WO2009109572A2 - Exposition sur phage monovalent de domaines variables simples - Google Patents
Exposition sur phage monovalent de domaines variables simples Download PDFInfo
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- WO2009109572A2 WO2009109572A2 PCT/EP2009/052499 EP2009052499W WO2009109572A2 WO 2009109572 A2 WO2009109572 A2 WO 2009109572A2 EP 2009052499 W EP2009052499 W EP 2009052499W WO 2009109572 A2 WO2009109572 A2 WO 2009109572A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- 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
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- 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
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- 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 present invention relates to methods of generating single variable domains, e.g. Nanobodies. of high affinity, to libraries or repertoires comprising said single variable domains on phages in a monovalent format and to plasmids, phagemids or phages (also simply referred herein as vector or vectors) designed to express said single variable domains in said libraries.
- single variable domains e.g. Nanobodies. of high affinity
- libraries or repertoires comprising said single variable domains on phages in a monovalent format and to plasmids, phagemids or phages (also simply referred herein as vector or vectors) designed to express said single variable domains in said libraries.
- E. Pavoni et al. in Gene 391 (2007) 120-129 describes a novel phagemid vector and method which allows a low-level expression of recombinant antibodies in order to express otherwise toxic proteins.
- E. Pavoni furthermore shows that an antibody library generated by applying said vector improves viability of bacteria harbouring harmful antibodies and thus counterbalances the probability of the antibodies, which are either neutral or harmful to bacterial host, to be selected from the library.
- Said novel vector has an amber codon positioned in a sequence encoding for the leader sequence of the scFv antibodies to be expressed.
- US 5,821,047 describes preparation and systematic selection of novel binding proteins, in particular conventional antibodies, having altered binding properties for a target molecule, in particular describes a method to produce a phagemid particle that rarely displays more than one copy of candidate binding proteins on the outer surface of the phagemid particle so that efficient selection of high affinity binding proteins can be achieved.
- One of the embodiments of the present invention is a method for obtaining single variable domains, e.g. Nanobodies, directed against a Target Molecule, preferably a multimeric Target Molecule, comprising the steps of: a) immunizing a mammal belonging to the Camelidae, preferably a Llama, with said Target Molecule, so as to raise an immune response and in particular to raise antibodies (and more in particular to raise heavy chain antibodies) against said Target Molecule; and b) obtaining an appropriate biological sample, e.g.
- the invention provides high affinity single variable domains, e.g. Nanobodies or dAbs, obtainable by the methods described in the above paragraph and further described herein.
- an embodiment of the present invention is a method for selecting single variable domains, e.g. Nanobodies and/or dAbs, said method comprises the steps of: a) constructing a replicable expression plasmid comprising a transcription regulatory element operably linked to DNA encoding single variable domains as herein described, wherein said DNA is fused to the DNA encoding at least a portion of a phage coat protein; and b) mutating the DNA encoding single variable domains as described herein at one or more selected positions thereby forming a family of related plasmids; and c) transforming suitable host cells with said plasmids; and d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein; and e) culturing the transformed infected host cells under conditions suitable for forming recombinant phage particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no
- the plasmids, phagemids. phages or vectors, expression vector will further contain a secretory signal sequences fused to the DNA encoding single variable domains, e.g. Nanobodies and/or dAbs as described herein, and the transcription regulatory element will be a promoter system.
- Preferred promoter systems are selected from; Lac Z, ⁇ PL, TAC, T7 polymerase, tryptophan, and alkaline phosphatase promoters and combinations thereof.
- a more preferred promoter system is the Lac Z promoter.
- Target Molecule will be a mammalian protein, preferably the protein will be selected from the list given below in the definition part and is in its preferred form a so called multimeric protein.
- FIG. 1 Schematic representation of standard methods and methods of the invention.
- the methods of the instant invention comprise a method for selecting binding single variable domains, e.g. Nanobodies and/or dAbs, having a desired, usually high, affinity for a Target Molecule from a library of single variable domains.
- the library of single variable domains, fused to a phage coat protein is produced preferably either de novo from a mammal, e.g. human or a mammal of the family of the Camelidae, preferably Llamas, or by mutagenesis and. preferably, a single copy of each single variable domain is displayed on the surface of a phagemid particle containing DNA encoding that polypeptide.
- phagemid particles are then contacted with a Target Molecule and those particles having the highest affinity for the Target Molecules are separated from those of lower affinity.
- the high affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained.
- the selected single variable domains produced by the method of this invention are useful per se as diagnostics, preventatives or therapeutics (e.g. agonists or antagonists) used in treatment of biological organisms. Structural analysis of the selected polypeptides may also be used to facilitate rational drug design.
- Target Molecule * or “Target Molecules'” is meant a protein with a biological function in an organism, preferably animal, more preferably mammal most preferred human, wherein said biological function may be involved in the initiation or progression or maintenance of a disease.
- said protein is selected from the group consisting of: human growth hormone (hGH). N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin. relaxin A-chain, relaxin B ⁇ chain. prorelaxin.
- glycoprotein hormones such as follicle stimulating hormones (FSH), thyroid stimulating hormone (TSH), and leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, a Nanobody.
- FSH follicle stimulating hormones
- TSH thyroid stimulating hormone
- LH leutinizing hormone
- glycoprotein hormone receptors calcitonin, glucagon, factor VIII, an antibody, a Nanobody.
- FSH follicle stimulating hormones
- TSH thyroid stimulating hormone
- LH leutinizing hormone
- glycoprotein hormone receptors calcitonin
- glucagon factor VIII
- an antibody a Nanobody.
- a molecule which is well tolerated by mammals in particularly humans and has a long half life when given systemically and/or locally e.g. poly glycol chains of different size, e.g. PEG-20.
- PEG-30 or PEG40 lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hematopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, a microbial protein, such as beta lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, receptors for hormones or growth factors; integrin, thrombopoietin, protein A or D, rheumatoid factors, nerve growth factors such as NGF- ⁇ .
- t-PA tissue-type plasminogen activator
- TGF transforming growth factors
- CD-4 DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferons such as interferon-alpha, -beta, and -gamma
- CSFs colony stimulating factors
- ILs interleukins
- ILs interleukins
- Target Molecule is a multimeric protein and even more preferred is a multimeric protein which subunits are selected from the group consisting of: von Willebrand Factor (vWF). IL-6, tumor necrosis factor-alpha and -beta and many others.
- vWF von Willebrand Factor
- IL-6 tumor necrosis factor-alpha and -beta and many others.
