Patent Application
20140213459
The present invention relates to methods for improving the folding stability of antibodies, to antibodies with improved folding stability, nucleic acid and vectors encoding such antibodies, and to uses of such antibodies, nucleic acid and vectors.
This invention relates to a novel approach for the stabilization of antibodies.
The biophysical stability of monoclonal antibodies is an important determinant of their usefulness and commercial value for several reasons (reviewed, among others, by Garber and Demarest, 2007, and by Honegger, 2008). First, high biophysical stability can result in high antibody expression yield in recombinant systems. High expression yield can facilitate antibody selection and screening, for example by enhancing the display level of antibodies on bacteriophages and by enhancing the soluble yield of antibodies in small-scale E. coli cultures, and therefore lead to the discovery of better antibody molecules. High expression yield can also be important in making the manufacturing of commercially available monoclonal antibodies economically viable by allowing a sufficiently small production scale suitable for the application. In monoclonal antibodies intended for therapeutic applications, high biophysical stability can be important as it can be associated with high solubility, therefore enabling antibodies to be efficiently formulated at high concentrations into drugs. Also in therapeutic monoclonal antibodies, high biophysical stability can be important for avoiding antibody aggregation during various manufacturing steps (including expression, purification, acid-mediated virus-deactivation and formulation) and during storage. The avoidance of aggregation is not only important for maximizing the economic viability of an antibody drug production process but is also thought to play an important role in minimizing the potential immunogenicity of antibody drugs in patients. Finally, also in therapeutic monoclonal antibodies, high biophysical stability is important in achieving a long antibody half-life both in patients and in disease models.
In recognition of the importance of high biophysical stability of monoclonal antibodies, researchers have aimed to improve the biophysical stability of monoclonal antibodies for the past several years. Research efforts have aimed to stabilize antibody constant domains, isolated antibody variable domains (especially autonomous VH domains known as VHH domains, VH domain antibodies, nanobodies or monobodies; but also autonomous VL domains), as well as heterodimeric antibodies comprising one VH and one VL domain. The read-out employed in such stabilization work has included increased expression yield, increased solubility of the expressed antibodies, increased levels of display in phage display libraries, increased resistance to denaturant-induced unfolding and increased resistance to heat-induced unfolding (known as thermal stability). In order to obtain antibody variable regions with improved biophysical stability, a variety of approaches has been taken that can be categorized as follows.
Researchers have employed specific antibody selection conditions, such as phage display with heat- or denaturant-induced stress during panning, to select antibody clones with superior biophysical properties, including reduced aggregation (Jung et al., 1999; Jespers et al., 2004(A); Dudgeon et al., 2008).
Researchers have employed specific antibody screening conditions, such as E. coli expression in the presence of reducing agents or fluorescent antigens able to permeate into the periplasm, or heating of secreted antibody clones in microtiter plates during antigen-specific ELISA screening, to select antibody clones with superior biophysical properties, including faster folding and greater thermal stability (Ribnicky et al., 2007; Martineau and Betton, 1999; Demarest et al., 2006).
Heterodimeric VH-VL antibody fragments have been stabilized by the addition of various entities such as chemical cross-linkers, peptide linkers to create single-chain Fv and single-chain Fab fragments, interchain disulphide bonds to create disulphide-stabilized Fv fragments, and heterodimeric coiled coils to create helix-stabilized Fv fragments (reviewed by Arndt et al., 2001).
Researchers have mutated framework residues at the VH-VL interface of heterodimeric VH-VL antibody fragments to obtain antibodies with greater resistance to denaturant-induced unfolding (Tan et al., 1998).
Heterodimeric VH-VL antibody fragments have been stabilized by increasing the hydrophilicity of solvent-exposed framework region residues. In some cases this has been done by disrupting hydrophobic patches at the antibody variable/constant domain interface (Nieba et al., 1997).
Autonomous VH domains have been stabilized by mutating positions otherwise contributing to the light chain interface, thereby improving solubility of this region that is buried in heterodimeric VH-VL antibodies (Davies and Riechmann, 1994; Riechmann, 1996; Bathelemy et al., 2007).
