
  • the purity of the protein was determined by SDS-PAGE (Laemmeli U.K., in: Proteases and biological control [Reich et al., ed.], Cold Spring Harbour Laboratory, pp. 661-676 (1975)).
  • sdAb preparation was further subjected to gel filtration chromatography using Superdex 75 column (Pharmacia) as described (Deng et al., Proc. Natl. Acad. Sci. USA., 4992-4996 (1995)) and the purified monomer species were used in binding studies by surface plasmon resonance.
  • Binding studies were performed using BIACORE 1000 (Jonsson et al., BioTechniques, 620-627 (1991)) available from Biacore Inc., Piscataway, N.J. Binding of the anti-Yst9.1 sdAbs to Yst9.1 scFv was assessed under the same conditions except that in this case sdAb was immobilized (540 RU) and the flow rate was set at 20 ⁇ l/min. For PTH binders 186 RU (PTH2) or 70 RU (control peptide) was immobilized and the flow rate was also set at 20 ⁇ l/min. Surface regeneration was achieved by washing the sensor chips with HBST buffer.
  • sdAb was passed over biotinylated p1779 (520 RU) or p1780 and p1781 control peptides (420 RU) which had been captured on a CM5 sensor chip coated with streptavidin (2260 RU).
  • Kinetic rate constants were determined using BIAevaluation software and fitting to 1:1 interaction model. Affinity constants were calculated from the kinetic rate constants and by Scatchard analysis of equilibrium binding data as described (MacKenzie et al., J. Biol. Chem, 1527-1533 (1996)).

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US20060246058A1 (en) 2006-11-02
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