- a multimeric protein is a protein which is associated (typically by non-covalent interactions) in biological organism such as humans with others as subunils in a multimeric structure and typically only in the multimeric format is able to unfold its biological function.
- the single variable domains that are present in the constructs of the invention may be any variable domain that forms a single antigen binding unit.
- such single variable domains will be amino acid sequences that essentially consist of 4 framework regions (FRl to FR4 respectively) and 3 complementarity determining regions (CDRl to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein).
- Such single variable domains and fragments are most preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.
- the single variable domain may for example comprise a light chain variable domain sequence (e.g. a V L -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V ⁇ -sequence or V HH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e.
- a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and ScFv fragments that need to interact with another variable domain - e.g. through a V J /V L interaction - to form a functional antigen binding domain).
- the single variable domain may be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” (or an amino acid sequence that is suitable for use as a dAb) or a NanobodyTM (as defined herein, and including but not limited to a V HH sequence); other single variable domains, or any suitable fragment of any one thereof.
- a domain antibody or an amino acid sequence that is suitable for use as a domain antibody
- a single domain antibody or an amino acid sequence that is suitable for use as a single domain antibody
- a “dAb” or an amino acid sequence that is suitable for use as a dAb
- NanobodyTM as defined herein, and including but not limited to a V HH sequence
- the amino acid sequence of the invention may be a NanobodyTM or a suitable fragment thereof.
- Nanobodies in particular V HH sequences and partially humanized Nanobodies
- Nanobodies including humanization and/or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non- limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO07/ 104529.
- high affinity as used herein is meant a dissociation constant for a monovalent binding Nanobody of (Kd) of ⁇ 100 nM and preferably 10 nM and more preferably InM and even more preferably ]00pM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art.
- high avidity' 1 as used herein is meant a dissociation constant for a bi ⁇ or multivalent binding Nanobody of (Kd) of ⁇ 100 nM and preferably 10 nM and more preferably InM and even more preferably lOOpM and most preferred 10 pM under physiological conditions and measured by standard procedures in the art.
- rigid secondary structure as used herein is meant any polypeptide segment exhibiting a regular repeated structure such as is found in; ⁇ -helices, 310 helices, ⁇ - helices, parallel and antiparallel ⁇ -sheets, and reverse turns.
- non-ordered structures that lack recognizable geometric order are also included in the definition of rigid secondary structure provided they form a domain or "patch" of amino acid residues capable of interaction with a target and that the overall shape of the structure is not destroyed by replacement of an amino acid within the structure. It is believed that some non-ordered structures are combinations of reverse turns.
- the geometry of these rigid secondary structures is well defined by ⁇ and psi torsional angles about the ⁇ -carbons of the peptide "backbone". The requirement that the secondary structure be exposed to the surface of the polypeptide is to provide a domain or "patch" of amino acid residues that can be exposed to and bind with a target molecule.
- leader sequence as used herein is meant a particular section of messenger
- immunoglobulin sequence whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VH H domains or V H /V L domains, respectively).
- sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “V HH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.
- nucleotide sequence * as used herein also encompasses a nucleic acid molecule with said nucleotide sequence, so that the terms "nucleotide sequence” and “nucleic acid” should be considered equivalent and are used interchangeably herein; h) Unless indicated otherwise, ail methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews Presta, Adv. Drug Deliv. Rev.
- an amino acid residue is referred to in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and/or at a pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged and/or uncharged at such a higher or lower pH, as will be clear to the skilled person.
- the degree of sequence identity between two or more nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBI Blast v2.0, using standard settings.
- a known computer algorithm for sequence alignment such as NCBI Blast v2.0
- Some other techniques, computer algorithms and settings for determining the degree of sequence identity are for example described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.
- the nucleotide sequence with the greatest number of nucleotides will be taken as the "first" nucleotide sequence, and the other nucleotide sequence will be taken as the "second" nucleotide sequence;
- the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [J 00%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered
- amino acid difference as defined herein.
- degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
- amino acid sequence with the greatest number of amino acid residues will be taken as the "first"' amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.
- amino acid substitutions which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide.
- Such conservative amino acid substitutions are well known in the art, for example from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 as well as WO 98/49185 and from the further references cited therein.
- Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues; Ala, Ser, Thr, Pro and GIy; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, GIu and GIn: (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, VaI and Cy s ; and (e) aromatic residues: Phe. Tyr and Trp.
- Particularly preferred conservative substitutions are as follows: Ala into GIy or into Ser; Arg into Lys; Asn into GIn or into His; Asp into GIu; Cys into Ser; GIn into Asn; GIu into Asp; GIy into Ala or into Pro; His into Asn or into GIn; Ue into Leu or into VaI; Leu into He or into VaI; Lys into Arg, into GIn or into GIu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser: Trp into Tyr; Tyr into Trp; and/or Phe into VaI. into He or into Leu.
- amino acid difference refers to an insertion, deletion or substitution of a single amino acid residue on a position of the first sequence, compared to the second sequence; it being understood that two amino acid sequences can contain one.
- nucleotide sequence or amino acid sequence is said to "comprise” another nucleotide sequence or amino acid sequence, respectively, or to “essentially consist of " another nucleotide sequence or amino acid sequence, this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the firstmentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the firstmentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the firstmentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein).
- a Nanobody of the invention when a Nanobody of the invention is said to comprise a CDR sequence, this may mean that said CDR sequence has been incorporated into the Nanobody of the invention, but more usually this generally means that the Nanobody of the invention contains within its sequence a stretch of amino acid residues with the same amino acid sequence as said CDR sequence, irrespective of how said Nanobody of the invention has been generated or obtained.
- the latter amino acid sequence when it has a specific biological or structural function, it preferably has essentially the same, a similar or an equivalent biological or structural function in the firstmentioned amino acid sequence (in other words, the firstmentioned amino acid sequence is preferably such that the latter sequence is capable of performing essentially the same, a similar or an equivalent biological or structural function).
- the CDR sequence and framework are preferably capable, in said Nanobody, of functioning as a CDR sequence or framework sequence, respectively.
- the firstmentioned nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the firstmentioned, larger nucleotide sequence).
- an expression product e.g. a polypeptide
- a nucleic acid sequence or amino acid sequence is considered to be "(in) essentially isolated (form/ ' - for example, compared to its native biological source and/or the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide. another biological component or macromolecule or at least one contaminant, impurity or minor component.
- a nucleic acid sequence or amino acid sequence is considered ''essentially isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100- fold, and up to 1000-fold or more.