Autonomous VH domains have been stabilized by engineering additional intradomain disulphide bonds within the framework region (Davies and Riechmann, 1996) or between CDRs (Tanha et al., 2001).
In autonomous human and camelid VH domains, the CDR3 has been engineered to compensate for the hydrophobicity of the former light chain interface and to obtain better solubility of these domains (Tanha et al., 2001; Jespers et al., 2004(B); Dottorini et al., 2004).
In autonomous VL domains, a position in CDR1 (residue 32) and two positions in CDR2 (residues 50 and 56) have been engineered to increase the stability of the isolated VL domains towards denaturant-induced unfolding and to improve the feasibility of their potential use as disulphide-free intrabodies (Steipe, 1994; Ohage et al., 1997; Proba et al., 1998; Ohage and Steipe, 1999); all numbering according to Kabat (Kabat and Wu, 1991).
Germline genes from which VH or VL antibody domains are derived have been identified and analysed, and their sequences compiled into databases (Lefranc et al., 1999; Retter et al., 2005), and family-specific key residues have been identified that are critical for the family-specific folding and side-chain-packing within the VH or within the VL domain (Ewert et al., 2003(A)). Then, by aligning germline genes, protein consensus sequences have been generated for variable domains that contain more of the family-specific key residues than variable domains derived from individual germline genes and as a result have potentially improved biophysical properties over variable domains derived from individual germline genes (Steipe et al., 1994; reviewed by Wörn and Plückthun, 2001). Resulting human variable domain consensus sequences have been used in the humanization of animal-derived monoclonal antibodies (for example, Carter et al., 1992) and in the construction of synthetic human antibody libraries (for example, Knappik et al., 2000).
Based on consensus sequences, human VH and VL germline families have been characterized, families with inferior or superior biophysical properties have been identified, and individual framework region residues responsible for the inferior or superior properties have been pin-pointed (for example, Ewert et al., 2003(A)). This has allowed researchers to generate antibodies with improved biophysical properties in several ways:
Human antibody clones of known specificity have been stabilized by human-to-human CDR grafting: Antigen-specific CDR loops and selected putative specificity-enhancing framework region residues from a donor clone derived from a human germline gene associated with inferior biophysical properties were transplanted onto a human acceptor framework associated with superior biophysical properties (Jung and Plückthun, 1997).
Human antibody clones of known specificity have been stabilized by framework-engineering: A set of framework region residues thought to be responsible for inferior properties of one germline family has been exchanged for a set of different framework region residues found in a germline family associated with superior properties, thereby improving the biophysical properties of the antibody clone while retaining most of the original framework region sequences and while retaining the specificity (Ewert et al., 2003(B)).
Based on the ranking of the biophysical properties of human germline family consensus genes in the context of VH-VL pairings, researchers have suggested that synthetic antibody libraries should be prepared in which only those germline families with a consensus that had shown superior biophysical properties in the VH-VL pairings (VH1, VH3 and VH5 as well as Vkappa1, Vkappa3 and Vlambda) should be represented (Ewert et al., 2003(A)).
Researchers have generated synthetic antibody libraries in which all germline families were represented, but all clones derived from a VH germline family associated with inferior biophysical properties (VH4) contained a point-mutation in the framework region designed to improve the biophysical properties of these clones (Rothe et al., 2008).
Researchers have generated synthetic libraries of VH-VL heterodimeric antibodies based on a single synthetic VH framework and a single synthetic VL framework (for example, Lee et al., 2004; Fellouse et al., 2007) or on a single synthetic VH framework and multiple synthetic VL frameworks (for example, Silacci et al., 2005) known for their favourable biophysical framework properties.
