EP2958939A1 - Charpente d'anticorps pour greffe de cdr - Google Patents

Charpente d'anticorps pour greffe de cdr

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Publication number
EP2958939A1
EP2958939A1 EP13705169.4A EP13705169A EP2958939A1 EP 2958939 A1 EP2958939 A1 EP 2958939A1 EP 13705169 A EP13705169 A EP 13705169A EP 2958939 A1 EP2958939 A1 EP 2958939A1
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EP
European Patent Office
Prior art keywords
immunobinder
framework
antibody
heavy chain
cdr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP13705169.4A
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German (de)
English (en)
Inventor
Dominik Escher
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Esbatech a Novartis Co LLC
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Esbatech a Novartis Co LLC
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Publication of EP2958939A1 publication Critical patent/EP2958939A1/fr
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]

Definitions

  • Non-human antibodies elicit a strong immune response in patients, usually following a single low dose injection (Schroff, 1985 Cancer Res 45:879-85, Shawler. J Immunol 1985 135: 1530- 5; Dillman, Cancer Biother 1994 9: 17-28). Accordingly, several methods for reducing the immunogenicity of murine and other rodent antibodies as well as technologies to make fully human antibodies using e.g. transgenic mice or phage display were developed.
  • Chimeric antibodies were engineered, which combine rodent variable regions with human constant regions (e.g., Boulianne Nature 1984 312:643- 6) reduced immunogenicity problems considerably (e.g., LoBuglio, Proc Natl Acad Sci 1989 86:4220-4; Clark, Immunol Today 2000 21:397-402).
  • Humanized antibodies were also engineered, in which the rodent sequence of the variable region itself is engineered to be as close to a human sequence as possible while preserving at least the original CDRs, or where the CDRs from the rodent antibody were grafted into framework of a human antibody (e.g., Riechmann, Nature 1988 332:323-7; US5,693,761).
  • Rabbit polyclonal antibodies are widely used for biological assays such as ELISAs or Western blots.
  • Polyclonal rabbit antibodies are oftentimes favored over polyclonal rodent antibodies because of their usually much higher affinity.
  • rabbit oftentimes are able to elicit good antibody responses to antigens that are poorly immunogenic in mice and/or which give not rise to good binders when used in phage display. Due to these well-known advantages of rabbit antibodies, they would be ideal to be used in the discovery and development of therapeutic antibodies. The reason that this is not commonly done is mainly due to technical challenges in the generation of monoclonal rabbit antibodies.
  • Rabbit antibodies like mouse antibodies are expected to elicit strong immune responses if used for human therapy, thus, rabbit antibodies need to be humanized before they can be used clinically.
  • the methods that are used to make humanized rodent antibodies cannot easily be extrapolated for rabbit antibodies due to structural differences between rabbit and mouse and, respectively, between rabbit and human antibodies.
  • the light chain CDR3 CDRL3 is often much longer than previously known CDRL3s from human or mouse antibodies.
  • WO 04/016740 describes a so-called "resurfacing” strategy.
  • the goal of a "resurfacing” strategy is to remodel the solvent-accessible residues of the non-human framework such that they become more human-like.
  • Similar humanization techniques for rabbit antibodies as described in WO 04/016740 are known in the art.
  • Both WO08/144757 and WO05/016950 disclose methods for humanizing a rabbit monoclonal antibody which involve the comparison of amino acid sequences of a parent rabbit antibody to the amino acid sequences of a similar human antibody. Subsequently, the amino acid sequence of the parent rabbit antibody is altered such that its framework regions are more similar in sequence to the equivalent framework regions of the similar human antibody. In order to gain good binding capacities, laborious development efforts need to be made for each immunobinder individually.
  • a potential problem of the above-described approaches is that not a human framework is used, but the rabbit framework is engineered such that it looks more human-like.
  • Such approach carries the risk that amino acid stretches that are buried in the core of the protein still might comprise immunogenic T cell epitopes.