- a nucleic acid sequence or amino acid sequence that is "'in essentially isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatograpbical technique, such as poiyacrylamide-gel electrophoresis; p)
- domain' ' as used herein generally refers to a globular region of an amino acid sequence (such as an antibody chain, and in particular to a globular region of a heavy chain antibody), or to a polypeptide that essentially consists of such a globular region.
- a domain will comprise peptide loops (for example 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds.
- the term '''binding domain'' refers to such a domain that is directed against an antigenic determinant (as defined herein); q)
- antigenic determinant refers to the epitope on the antigen recognized by the antigen-binding molecule (such as a Nanobody or a polypeptide of the invention) and more in particular by the antigen-binding site of said molecule.
- the terms '' 'antigenic determinant ⁇ and "epitope” may also be used interchangeably herein.
- An amino acid sequence (such as a Nanobody, an antibody, a polypeptide of the invention, or generally an antigen binding protein or polypeptide or a fragment thereof) that can (specifically) bind to, that has affinity for and/or that has specificity for a specific antigenic determinant, epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against” or “directed against” ' said antigenic determinant, epitope, antigen or protein,
- the term “'specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding molecule or antigen- binding protein (such as a Nanobody or a polypeptide of the invention) molecule can bind.
- the specificity of an antigen-binding protein can be determined based on affinity and/or avidity.
- the affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (K D )- is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein: the lesser the value of the K D , the stronger the binding strength between an antigenic determinant and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (K A ), which is 1 /K D ).
- affinity can be determined in a manner known per se. depending on the specific antigen of interest.
- Avidity is the measure of the strength of binding between an antigen-binding molecule (such as a Nanobody or polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen -binding molecule.
- antigen-binding proteins such as the amino acid sequences, Na ⁇ obodies and/or polypeptides of the invention
- K D dissociation constant
- K A association constant
- the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned herein. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more then 10 "4 moles/liter or 10 " moles/liter (e.g., of 10 "2 moles/liter).
- the affinity denotes the strength or stability of a molecular interaction.
- the affinity is commonly given as by the K D , or dissociation constant, which has units of moi/liter (or M).
- the affinity can also be expressed as an association constant.
- K A which equals 1/K D and has units of (mol/liter) "1 (or M "1 ).
- the K D - value characterizes the strength of a molecular interaction also in a thermodynamic sense as it is related to the free energy (DG) of binding by the well known relation DG-RT.ln(Ko) (equivalently DG-RT-IH(KA)), where R equals the gas constant, T equals the absolute temperature and In denotes the natural logarithm.
- the K D for biological interactions which are considered meaningful are typically in the range of 10 "10 M (0.1 nM) to 10 "5 M (10000 nM). The stronger an interaction is, the lower is its KQ.
- the off-rate k o ff has units s "1 (where s is the SI unit notation of second).
- the on-rate Ic 0n has units M -1 S "1 .
- the on- rate may vary between 10 2 M -1 S “1 to about 10 7 M -1 S “1 , approaching the diffusion- limited association rate constant for bimolecular interactions.
- the affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well known surface plasmon resonance (SPR) biosensor technique (see for example Ober et at, Intern. Immunology, 13, 1551-1559. 2001) where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding It 0n , k o ⁇ - measurements and hence K D (or K A ) values.
- SPR surface plasmon resonance
- the measured K D may correspond to the apparent K D if the measuring process somehow influences the intrinsic binding affinity of the implied molecules for example by artefacts related to the coating on the biosensor of one molecule. Also, an apparent K D may be measured if one molecule contains more than one recognition sites for the other molecule. In such situation the measured affinity may be affected by the avidity of the interaction by the two molecules.
- K D K D and apparent K D should be treated with equal importance or relevance.
- the experienced scientist may judge it to be convenient to determine the binding affinity relative to some reference molecule.
- a reference molecule C that is known to bind to B and that is suitably labelled with a fluorophore or chromophore group or other chemical moiety, such as biotin for easy detection in an ELISA or FACS (Fluorescent activated cell sorting) or other format (the fluorophore for fluorescence detection, the chromophore for light absorption detection, the biotin for streptavidin-mediated ELISA detection).
- the reference molecule C is kept at a fixed concentration and the concentration of A is varied for a given concentration or amount of B.
- an IC 50 value is obtained corresponding to the concentration of A at which the signal measured for C in absence of A is halved.
- Ko ref - the KQ of the reference molecule, is known, as well as the total concentration c ref of the reference molecule
- the apparent K D for the interaction A-B can be obtained from following formula: K D ⁇ IC ⁇ O /( ⁇ +C K ⁇ / Koi ef )- Note that if c re r « Kp r ef ⁇ K- D ⁇ IC 5 O.
- the half-life of an amino acid sequence, compound or polypeptide of the invention can generally be defined as the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.
- the in vivo half-life of an amino acid sequence, compound or polypeptide of the invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering to a warm-blooded animal (i.e.
- a human or to another suitable mammal such as a mouse, rabbit, rat, pig, dog or a primate, for example monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatto)) and baboon [Papio ursinus)) a suitable dose of the amino acid sequence, compound or polypeptide of the invention; collecting blood samples or other samples from said animal; determining the level or concentration of the amino acid sequence, compound or polypeptide of the invention in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence, compound or polypeptide of the invention has been reduced by 50% compared to the initial level upon dosing, Reference is for example made to the Experimental Part belovv.
- the half-life can be expressed using parameters such as the 11/2 -alpha, 11/2 -beta and the area under the curve (AUC).
- an "increase in half-life” refers to an increase in any one of these parameters, such as any two of these parameters, or essentially all three these parameters.
- increase in half-life or “increased half- life' " in particular refers to an increase in the tl/2-beta. either with or without an increase in the tl/2-alpha and/or the AUC or both.
- modulating or “to modulate'” generally means either reducing or inhibiting the activity of. or alternatively increasing the activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay.
- '"modulating” or “'to modulate” may mean either reducing or inhibiting the activity of, or alternatively increasing a (relevant or intended) biological activity of, a target or antigen, as measured using a suitable in vitro, cellular or in vivo assay (which will usually depend on the target or antigen involved), by at least 1%.
- At least 5% such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to activity of the target or antigen in the same assay under the same conditions but without the presence of the construct of the invention.
- moduleating may also involve effecting a change (which may either be an increase or a decrease) in affinity, avidity, specificity and/or selectivity of a target or antigen for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates: and/or effecting a change (which may either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of the construct of the invention, As will be clear to the skilled person, this may again be determined in any suitable manner and/or using any suitable assay known per se, depending on the target or antigen involved.