Efforts have been made to obtain naturally occurring and therefore potentially stable CDR conformations in synthetic libraries of single domain antibodies and VH-VL heterodimeric antibodies, in order to give the antibodies nature-like and good, albeit not especially improved, biophysical properties. To this end, some CDR positions that are known to be determinants of specific canonical CDR structures (Chothia et al., 1992; Tomlinson et al., 1995; Al-Lazikani et al., 1997) have been left undiversified or subjected to restricted diversification in many published synthetic antibody libraries, maintaining them as the dominant residue or residues most frequently found in the germline family context of the particular VH or VL domain on which the library is based. Among such positions that bear canonical-structure-determining CDR residues and that have been left undiversified or subjected to restricted diversification in published antibody libraries are positions 27, 29 and 34 in HCDR1, positions 52a, 54 and 55 in HCDR2, and positions 94 and 101 in HCDR3, as well as positions 90 and 95 in the LCDR3 of Vkappa domains (all numbering according to Kabat (Kabat and Wu, 1991)).
Also in efforts to obtain natural and potentially stable CDR conformations in synthetic libraries of single domain antibodies and VH-VL heterodimeric antibodies, other CDR residues buried within the VH domain or within the VL domain, which are naturally conserved independently of different specific canonical CDR structures, have been left undiversified or subjected to restricted diversification in some synthetic antibody libraries, maintaining them as the dominant amino acid most frequently found in nature (for example, VH position 51 in HCDR2 has been kept undiversified in several published synthetic human antibody libraries, bearing an invariant Ile).
In contrast to the attention the naturally conserved residues described above have received in synthetic antibody library designs, very little work has been done with VH-VL heterodimeric antibodies on engineering the many, widely divergent CDR residues, which are not buried within the VH domain or within the VL domain and are not determinants of any specific canonical structure, towards superior biophysical properties of the final heterodimeric antibody. Instead, in published library designs, these residues have usually been diversified with the aim of maximizing antigen binding. This has typically been done either by complete or near-complete diversification to all naturally occurring amino acids, or by diversification regimens that aimed to reflect a natural distribution of amino acids in a particular CDR position, or by restricted diversification that maximized representation of amino acids known to be statistically important to antigen binding whilst minimizing library complexity. Examples of diversification regimens employed by previous researchers for these positions include the following:
Except for a few exceptions (see below), very little work has been done with VH-VL heterodimeric antibodies in relation to engineering the many, widely divergent CDR residues, which are not buried within the VH domain or within the VL domain and are not determinants of any specific canonical structure, towards superior biophysical properties of the final heterodimeric antibody.
Although not in the context of biophysical stability, Ueda et al. (1995) have observed that residue 95 in CDR3 of human VH domains, which is neither canonical-structure-determining nor necessarily buried within the VH domain, has an impact on the affinity of the heavy chain towards light chains in the context of VH-VL pairings. The investigators speculated that this residue may play a role in determining the shape of the VH CDR3 loop and observed that VH domains with the flexible residue Gly in position 95 appeared to exhibit the highest average affinity for light chains.
In work aimed at selecting stabilized antibodies by phage display, Jung et al. (1999) selected a mutant of the single chain Fv fragment 4D5Flu, which carried the two point mutations His to Asn in position 27d of LCDR1 and Phe to Val in position 55 of LCDR2. The investigators found that this mutant was more highly expressed and its thermal stability in DSC measurements was increased from 62.3° C. to 66.2° C. in PBS. The investigators suggested that single mutants should be analyzed to delineate the effects of the two mutations.
In work aimed at stabilizing a human tetanus toxoid-specific Fab fragment, Demarest et al. (2006) have observed that mutating residue 50 in LCDR1 of a human Vkappa domain (which is neither canonical-structure-determining nor usually buried within the Vkappa domain) from the wild-type residue Trp to smaller residues His and Ala significantly increased the biophysical stability of a heterodimeric VH-VL antibody against tetanus toxoid. The investigators speculated that the large native residue Trp likely causes a steric clash with HCDR3 residues of the antibody clone under investigation.
However, despite the fact that many attempts have been made to provide stable antibody frameworks and/or to stabilize existing antibodies, so far these attempts have had limited success.
Thus, there was still a large unmet need to provide novel methods for the stabilization of antibodies and novel stable antibody frameworks for the generation of antibody libraries or for CDR-grafting and/or humanization approaches.