  • the grafting of non-human CDRs antibodies into a human framework is a major protein engineering task.
  • the transfer of antigen binding loops from a naturally evolved framework to a different artificially selected human framework must be performed so that native loop conformations are retained for antigen binding. Often antigen binding affinity is greatly reduced or abolished after loop grafting.
  • the use of carefully selected human frameworks in grafting the antigen binding loops maximizes the probability of retaining binding affinity in the humanized molecule (Roguzka et al 1996).
  • the many grafting experiments available in the literature provide a rough guide for CDR grafting, it is not possible to generalize a pattern. Typical problems consist in loosing the specificity, stability or producibility after grafting the CDR loops.
  • the invention provides the heavy chain variable regions of a particular human antibody (the so called, "a72" VH framework sequence) which is especially suitable as acceptor for CDRs from a variety of antibodies, in particular from rabbit antibodies, of different binding specificities, independent of whether a disulfide bridge is present in a CDR or not.
  • Humanized immunobinders generated by the grafting of rabbit CDRs into this highly compatible variable chain framework consistently and reliably retain the spatial orientation of the rabbit antibodies from which the donor CDRs are derived. Therefore, no structurally relevant positions of the donor immunobinder need to be introduced into the acceptor framework. Due to these advantages, high-throughput humanization of rabbit antibodies with no or little optimization of the binding capacities can be achieved.
  • the invention provides methods for grafting rabbit and other non-human CDRs, into the soluble and stable light chain and/or heavy chain human antibody framework sequences disclosed herein, thereby generating humanized antibodies with superior biophysical properties.
  • immunobinders generated by the methods of the invention exhibit superior functional properties such as solubility and stability.
  • Figure 1 depicts the CDR HI definition used herein for grafting antigen binding sites from rabbit monoclonal antibodies into the highly soluble and stable human antibody frameworks.
  • Figure 2 An analysis of rabbit antibody sequences extracted from the Kabat database confirms that CDR3 of the variable heavy chain is typically by three amino acids longer than its murine counterpart.
  • antibody refers to whole antibodies and any antigen binding fragment.
  • antigen binding polypeptide and “immunobinder” are used simultaneously herein.
  • An “antibody” refers to a protein, optionally glycosylated, comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system ⁇ e.g. , effector cells) and the first component (Clq) of the classical complement system.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen ⁇ e.g. , TNF). It has been shown that the antigen -binding function of an antibody can be performed by fragments of a full-length antibody.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and CHI domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V H domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker.
  • CDR complementarity determining region
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotype, for example, an IgG ⁇ e.g., an IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.
  • immunobinder refers to a molecule that contains all or a part of the antigen binding site of an antibody, e.g. all or part of the heavy and/or light chain variable domain, such that the immunobinder specifically recognizes a target antigen.
  • Non-limiting examples of immunobinders include full-length immunoglobulin molecules and scFvs, as well as antibody fragments, including but not limited to (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C H I domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3.sup.rd ed.
  • single chain antibody refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker.
  • Such scFv molecules can have the general structures: NH 2 -VL-linker-VH-COOH or NH 2 - VH-linker-VL-COOH.
  • a suitable state of the art linker consists of repeated GGGGS amino acid sequences or variants thereof.
  • a (GGGGS)4 linker of the amino acid sequence set forth in SEQ ID NO: 8 is used, but variants of 1-3 repeats are also possible (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers that can be used for the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • the term "functional property" is a property of a polypeptide (e.g., an immunobinder) for which an improvement (e.g., relative to a conventional polypeptide) is desirable and/or advantageous to one of skill in the art, e.g., in order to improve the manufacturing properties or therapeutic efficacy of the polypeptide.
  • the functional property is stability (e.g., thermal stability).
  • the functional property is solubility (e.g., under cellular conditions).
  • the functional property is aggregation behavior.
  • the functional property is protein expression (e.g., in a prokaryotic cell).
  • the functional property is refolding behavior following inclusion body solubilization in a manufacturing process.