- Modulating may also mean effecting a change (i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect) with respect to one or more biological or physiological mechanisms, effects, responses, functions, pathways or activities in which the target or antigen (or in which its substrate(s), ligand(s) or path way (s) are involved, such as its signalling pathway or metabolic pathway and their associated biological or physiological effects) is involved.
- a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
- a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
- a change i.e. an activity as an agonist, as an antagonist or as a reverse agonist, respectively, depending on the target or antigen and the desired biological or physiological effect
- an action as an agonist or antagonist may be such that an intended biological or physiological activity is increased or decreased, respectively, by at least 1%, preferably at least 5%, such as at least 10% or at least 25%, for example by at least 50%, at least 60%, at least 70%, at least 80%, or 90% or more, compared to the biological or physiological activity in the same assay under the same conditions but without the presence of the construct of the invention.
- Modulating may for example also involve allosteric modulation of the target or antigen; and/or reducing or inhibiting the binding of the target or antigen to one of its substrates or ligands and/or competing with a natural ligand, substrate for binding to the target or antigen.
- interaction site on the target or antigen means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is a site for binding to a ligand, receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerization (such as homomerization or heterodimerization) of the target or antigen: or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen that is involved in a biological action or mechanism of the target or antigen.
- an '"interaction site can be any site, epitope, antigenic determinant, part, domain or stretch of amino acid residues on the target or antigen to which an amino acid sequence or polypeptide of the invention can bind such that the target or antigen (and/or any pathway, interaction, signalling, biological mechanism or biological effect in which the target or antigen is involved) is modulated (as defined herein), w)
- An amino acid sequence or polypeptide is said to be "specific for" a first target or antigen compared to a second target or antigen when is binds to the first antigen with an affinity (as described above, and suitably expressed as a K D value, K A value, K off rate and/or K 0n rate) that is at least 10 times, such as at least 100 times, and preferably at least 1000 times, and up to 10,000 times or more better than the affinity with which said amino acid sequence or polypeptide binds to the second target or polypeptide.
- an affinity as described above, and suitably expressed as a K D
- the first antigen may bind to the target or antigen with a K D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10,000 times less or even less than that, than the K D with which said amino acid sequence or polypeptide binds to the second target or polypeptide.
- a K D value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 times less, such as 10,000 times less or even less than that, than the K D with which said amino acid sequence or polypeptide binds to the second target or polypeptide.
- an amino acid sequence or polypeptide is "specific for" a first target or antigen compared to a second target or antigen, it is directed against (as defined herein) said first target or antigen, but not directed against said second target or antigen.
- Another suitable quantitative cross-blocking assay uses an ELISA-based approach to measure competition between amino acid sequence or another binding agents in terms of their binding to the target.
- the following generally describes a suitable Biacore assay for determining whether an amino acid sequence or other binding agent cross-blocks or is capable of cross- blocking according to the invention. It will be appreciated that the assay can be used with any of the amino acid sequence or other binding agents described herein.
- the Biacore machine (for example the Biacore 3000) is operated in line with the manufacturer's recommendations.
- the target protein is coupled to a CM5 Biacore chip using standard amine coupling chemistry to generate a surface that is coated with the target.
- test amino acid sequences (termed A* and B*) to be assessed for their ability to cross- block each other are mixed at a one to one molar ratio of binding sites in a suitable buffer to create the test mixture.
- concentrations on a binding site basis the molecular weight of an amino acid sequence is assumed to be the total molecular weight of the amino acid sequence divided by the number of target binding sites on that amino acid sequence.
- the concentration of each amino acid sequence in the test mix should be high enough to read ⁇ y saturate the binding sites for that amino acid sequence on the target molecules captured on the Biacore chip.
- the amino acid sequences in the mixture are at the same molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1.5 micromolar (on a binding site basis).
- Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix.
- the test mixture is passed over the target- coated Biacore chip and the total amount of binding recorded. The chip is then treated in such a way as to remove the bound amino acid sequences without damaging the chip-bound target.
- a cross-blocking amino acid sequence or other binding agent according to the invention is one which will bind to the target in the above Biacore cross-blocking assay such that during the assay and in the presence of a second amino acid sequence or other binding agent of the invention the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the maximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the maximum theoretical binding, and more specifically between 70% and 0.1 % (e.g. 70% to 4%) of maximum theoretical binding (as just defined above) of the two amino acid sequences or binding agents in combination.
- the Biacore assay described above is a primary assay used to determine if amino acid sequences or other binding agents cross-block each other according to the invention. On rare occasions particular amino acid sequences or other binding agents may not bind to target coupled via amine chemistry to a CM5 Biacore chip (this usually occurs when the relevant binding site on target is masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a tagged version of the target, for example a N-terminal His-tagged version (R & D Systems, Minneapolis, MN. USA; 2005 cat# 1406-ST-025).
- an anti- His amino acid sequence would be coupled to the Biacore chip and then the His- tagged target would be passed over the surface of the chip and captured by the anti- His amino acid sequence.
- the cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His-tagged target would be loaded back onto the anti-His amino acid sequence coated surface.
- C-terminal His-tagged target could alternatively be used.
- various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. HA tag with anti-HA antibodies; FLAG tag with anti-FLAG antibodies: biotin tag with streptavidin).
- the general principal of the assay is to have an amino acid sequence or binding agent that is directed against the target coated onto the wells of an ELISA plate. An excess amount of a second, potentially cross-blocking, anti-target amino acid sequence is added in solution (i.e. not bound to the ELISA plate). A limited amount of the target is then added to the wells. The coated amino acid sequence and the amino acid sequence in solution compete for binding of the limited number of target molecules.
- the plate is washed to remove excess target that has not been bound by the coated amino acid sequence and to also remove the second, solution phase amino acid sequence as well as any complexes formed between the second, solution phase amino acid sequence and target.
- the amount of bound target is then measured using a reagent that is appropriate to detect the target.
- An amino acid sequence in solution that is able to cross-block the coated amino acid sequence will be able to cause a decrease in the number of target molecules that the coated amino acid sequence can bind relative to the number of target molecules that the coated amino acid sequence can bind in the absence of the second, solution phase, amino acid sequence.
- the first amino acid sequence e.g.
- an Ab-X is chosen to be the immobilized amino acid sequence, it is coated onto the wells of the ELlSA plate, after which the plates are blocked with a suitable blocking solution to minimize non-specific binding of reagents that are subsequently added.
- An excess amount of the second amino acid sequence, i.e. Ab-Y is then added to the ELISA plate such that the moles of Ab-Y [target] binding sites per well are at least 10 fold higher than the moles of Ab-X [target] binding sites that were used, per well, during the coating of the ELISA plate, [target] is then added such that the moles of [target] added per well are at least 25-fold lower than the moles of Ab-X [target] binding sites that were used for coating each well.