The solution for this problem that has been provided by the present invention, i.e. the modification of particular residues in the CDR regions and/or conserved framework residues, has so far not been achieved or suggested by the prior art.
The present invention relates to a method for improving the folding stability of antibodies and to antibodies with improved folding stability.
In a first aspect, the present invention relates to a method for modifying a parental antibody variable domain comprising a variable heavy (VH) chain domain and a variable light (VL) chain domain, comprising the steps of:
In a second aspect, the present invention relates to a method for modifying a parental antibody variable domain, comprising the step of:
In a third aspect, the present invention relates to an antibody variable domain comprising at least one VL or VH domain selected from the group of:
In a fourth aspect, the present invention relates to a method for modifying an antibody variable domain, comprising the step of:
In a fifth aspect, the present invention relates to the use of an antibody variable domain according to the present invention, or an antibody variable domain modified according to the present invention, in the construction of a diverse collection of antibody variable domains, comprising the step of:
In a sixth aspect, the present invention relates to a method for construction of a diverse collection of antibody variable domains, comprising the step of (a) diversifying one or more amino acid positions in one or more CDR regions of an antibody variable domain according to claim 1, or an antibody variable domain modified according to the method of claim 2, provided that
In a seventh aspect, the present invention relates to a diverse collection of antibody variable domains, wherein said collection comprises one or more diverse collections of amino acid residues at one or more positions in one or more CDR regions, provided that
In an eighth aspect, the present invention relates to an antibody that has a melting temperature of significantly above 92° C. when analyzed by differential scanning calorimetry in pure 1× phosphate buffered saline pH 7.4, (containing 1.06 mM KH2PO4, 2.97 mM Na2HPO4×7H2O, 155.17 mM NaCl and no other supplements) using a scan-rate of 60° C. per hour, no gain and a scan range of 32° C. to 115° C.
In a ninth aspect, the present invention relates to nucleic acid sequence encoding the antibody or functional fragment thereof according to the present invention.
In a tenth aspect, the present invention relates to a vector comprising the nucleic acid sequence according to the present invention.
In an eleventh aspect, the present invention relates to a host cell comprising the nucleic acid sequence according to the present invention, or the vector according to the present invention.
In a twelfth aspect, the present invention relates to a method for generating the antibody or functional fragment thereof according to the present invention, comprising the step of expressing the nucleic acid sequence according to the present invention, or the vector according to the present invention, either in vitro of from an appropriate host cell, including the host cell according to the present invention.
In a thirteenth aspect, the present invention relates to pharmaceutical compositions comprising an antibody molecule or functional fragment thereof, and optionally a pharmaceutically acceptable carrier and/or excipient. The compositions may be formulated e.g. for once-a-day administration, twice-a-day administration, or three times a day administration.
The peculiarity of this invention compared to former approaches for stabilizing antibodies is the so far unknown effect of modifications to CDR residues and to highly conserved residues in the framework regions, which results in antibodies with unprecedented stabilities.
In a first aspect, the present invention relates to a method for modifying a parental antibody variable domain comprising a variable heavy (VH) chain domain and a variable light (VL) chain domain, comprising the steps of
As used herein, the term “antibody” refers to an immunoglobulin (Ig) molecule that is defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), which includes all conventionally known antibodies and functional fragments thereof. A “functional fragment” of an antibody/immunoglobulin molecule hereby is defined as a fragment of an antibody/immunoglobulin molecule (e.g., a variable region of an IgG) that retains the antigen-binding region. An “antigen-binding region” of an antibody typically is found in one or more hypervariable region(s) (or complementarity-determining region, “CDR”) of an antibody molecule, i.e. the CDR-1, -2, and/or -3 regions; however, the variable “framework” regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs. Preferably, the “antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 113 of VH; numbering according to WO 97/08320). A preferred class of antibody molecules for use in the present invention is IgG.