  • the functional property is not an improvement in antigen binding affinity.
  • the improvement of one or more functional properties has no substantial effect on the binding affinity of the immunobinder.
  • CDR refers to one of the six hypervariable regions within the variable domains of an antibody that mainly contribute to antigen binding.
  • One of the most commonly used definitions for the six CDRs was provided by Kabat E.A. et ah, (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • CDR HI or HI the residue positions (Kabat numbering) starting with position 26 and ending prior to position 36. This definition is basically a fusion of CDR HI as differently defined by Kabat and Chotia (see also Figure 1 for illustration).
  • antibody framework refers to the part of the variable domain, either VL or VH, which serves as a scaffold for the antigen binding loops (CDRs) of this variable domain. In essence it is the variable domain without the CDRs.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the TNF molecule).
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
  • binding refers to antibody binding to an epitope on a predetermined antigen.
  • the antibody binds with an affinity (K D ) of approximately less than 10 - " 7 M, such as approximately less than 10 - " 8 M, 10 - " 9 M or 10 - " 10 M or even lower.
  • K D or "Kd” refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the antibodies of the invention bind to TNF with a dissociation equilibrium constant (K D ) of less than approximately 10 - " 7 M, such as less than approximately 10 - " 8 M, 10 - “ 9 M or 10 - “ 10 M or even lower, for example, as determined using surface plasmon resonance (SPR) technology in a BIACORE instrument.
  • SPR surface plasmon resonance
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single- stranded or double- stranded, but preferably is double- stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • the vectors disclosed herein can be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells include bacterial, microbial, plant or animal cells, preferably, Escherichia coli, Bacillus subtilis; Saccharomyces cerevisiae, Pichia pastoris, CHO (Chinese Hamster Ovary lines) or NSO cells.
  • lagomorphs refers to members of the taxonomic order Lagomorpha, comprising the families Leporidae (e.g. hares and rabbits), and the Ochotonidae (pikas). In a most preferred embodiment, the lagomorphs is a rabbit.
  • the term "rabbit” as used herein refers to an animal belonging to the family of the leporidae.
  • identity refers to the sequence matching between two polypeptides, molecules or between two nucleic acids. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (for instance, if a position in each of two polypeptides is occupied by a lysine), then the respective molecules are identical at that position.
  • the "percentage identity” between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Generally, a comparison is made when two sequences are aligned to give maximum identity.
  • Such alignment can be provided using, for instance, the method of the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • AHo numbering scheme The AHo numbering system is described further in Honegger, A. and Pluckthun, A. (2001) J. Mol. Biol. 309:657-670).
  • the Kabat numbering system as described further in Kabat et al. (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) may be used. Conversion tables for the two different numbering systems used to identify amino acid residue positions in antibody heavy and light chain variable regions are provided in A. Honegger, J.Mol.Biol. 309 (2001) 657-670.
  • the present invention provides a human acceptor framework sequence for the grafting of CDRs from lagomorph species, for example, from rabbit.
  • the human single-chain VH framework a72 (SEQ ID NO: 1) was surprisingly found to be in essence highly compatible with the antigen-binding sites of rabbit antibodies. Therefore, the a72 VH represents a suitable scaffold to construct stable humanized scFv antibody fragments derived from grafting of rabbit loops.
  • the invention provides an immunobinder acceptor framework, comprising a VH sequence having at least 70 % identity to SEQ ID No. 1.
  • Said sequence may be combined with any other suitable variable light chain.
  • a preferred variable light chain is SEQ ID NO: 2 which was also disclosed in WO03/097697 and designated KI27, or any other VL sequence as disclosed in WO03/097697.
  • variable heavy chain framework is linked to a variable light chain framework via a linker.
  • the linker may be any suitable linker, for example a linker comprising 1 to 4 repeats of the sequence GGGGS (SEQ ID NO: 5), preferably a (GGGGS) 4 peptide (SEQ ID NO: 4), or a linker as disclosed in Alfthan et al. (1995) Protein Eng. 8:725-731.