- the ELlSA plate is washed and a reagent for detecting the target is added to measure the amount of target specifically bound by the coated anti-[target] amino acid sequence (in this case Ab-X).
- the background signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab-X).
- second solution phase amino acid sequence in this case Ab-Y).
- [target] buffer only i.e. no target
- target detection reagents The positive control signal for the assay is defined as the signal obtained in wells with the coated amino acid sequence (in this case Ab- X), second solution phase amino acid sequence buffer only (i.e. no second solution phase amino acid sequence), target and target detection reagents.
- Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or in format 2, the solution phase anti-target amino acid sequence is able to cause a reduction of between 60% and 100%, specifically between 70% and 100%, and more specifically between 80% and 100%, of the target detection signal (i.e. the amount of target bound by the coated amino acid sequence) as compared to the target detection signal obtained in the absence of the solution phase anti- target amino acid sequence (i.e. the positive control wells).
- the total number of amino acid residues in a Nanobody can be in the region of 110-120, is preferably 112-1 15, and is most preferably 113.
- parts, fragments, analogs or derivatives (as further described herein) of a Nanobody are not particularly limited as to their length and/or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein and are also preferably suitable for the purposes described herein; z)
- the amino acid residues of a Nanobody are numbered according to the general numbering for V H domains given by Kabat et al. (''Sequence of proteins of immunological interest", US Public Health Services, NlH Bethesda, MD, Publication No. 91), as applied to V HH domains from Camelids in the article of Riechma ⁇ n and Muyldermans, J. Immunol.
- FRl of a Nanobody comprises the amino acid residues at positions 1-30
- CDRl of a Nanobody comprises the amino acid residues at positions 31-35
- FR2 of a Nanobody comprises the amino acids at positions 36-49
- CDR2 of a Nanobody comprises the amino acid residues at positions 50-65
- FR3 of a Nanobody comprises the amino acid residues at positions 66-94
- CDR3 of a Nanobody comprises the amino acid residues at positions 95-102
- FR4 of a Nanobody comprises the amino acid residues at positions 103-113.
- the total number of amino acid residues in each of the CDR' s may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
- the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
- Promoters most commonly used in prokaryotic vectors include the lac Z promoter system, the alkaline phosphatase pho A promoter, the bacteriophage ⁇ PL promoter (a temperature sensitive promoter), the tac promoter (a hybrid trp-lac promoter that is regulated by the lac repressor), the tryptophan promoter, and the bacteriophage T7 promoter. While these are the most commonly used promoters, other suitable microbial promoters may be used as well.
- Preferred promoters for practicing this invention are those that can be tightly regulated such that expression of the fusion gene can be controlled. It is believed that the problem that went unrecognized in the prior art was that display of multiple copies of the fusion protein comprising a single variable domain, e.g. Nanobody, and said fusion protein situated on the surface of the phage particle lead to multipoint attachment of the phage with the target. It is believed this effect is particularly of importance when selection for binders to a muJtimeric Target Molecule is done. Here it is reported for the first time that phage display of a single variable domain, e.g. Nanobody, using conventional and so far described and known methods of single variable domain, e.g. Nanobody.
- said single variable domain e.g. Nanobody. fusion proteins.
- This effect results in selection of high avidity single variable domain, e.g. Nanobody, and show typically in a monovalent setting moderate affinity.
- Said selected single variable domain, e.g. Nanobody may show high avidity when formatted e.g. in bivalent or multivalent format, in particular when binding to multimeric Target Molecules.
- the effective or apparent Kd may be as high as the product of the individual Kds for each copy of the displayed fusion protein. This effect may be the reason why persons skilled in the art of single variable domain, e.g. Nanobody. selection are sometimes unable to separate moderate affinity peptides from higher affinity peptides in particular when selection for multimeric Target Molecules is performed.
- Preferred promoters used to practice this invention are the lac Z promoter.
- the lac Z promoter is regulated by the lac repressor protein iac i, and thus transcription of the fusion gene can be controlled by manipulation of the level of the lac repressor protein.
- the phagemid containing the lac Z promoter is grown in a cell strain that contains a copy of the lac i repressor gene, a repressor for the lac Z promoter.
- Exemplary cell strains containing the lac i gene include JM 101 and XLl -blue.
- the host cell can be cotransfected with a plasmid containing both the repressor lac i and the lac Z promoter.
- One other useful component of vectors used to practice this invention is a signal sequence.
- This sequence is typically located immediately 5' to the gene encoding the fusion protein, and will thus be transcribed at the amino terminus of the fusion protein. However, in certain cases, the signal sequence has been demonstrated to be located at positions other 5 1 to the gene encoding the protein to be secreted. This sequence targets the protein to which it is attached across the inner membrane of the bacterial cell.
- the DNA encoding the signal sequence may be obtained as a restriction endonuclease fragment from any gene encoding a protein that has a signal sequence. Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, LamB or OmpF, MaIE, PhoA and other genes.
- phenotypic selection genes are those encoding proteins that confer antibiotic resistance upon the host cell.
- ampicillin resistance gene (amp) and the tetracycline resistance gene (tet) are readily employed for this purpose.
- Construction of suitable vectors comprising the aforementioned components as well as the gene encoding the desired single variable domain, e.g. Nanobody, is prepared using standard recombinant DNA procedures. Isolated DNA fragments to be combined to form the vector are cleaved, tailored, and ligated together in a specific order and orientation to generate the desired vector.
- Particularly preferred vectors are pUl 19 vectors or variants or close derivatives thereof. E.g.
- the DNA is cleaved using the appropriate restriction enzyme or enzymes in a suitable buffer.
- a suitable buffer In general, about 0.2-1 ⁇ g of plasmid or DNA fragments is used with about 1 -2 units of the appropriate restriction enzyme in about 20 ⁇ l of buffer solution.
- Appropriate buffers, DNA concentrations, and incubation times and temperatures are specified by the manufacturers of the restriction enzymes. Generally, incubation times of about one or two hours at 37° C are adequate, although several enzymes require higher temperatures. After incubation, the enzymes and other contaminants are removed by extraction of the digestion solution with a mixture of phenol and chloroform, and the DNA is recovered from the aqueous fraction by precipitation with ethanol.
- the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after eiidonuclease digestion. However it may be necessary to first convert the sticky ends commonly produced by endonuclease digestion to blunt ends to make them compatible for ligation. To blunt the ends, the DNA is treated in a suitable buffer for at least 15 minutes at 15° C with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in. the presence of the four deoxynucleotide triphosphates. The DNA is then purified by phenol- chloroform extraction and ethanol precipitation.