“Functional fragments” of the invention include the domain of a F(ab′)2 fragment, a Fab fragment, scFv or constructs comprising single immunoglobulin variable domains or single domain antibody polypeptides, e.g. single heavy chain variable domains or single light chain variable domains. The F(ab′)2 or Fab may be engineered to minimize or completely remove the intermolecular disulphide interactions that occur between the CH1 and CL domains.
An antibody may be derived from immunizing an animal, or from a recombinant antibody library, including an antibody library that is based on amino acid sequences that have been designed in silico and encoded by nucleic acids that are synthetically created. In silico design of an antibody sequence is achieved, for example, by analyzing a database of human sequences and devising a polypeptide sequence utilizing the data obtained therefrom. Methods for designing and obtaining in silico-created sequences are described, for example, in Knappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol. Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issued to Knappik et al.
As used herein, a binding molecule is “specific to/for”, “specifically recognizes”, or “specifically binds to” a target, such as a target biomolecule (or an epitope of such biomolecule), when such binding molecule is able to discriminate between such target biomolecule and one or more reference molecule(s), since binding specificity is not an absolute, but a relative property. In its most general form (and when no defined reference is mentioned), “specific binding” is referring to the ability of the binding molecule to discriminate between the target biomolecule of interest and an unrelated biomolecule, as determined, for example, in accordance with a specificity assay methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA tests and peptide scans. For example, a standard ELISA assay can be carried out. The scoring may be carried out by standard colour development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogenperoxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (=negative reaction) may be about 0.1 OD; typical positive reaction may be about 1 OD. This means the ratio between a positive and a negative score can be 10-fold or higher. Typically, determination of binding specificity is performed by using not a single reference biomolecule, but a set of about three to five unrelated biomolecules, such as milk powder, BSA, transferrin or the like.
In the context of the present invention, the term “about” or “approximately” means between 90% and 110% of a given value or range.
However, “specific binding” also may refer to the ability of a binding molecule to discriminate between the target biomolecule and one or more closely related biomolecule(s), which are used as reference points. Additionally, “specific binding” may relate to the ability of a binding molecule to discriminate between different parts of its target antigen, e.g. different domains, regions or epitopes of the target biomolecule, or between one or more key amino acid residues or stretches of amino acid residues of the target biomolecule.
In certain embodiments, the antibody or functional fragment of the present invention is selected from a single chain Fv fragment, a Fab fragment and an IgG.
Functional fragments according to the present invention may be Fv (Skerra, A. & Plückthün (1988). Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041), scFv (Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S. & Whitlow, M. (1988). Single-chain antigen-binding proteins. Science 242, 423-426.; Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R. & Oppermann, H. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879-5883.), disulfide-linked Fv (Glockshuber, R., Malia, M., Pfitzinger, I. & Plückthun, A. (1992). A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362-1367.; Brinkmann, U., Reiter, Y., Jung, S., Lee, B. & Pastan, I. (1993). A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542.), Fab, (Fab′) 2 fragments, single VH domains or other fragments well-known to the practitioner skilled in the art, which comprise at least one variable domain of an immunoglobulin or immunoglobulin fragment and have the ability to bind to a target.
In particular embodiments, steps (c) and optionally (d) are performed by modifying one or more nucleic acid sequences encoding the parental antibody variable domain.
In particular embodiments, the method of the present invention comprises the additional step of:
In particular embodiments, the method comprises the additional steps of:
In particular embodiments, the method comprises the additional step of:
In particular embodiments, the invention relates to a method, wherein in step (c) or (d) at least one amino acid residue is changed from an amino acid being the consensus amino acid for that position in the family of antibody sequences the parental antibody variable domain belongs to a non-consensus amino acid.