- Klenow Klenow fragment of DNA polymerase I
- the cleaved DNA fragments may be size- separated and selected using DNA gel electrophoresis.
- the DNA may be electrophoresed through either an agarose or a poly aery 1 amide matrix. The selection of the matrix will depend on the size of the DNA fragments to be separated.
- the DNA is extracted from the matrix by electroelution, or, if low-melting agarose has been used as the matrix, by melting the agarose and extracting the DNA from it.
- the DNA fragments that are to be ligated together are put in solution in about equimolar amounts.
- the solution will also contain ATP, ligase buffer and a ligase such as T4 DNA ligase at about 10 units per 0.5 ⁇ g of DNA.
- the vector is at first linearized by cutting with the appropriate restriction endonuclease(s).
- the linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. The phosphatasing prevents self-ligation of the vector during the ligation step.
- Prokaryotes are the preferred host cells for this invention.
- Suitable prokaryotic host cells include E. coli strain JMlOl, E. coli K12 strain 294 (ATCC number 31,446), E. coli strain W31 10 (ATCC number 27,325).
- E. coli X1776 ATCC number 31,537), E. coli XL-I Blue (Stratagene), and E. coli B; however many other strains of E. coli. such as HBlOl. NM 522, NM538, NM539, and many other species and genera of prokaryotes may be used as well.
- bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may all be used as hosts. Transformation of prokaryotic cells is readily accomplished using the calcium chloride method as described in section 1.82 of Sambrook et al., Molecular Biology: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 1989. Alternatively, electroporation (Neumann et al.. EMBO J., 1 :841 1982) may be used to transform these cells. The transformed cells are selected by growth on an antibiotic, commonly tetracycline (tet) or ampicillin (amp), to which they are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
- an antibiotic commonly tetracycline (tet) or ampicillin (amp)
- Plasmid DNA can be isolated using methods known in the art. Two suitable methods are the small scale preparation of DNA and the large-scale preparation of DNA as described in sections 1.25- 1.33 of Sambrook et al., supra. The isolated DNA can be purified by methods known in the art such as that described in section 1.40 of Sambrook et al., supra. This purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing. DNA sequencing is generally performed by either the method of Messing et al. Nucleic Acids Res., 9:309 1981 or by the method of Maxam et al. Meth. EnzymoL, 65:499 1980.
- This invention contemplates fusing the gene enclosing the desired polypeptide (gene 1) to a second gene (gene 2) such that a fusion protein is generated during transcription.
- Gene 2 is typically a coat protein gene of a phage, and preferably it is the phage Ml 3 gene III coat protein, or a fragment thereof. Fusion of genes 1 and 2 may be accomplished by inserting gene 2 into a particular site on a plasmid that contains gene 1, or by inserting gene 1 into a particular site on a plasmid that contains gene 2.
- Insertion of a gene into a plasmid requires that the plasmid be cut at the precise location that the gene is to be inserted. Thus, there must be a restriction endonuclease site at this location (preferably a unique site such that the plasmid will only be cut at a single location during restriction endonuclease digestion).
- the plasmid is digested, phosphatased, and purified as described above.
- the gene is then inserted into this linearized plasmid by ligating the two DNAs together. Ligation can be accomplished if the ends of the plasmid are compatible with the ends of the gene to be inserted.
- the DNAs can be ligated together directly using a ligase such as bacteriophage T4 DNA Jigase and incubating the mixture at 16° C for 1-4 hours in the presence of ATP and ligase buffer as described in section 1.68 of Sambrook et al., supra. If the ends are not compatible, they must first be made blunt by using the Klenow fragment of DNA polymerase I or bacteriophage T4 DNA polymerase, both of which require the four deoxyribonucleotide triphosphates to fill-in overhanging single-stranded ends of the digested DNA.
- a ligase such as bacteriophage T4 DNA Jigase
- the ends may be blunted using a nuclease such as nuclease Sl or mung-bean nuclease, both of which function by cutting back the overhanging single strands of DNA.
- the DNA is then religated using a ligase as described above.
- oligonucleotide linkers may be used. The linkers serve as a bridge to connect the plasmid to the gene to be inserted. These linkers can be made synthetically as double stranded or single stranded DNA using standard methods.
- the linkers have one end that is compatible with the ends of the gene to be inserted; the linkers are first ligated to this gene using ligation methods described above.
- the other end of the linkers is designed to be compatible with the plasmid for ligation.
- care must be taken to not destroy the reading frame of the gene to be inserted or the reading frame of the gene contained on the plasmid.
- it may be necessary to design the linkers such that they code for part of an amino acid, or such that they code for one or more amino acids.
- DNA encoding a termination codon may be inserted at any suitable position upstream the Nanobody gene, preferably in the leader sequence.
- termination codons are UAG (amber).
- the termination codon introduced in the wild type host cell results in the synthesis of very few maximal one copy of the fusion Nanobody product per phage.
- growth in a suppressor host cell results in the synthesis of large quantities of fused protein.
- Such suppressor host cells contain a tRNA modified to insert an amino acid in the termination codon position of the mRNA thereby resulting in production of normal amounts of the fusion protein.
- suppressor host cells are well known and described, such as E. coll suppressor strain (Bullock et al., Bio Techniques 5, 376-379, 1987). Any acceptable method may be used to place such a termination codon into the mRNA encoding the fusion polypeptide.
- the leader sequence usually contains a ribosome binding site (RBS), which in bacteria also known as the Shine-Dai garno sequence (AGGAGGU).
- RBS ribosome binding site
- AGGAGGU Shine-Dai garno sequence
- Alteration (mutation) of single variable domain e.g. Nanobody. gene at Selected Positions
- the single variable domain, e.g. Nanobody or dAbs. gene may be altered at one or more selected codons.
- An alteration is defined as a substitution, deletion, or insertion of one or more codons in the gene encoding the polypeptide that results in a change in the amino acid sequence of the polypeptide as compared with the unaltered or native sequence of the same polypeptide.
- the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule.
- the alterations may be produced be a variety of methods known in the art. These methods include but are not limited to oligonucleotide-mediated mutagenesis and cassette mutagenesis.
- Oligonucleotide-mediated mutagenesis is preferred method for preparing substitution, deletion, and insertion variants of gene 1. This technique is well known in the art as described by Zoller et al. Nucleic Acids Res. 10:6487-6504 1987. Briefly, the Nanobody gene is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single- stranded form of the plasmid containing the unaltered or native DNA sequence of the single variable domain, e.g. Nanobody or dAbs, gene.
- a DNA template where the template is the single- stranded form of the plasmid containing the unaltered or native DNA sequence of the single variable domain, e.g. Nanobody or dAbs, gene.
- a DNA polymerase is used to synthesize an entire second complementary strand of the template and will thus incorporate the oligonucleotide primer, and thus will code for the selected alteration in the single variable domain, e.g. Nanobody or dAbs, gene.
- oligonucleotides of at least 25 nucleotides in length are used.
- An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule.
- oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. Proc. Natl. Acad. Sci. USA, 75:5765 1978.
- the oligonucleotide is hybridized to the single stranded template under suitable hybridization conditions.
- a DNA polymerizing enzyme usually the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis.
- a heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of the single variable domain, e.g. Nanobody or dAbs.
- This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. CoIi JMlOl . After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.
- a suitable host cell usually a prokaryote such as E. CoIi JMlOl .
- After growing the cells they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.
- the method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s).
- the modifications are as follows:
- the single-stranded oligonucleotide is annealed to the single-stranded template as described above.
- a mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothyrmidine (dTTP) is combined with a modified thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham).
- dCTP-(aS) which can be obtained from Amersham
- this new strand of DNA will contain dCTP-(aS) instead of dCTP. which serves to protect it from restriction endonuclease digestion.
- the template strand of the double- stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave a molecule that is only partially single-stranded.
- a complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable hose cell such as E. coli JMlOi, as described above.
- Mutants with more than one amino acid to be substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If, however, the amino acids are located some distance from each other (separated by more than about ten amino acids), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed.
- This method is also a preferred method for preparing substitution, deletion, and insertion variants of single variable domain, e.g. Nanobody or dAbs, gene.
- the method is based on that described by Wells et al. Gene, 34:335 1985.
- the starting material is the plasmid (or other vector) comprising the single variable domain, e.g. Nanobody or dAbs, gene, the gene to be mutated.
- the codon(s) in the single variable domain, e.g. Nanobody or dAbs, gene to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s).
- restriction sites may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in single variable domain, e.g. Nanobody or dAbs, gene.
- the restriction sites After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it.
- a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette.
- This cassette is designed to have 3' and 5' ends that are compatible with the ends of the linearized plasmid. such that it can be directly ligated to the plasmid.
- This plasmid now contains the mutated DNA sequence of single variable domain, e.g. Nanobody or dAbs, gene.
- Target Molecules such as described above, may be isolated from natural sources or prepared by recombinant methods by procedures known in the art.
- glycoprotein hormone receptors may be prepared by the technique described by McFarland et al, Science 245:494-499 1989. nonglycosylated forms expressed in E. coli are described by Fuh et al. J. Biol. Chem 265:31 1 1-3115, 1990.
- Other receptors can be prepared by standard methods.
- the purified Target Molecules may be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44. 1976. or by other means known in the art.
- the immobilized target is contacted with the library of phagemid/phage particles under conditions suitable for binding of at least a portion of the phagemid particles with the immobilized target.
- the conditions including pH, ionic strength, temperature and the like will mimic physiological conditions.
- Binders Bound phagemid or phage particles having high affinity for the immobilized Target Molecules are separated from those having a low affinity (and thus do not bind to the target) by washing. Binders may be dissociated from the immobilized Target Molecules by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
- Suitable host cells are infected with the binders and helper phage, and the host cells are cultured under conditions suitable for amplification of the phagemid particles.
- the phagemid particles are then collected and the selection process is repeated one or more times until binders having the desired affinity for the target molecule are selected.
- the library of phagemid particles may be sequentially contacted with more than one immobilized Target Molecule to improve selectivity for a particular Target Molecule. For example, it is often the case that a Target Molecule has more than one natural receptor.
- GDF-8 used as the Target Molecule, both GDF-8 and GDF-11 share common amino acid sequence. It may be desirable to improve the selectivity of single variable domain, e.g.
- Nanobody or dAbs binders for e.g. GDF-8 over GDF-11. This can be achieved by first contacting the library of phagemid particles with immobilized GDF-11, eluting those with a low affinity for the GDF-11 and then contacting the low affinity GDF-11 "binders" or non-binders with the immobilized GDF-8. and selecting for high affinity GDF-8 binders. This same strategy may be e.g. employed to improve selectivity of a particular hormone or protein for its primary function receptor over its clearance receptor.
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Abstract
La présente invention concerne des procédés de création de domaines variables simples de grande affinité, des bibliothèques ou des répertoires comprenant lesdits domaines variables simples sur des phages à un format monovalent, et des plasmides, des phagemides ou des phages adaptés pour exprimer lesdits domaines variables simples dans lesdites bibliothèques.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012066058A1 (fr) | 2010-11-16 | 2012-05-24 | Boehringer Ingelheim International Gmbh | Agents et méthodes de traitement de maladies qui sont corrélées à une expression de bcma |
WO2012103797A1 (fr) | 2011-01-31 | 2012-08-09 | 艾比玛特生物医药(上海)有限公司 | Procédé de préparation d'anticorps, anticorps et bibliothèque d'anticorps ainsi préparés |