In a second aspect, the present invention relates to a method for modifying a parental antibody variable domain, comprising the step of:
In a further aspect, the present invention relates to a method for modifying a parental antibody variable domain, comprising the step of:
In the context of the present invention, the reference “H(103 minus 5)” refers to the fifth amino acid residue before the conserved residue H103:W. Such a nomenclature is necessary, since CDR3 of VH is of considerable length variability, so that the number, including any letter following a number (see
In a further aspect, the present invention relates to a method for modifying a parental antibody variable domain, comprising the steps presented in sections [0061] and [0062]:
In the context of the present invention, the terms “Vlambda1”, and “VH3” refer to the subclasses of human antibody variable light (VL) and heavy (VH) chain domains as defined in WO 97/08320 (VH1a, VH1b, VH2, VH3, VH4, VH5, and VH6; Vkappa1, Vkappa2, Vkappa3 and Vkappa4; Vlambda1, Vlambda2 and Vlambda3). In this context, the term “subclass” refers to a group of variable domains sharing a high degree of identity and similarity, which can be represented by a consensus sequence for a given subclass. In the context of the present invention, the term “consensus sequence” refers to the HuCAL consensus genes as defined in WO 97/08320. The determination whether a given VL or VH domain belongs to a given VL or VH subclass is made by alignment of the respective variable domain with all known human germline segments (VBASE, Cook, G. P. & Tomlinson, I. M. (1995). The human immunoglobulin V-H repertoire. Immunology Today 16, 237-242) and determination of the highest degree of homology using a homology search matrix such as BLOSUM (Henikoff, S. & Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 89, 10915-10919). Methods for determining homologies and grouping of sequences according to homologies are well known to one of ordinary skill in the art. The grouping of the individual germline sequences into subclasses is done according to WO 97/08320.
In particular embodiments, said parental antibody variable domain is modified by making or causing at least one of the changes listed in (i)(a), (ii)(a) and (iii)(a).
In particular embodiments, the invention relates to a method, wherein at least two of said changes are made or caused, particularly wherein at least three of said changes are made or caused.
In certain embodiments, no change is made or caused at position L55. In certain other embodiments, no change is made or caused at position H95.
In a third aspect, the present invention relates to an antibody variable domain comprising at least one VL or VH domain selected from the group of:
In certain embodiments, the antibody variable domain comprises a VH and/of VL domain comprising at least three amino acid changes independently selected from the groups listed in (i)(B), (ii)(B) and (iii)(B).
In a fourth aspect, the present invention relates to a method for modifying an antibody variable domain, comprising the step of:
In an additional aspect, the present invention relates to a method for modifying an antibody variable domain, comprising the step of:
In certain embodiments, no change is made or caused at position L55. In certain other embodiments, no change is made or caused at position H95.
In certain embodiments, the method comprises to make or cause at least three amino acid changes independently selected from the groups listed in (i), (ii) and (iii).
In a fifth aspect, the present invention relates to the use of an antibody variable domain according to the present invention, or an antibody variable domain modified according to the present invention, in the construction of a diverse collection of antibody variable domains, comprising the step of:
In particular embodiments, none of the following CDR positions is diversified: Vkappa1: L55, L94, L96; Vlambda1: L96; VH3: H50, H60, H63, H64, and H95.
In particular additional embodiments, the following diversification schemes are used for Vkappa libraries:
In particular additional embodiments, the following diversification schemes are used for Vlambda libraries:
In particular additional embodiments, the following diversification schemes are used for VH libraries:
In a sixth aspect, the present invention relates to a method for construction of a diverse collection of antibody variable domains, comprising the step of (a) diversifying one or more amino acid positions in one or more CDR regions of an antibody variable domain according to claim 1, or an antibody variable domain modified according to the method of claim 2, provided that
In particular embodiments, none of the following CDR positions is diversified: Vkappa1: L55, L94, L96; Vlambda1: L96; VH3: H50, H60, H63, H64, and H95.
In a seventh aspect, the present invention relates to a diverse collection of antibody variable domains, wherein said collection comprises one or more diverse collections of amino acid residues at one or more positions in one or more CDR regions, provided that
In particular embodiments, none of the following CDR positions is diversified: Vkappa1: L55, L94, L96; Vlambda1: L96; VH3: H50, H60, H63, H64, and H95.