WO2015200626A1 (fr) * | 2014-06-25 | 2015-12-30 | The Rockefeller University | Compositions et procédés pour la production rapide de répertoires polyvalents de nanocorps |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992009690A2 (fr) * | 1990-12-03 | 1992-06-11 | Genentech, Inc. | Methode d'enrichissement pour des variantes de l'hormone de croissance avec des proprietes de liaison modifiees |
WO2001021817A1 (fr) * | 1999-09-24 | 2001-03-29 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Phages recombinants capables de penetrer dans des cellules hotes via une interaction specifique avec un recepteur artificiel |
EP1134231A1 (fr) * | 2000-03-14 | 2001-09-19 | Unilever N.V. | Domaines variables de la chaine lourde d'anticorps contre des enzymes humaines alimentaires et leurs utilisations |
WO2001090190A2 (fr) * | 2000-05-26 | 2001-11-29 | National Research Council Of Canada | Fragments d'anticorps de fixation d'antigenes monodomaines, derives d'anticorps de lamas |
WO2002051870A2 (fr) * | 2000-12-22 | 2002-07-04 | GRAD, Carole Legal Representative of KAPLAN, Howard | Bibliotheques d'affichage de phages de fragments vh humains |
WO2002057445A1 (fr) * | 2000-05-26 | 2002-07-25 | National Research Council Of Canada | Anticorps cibles sur le cerveau a domaine unique, derives d'anticorps de lama |
EP1452599A1 (fr) * | 1991-03-01 | 2004-09-01 | Dyax Corp. | Phage amélioré pour la visualisation d'un déterminant antigènique |
GB2428293A (en) * | 2005-07-13 | 2007-01-24 | Domantis Ltd | Phage display libraries |
EP1803814A1 (fr) * | 2005-12-27 | 2007-07-04 | SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. | Procédé d'amélioration la capacité de sélection d'anticorps dans une bibliothèque d'affichage de phages |
WO2007104529A2 (fr) * | 2006-03-13 | 2007-09-20 | Ablynx N.V. | Séquences d'acides aminés dirigées contre il-6 et polypeptides incluant lesdites séquences dans le traitement de maladies et de troubles associés au signalement faisant intervenir il-6 |
-
2009
- 2009-03-03 WO PCT/EP2009/052499 patent/WO2009109572A2/fr active Application Filing
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992009690A2 (fr) * | 1990-12-03 | 1992-06-11 | Genentech, Inc. | Methode d'enrichissement pour des variantes de l'hormone de croissance avec des proprietes de liaison modifiees |
EP1452599A1 (fr) * | 1991-03-01 | 2004-09-01 | Dyax Corp. | Phage amélioré pour la visualisation d'un déterminant antigènique |
WO2001021817A1 (fr) * | 1999-09-24 | 2001-03-29 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Phages recombinants capables de penetrer dans des cellules hotes via une interaction specifique avec un recepteur artificiel |
EP1134231A1 (fr) * | 2000-03-14 | 2001-09-19 | Unilever N.V. | Domaines variables de la chaine lourde d'anticorps contre des enzymes humaines alimentaires et leurs utilisations |
WO2001090190A2 (fr) * | 2000-05-26 | 2001-11-29 | National Research Council Of Canada | Fragments d'anticorps de fixation d'antigenes monodomaines, derives d'anticorps de lamas |
WO2002057445A1 (fr) * | 2000-05-26 | 2002-07-25 | National Research Council Of Canada | Anticorps cibles sur le cerveau a domaine unique, derives d'anticorps de lama |
WO2002051870A2 (fr) * | 2000-12-22 | 2002-07-04 | GRAD, Carole Legal Representative of KAPLAN, Howard | Bibliotheques d'affichage de phages de fragments vh humains |
GB2428293A (en) * | 2005-07-13 | 2007-01-24 | Domantis Ltd | Phage display libraries |
EP1803814A1 (fr) * | 2005-12-27 | 2007-07-04 | SIGMA-TAU Industrie Farmaceutiche Riunite S.p.A. | Procédé d'amélioration la capacité de sélection d'anticorps dans une bibliothèque d'affichage de phages |
WO2007104529A2 (fr) * | 2006-03-13 | 2007-09-20 | Ablynx N.V. | Séquences d'acides aminés dirigées contre il-6 et polypeptides incluant lesdites séquences dans le traitement de maladies et de troubles associés au signalement faisant intervenir il-6 |
Non-Patent Citations (8)
Title |
---|
BASS S ET AL: "HORMONE PHAGE: AN ENRICHMENT METHOD FOR VARIANT PROTEINS WITH ALTERED BINDING PROPERTIES" PROTEINS: STRUCTURE, FUNCTION AND GENETICS, JOHN WILEY & SONS, INC, US, vol. 8, no. 4, 1 January 1990 (1990-01-01), pages 309-314, XP000938404 ISSN: 0887-3585 * |
HOLT L J ET AL: "Domain antibodies: proteins for therapy" TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 21, no. 11, 1 November 2003 (2003-11-01), pages 484-490, XP004467495 ISSN: 0167-7799 * |
LOWMAN H B ET AL: "SELECTING HIGH-AFFINITY BINDING PROTEINS BY MONOVALENT PHAGE DISPLAY" BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, EASTON, PA.; US, vol. 30, no. 45, 12 November 1991 (1991-11-12), pages 10832-10838, XP002042460 ISSN: 0006-2960 * |
MATTHEWS DAVID J ET AL: "Substrate phage: Selection of protease substrates by monovalent phage display" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, WASHINGTON, DC, vol. 260, no. 5111, 21 May 1993 (1993-05-21), pages 1113-1117, XP002145212 ISSN: 0036-8075 * |
O'CONNELL DAVID ET AL: "PHAGE VERSUS PHAGEMID LIBRARIES FOR GENERATION OF HUMAN MONOCLONAL ANTIBODIES" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 321, no. 1, 2 August 2002 (2002-08-02), pages 49-56, XP009081320 ISSN: 0022-2836 * |
PAVONI ET AL: "New display vector reduces biological bias for expression of antibodies in E. coli" GENE, ELSEVIER, AMSTERDAM, NL, vol. 391, no. 1-2, 8 March 2007 (2007-03-08), pages 120-129, XP005917921 ISSN: 0378-1119 * |
SCOTT J K ET AL: "Phage display: A laboratory manual, PHAGE-DISPLAY VECTORS" PHAGE DISPLAY. A LABORATORY MANUAL, XX, XX, 1 January 2001 (2001-01-01), pages 2-1, XP002378612 * |
VAN KONINGSBRUGGEN S ET AL: "Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy." JOURNAL OF IMMUNOLOGICAL METHODS, vol. 279, no. 1-2, August 2003 (2003-08), pages 149-161, XP002530784 ISSN: 0022-1759 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012066058A1 (fr) | 2010-11-16 | 2012-05-24 | Boehringer Ingelheim International Gmbh | Agents et méthodes de traitement de maladies qui sont corrélées à une expression de bcma |
EP3974453A2 (fr) | 2010-11-16 | 2022-03-30 | Amgen Inc. | Agents et procédés pour traiter les maladies en corrélation avec l'expression bcma |
WO2012103797A1 (fr) | 2011-01-31 | 2012-08-09 | 艾比玛特生物医药(上海)有限公司 | Procédé de préparation d'anticorps, anticorps et bibliothèque d'anticorps ainsi préparés |
WO2015200626A1 (fr) * | 2014-06-25 | 2015-12-30 | The Rockefeller University | Compositions et procédés pour la production rapide de répertoires polyvalents de nanocorps |
US20170212130A1 (en) * | 2014-06-25 | 2017-07-27 | The Rockefeller University | Compositions and methods for rapid production of versatile nanobody repertoires |
EP3399052A3 (fr) * | 2014-06-25 | 2018-12-26 | The Rockefeller University | Compositions et procédés pour la production rapide de répertoires polyvalents de nanocorps |
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WO2009109572A3 (fr) | 2009-11-12 |
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