In an eighth aspect, the present invention relates to an antibody that has a melting temperature of the Fab fragment of significantly above 92° C. when analyzed by differential scanning calorimetry in pure 1× phosphate buffered saline pH 7.4, (containing 1.06 mM KH2PO4, 2.97 mM Na2HPO4×7 H2O, 155.17 mM NaCl and no other supplements) using a scan-rate of 60° C. per hour, no gain and a scan range of 32° C. to 115° C.
In one embodiment, the melting temperature of the Fab fragment is above 100° C.
In a ninth aspect, the present invention relates to a nucleic acid sequence encoding the antibody or functional fragment thereof according to the present invention.
In a tenth aspect, the present invention relates to a vector comprising the nucleic acid sequence according to the present invention.
In an eleventh aspect, the present invention relates to a host cell comprising the nucleic acid sequence according to the present invention, or the vector according to the present invention.
In a twelfth aspect, the present invention relates to a method for generating the antibody or functional fragment thereof according to the present invention, comprising the step of expressing the nucleic acid sequence according to the present invention, or the vector according to the present invention, either in vitro or in an appropriate host cell, including the host cell according to the present invention.
In a thirteenth aspect, the present invention relates to pharmaceutical compositions comprising an antibody molecule or functional fragment thereof, and optionally a pharmaceutically acceptable carrier and/or excipient. The compositions may be formulated e.g. for once-a-day administration, twice-a-day administration, or three times a day administration.
The phrase “pharmaceutically acceptable”, as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., human). The term “pharmaceutically acceptable” may also mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
In the context of the present invention, the term “about” or “approximately” means between 90% and 110% of a given value or range.
The term “carrier” applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which an active compound (e.g., a bispecific antibody fragment) is administered. Such pharmaceutical carriers may be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by A. R. Gennaro, 20th Edition.
The active ingredient (e.g., a modified antibody fragment) or the composition of the present invention may be used for the treatment of at least one disease or disorders, wherein the treatment is adapted to or appropriately prepared for a specific administration as disclosed herein (e.g., to once-a-day, twice-a-day, or three times a day administration). For this purpose the package leaflet and/or the patient information contains corresponding information.
The active ingredient (e.g., the modified antibody molecule or fragment thereof) or the composition of the present invention may be used for the manufacture of a medicament for the treatment of at least one disease or disorder, wherein the medicament is adapted to or appropriately prepared for a specific administration as disclosed herein (e.g., to once-a-day, twice-a-day, or three times a day administration). For this purpose the package leaflet and/or the patient information contains corresponding information.
The following examples illustrate the invention without limiting its scope.
Antibody genes were designed based on the desired amino acid sequence and purchased as synthetic genes or synthetic gene fragments from GeneArt or DNA2.0. Genes encoding antibody variants with point mutations were generated by PCR or overlap PCR, using the polymerase Pwo Master, purchased from Roche, and synthetic oligonucleotides encoding the desired point mutations, purchased from Thermo Fisher Scientific, according to manufacturer's instructions. An E. coli Fab expression vector was generated by modification of the plasmid pUC19, which was purchased from New England Biolabs. The pUC19 backbone was modified by the addition of two synthetic ribosome binding sites driving expression of antibody heavy and light chains, two synthetic signal peptide sequences driving the secretion of antibody chains into the E. coli periplasm and one M13 phage origin potentially enabling single strand production. Synthetic antibody genes, synthetic fragments of antibody genes and PCR-generated variants of antibody genes encoding point mutations were cloned into this E. coli Fab expression vector by restriction digestion, using restriction endonucleases purchased from Roche, followed by ligation, using LigaFast purchased from Promega, according to manufacturer's instructions. Ligation reactions were transformed into competent TG1 E. coli cells purchased from Stratagene or Zymoresearch.
TG1 E. coli clones bearing Fab expression constructs were grown in LB and TB solid and liquid media, purchased from Carl Roth, which were supplemented with Carbenicillin and glucose, purchased from VWR. Antibody expression in liquid cultures was performed overnight in Erlenmeyer flasks in a shaking incubator and was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG), purchased from Carl Roth, to the growth medium. Culture supernatants containing secreted Fab fragments were clarified by centrifugation of the expression cultures. Clarified culture supernatants were supplemented with a 1% volume of Streptomycin/Penicillin solution, purchased from PAA Laboratories, a 2% volume of 1M Tris pH8.0, purchased from VWR, and a 0.4% volume of STREAMLINE rProtein A resin, purchased from GE Healthcare. The supplemented culture supernatants were incubated on a rolling incubator for 3 hours or overnight to achieve binding of Fab fragments to the protein A resin. Resins were then transferred into gravity flow columns, washed once using 30 bedvolumes of 2×PBS pH 7.4, purchased from Invitrogen, washed once using 5 bedvolumes of a buffer containing 10 mM Tris pH 6.8 and 100 mM NaCl, purchased from VWR, and eluted using a buffer containing 10 mM citric acid pH3 and 100 mM NaCl, purchased from VWR. Eluted Fab fragments were neutralized by adding an 8% volume of 1M Tris pH 8.0. Neutralized purified Fab fragments were buffer exchanged into pure 1×PBS pH 7.4 (containing 1.06 mM KH2PO4, 2.97 mM Na2HPO4×7H2O, 155.17 mM NaCl and no other supplements; Invitrogen catalogue No. 10010056), using illustra NAP-5 desalting columns from GE Healthcare, according to manufacturer's instructions.
The biophysical stability of purified, buffer-exchanged Fab fragments was determined in 1×PBS pH 7.4 (Invitrogen catalogue No. 10010056) using differential scanning calorimetry (DSC). For all measurements, a capillary cell microcalorimeter equipped with autosampler and controlled by VPViewer2000 CapDSC software from MicroCal was used. All Fab fragments were scanned against pure buffer containing no antibody (1×PBS pH 7.4; Invitrogen catalogue No. 10010056). The scan parameters were set to analyse a temperature window from 32° C. to between 105° C. and 115° C., with a pre-scan thermostat of 2 minutes, a post-scan thermostat of 0 minutes and no gain. The scan rate was set to 250° C. per hour for screening applications and to 60° C. per hour for re-analysis of the most stable combination mutants. The absolute melting temperature of the Fab fragments determined in screening mode (scan-rate 250° C. per hour) was 3.7° C. to 4.5° C. higher than in re-analysis mode (scan-rate 60° C. per hour), but ranking of clones was the same in both modes. Melting temperatures of Fab fragments were determined after PBS reference subtraction, using Origin 7.0 software from MicroCal.
The following Table 1 shows a compilation of experimental data obtained with mutants of Vkappa1/VH3 and Vlambda1/VH3 Fab fragments.
Combining several of the improved sequence features identified in this invention allows generation of exceptionally thermostable Fab fragments. In some instances, melting temperatures of such combination mutants can exceed 100° C., even at a slow scan rate of 1° C. per min. For example, antibody clone KRd15.6 with the VH domain amino acid sequence EAQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWIRQAPGKGLEWIGQIS GSGGSTYYNDSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARDSGYFD IWGQGTLVTVSS and the VK domain amino acid sequence AIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYAASSL YSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSLPYTFGQGTKVEIK R was analysed as follows.
The Fab fragment was expressed in E. coli, affinity-purified on protein A resin and buffer-exchanged into 1×PBS pH 7.4 (Invitrogen catalogue No. 10010056, containing 1.06 mM KH2PO4, 2.97 mM Na2HPO4×7H2O, 155.17 mM NaCl and no other supplements). For DSC measurements, a capillary cell microcalorimeter equipped with autosampler and controlled by VPViewer2000 CapDSC software from MicroCal was used. The Fab fragment was scanned against pure buffer containing no antibody (1×PBS pH 7.4; Invitrogen catalogue No. 10010056). The scan parameters were set to analyze a temperature window from 32° C. to 115° C., with a pre-scan thermostat of 2 min, a post-scan thermostat of 0 min and no gain. The scan rate was set to 60° C. per h.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
To the extent possible under the respective patent law, all patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11004371.8 | May 2011 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/002278 | 5/29/2012 | WO | 00 | 3/21/2014 |