  • an immunobinder acceptor framework comprising
  • variable heavy chain framework having at least 70% identity, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity, to SEQ ID NO: 1;
  • variable light chain framework having at least 70% identity, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity, to SEQ ID NO: 2.
  • the invention provides an immunobinder, having a sequence with at least 60%, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, identity to SEQ ID NO: 3.
  • the framework is compatible with virtually any rabbit CDRs. Containing different rabbit CDRs, it is well expressed and good produced contrary to the rabbit wild type single chains and still almost fully retains the affinity of the original donor rabbit antibodies.
  • the immunobinder acceptor frameworks as described herein may comprise solubility enhancing substitution in the heavy chain framework, preferably at positions 12, 103 and 144 (AHo numbering).
  • a hydrophobic amino acid is substituted by a more hydrophilic amino acid.
  • Hydrophilic amino acids are e.g. Arginine (R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glycine (G), Histidine (H), Lysine (K), Serine (S) and Threonine (T).
  • the heavy chain framework comprises (a) Serine (S) at position 12; (b) Serine (S) or Threonine (T) at position 103 and/or (c) Serine (S) or Threonine (T) at position 144.
  • stability enhancing amino acids may be present at one or more positions 1, 3, 4, 10, 47, 57, 91 and 103 of the variable light chain framework (AHo numbering).
  • variable light chain framework comprises glutamic acid (E) at position 1, valine (V) at position 3, leucine (L) at position 4, Serine (S) at position 10; Arginine (R) at position 47, Serine (S) at position 57, phenylalanine (F) at position 91 and/or Valine (V) at position 103.
  • the VH comprises at position 141 a glycine (G). This substitution may improve long- term storage of the protein.
  • the acceptor frameworks disclosed herein can be used to generate a human or humanized antibody which retains the binding properties of the non-human antibody from which the non-human CDRs are derived.
  • the invention encompasses an immunobinder acceptor framework as disclosed herein, further comprising heavy chain CDRl, CDR2 and CDR3 and/or light chain CDRl, CDR2 and CDR3 from a donor immunobinder, preferably from a mammalian immunobinder, more preferably from a lagomorph immunobinder and most preferably from a rabbit.
  • the invention provides an immunobinder specific to a desired antigen comprising
  • variable light chain CDRs of a lagomorph (i) variable light chain CDRs of a lagomorph
  • a human variable heavy chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, and most preferably 100% identity to SEQ ID NO: 1.
  • the lagomorph is a rabbit.
  • the immunobinder comprises heavy chain CDRl, CDR2 and CDR3 and light chain CDRl, CDR2 and CDR3 from the donor immunobinder.
  • the grafting of the non-human CDRs onto the frameworks disclosed herein yields a molecule wherein the CDRs are displayed in a proper conformation.
  • the affinity of the immunobinder may be improved by grafting antigen interacting framework residues of the non-human donor immunobinder. These positions may e.g. be identified by
  • step (ii) generating a sequence alignment of donor variable domain sequences with germ line progenitor sequence or consensus sequence of step (i);
  • the present invention provides an immunobinder which comprises the immunobinder acceptor framework described herein.
  • Said immunobinder may e.g. be a scFv antibody, a full-length immunoglobulin, a Fab fragment, a Dab or a Nanobody.
  • the immunobinder is attached to one or more molecules, for example a therapeutic agent such as a cytotoxic agent, a cytokine, a chemokine, a growth factor or other signaling molecule, an imaging agent or a second protein such as a transcriptional activator or a DNA-binding domain.
  • a therapeutic agent such as a cytotoxic agent, a cytokine, a chemokine, a growth factor or other signaling molecule
  • an imaging agent such as a transcriptional activator or a DNA-binding domain.
  • the immunobinder as disclosed herein may e.g. be used in diagnostic applications, therapeutic application, target validation or gene therapy.
  • the invention further provides an isolated nucleic acid encoding the immunobinder acceptor framework disclosed herein or the immunobinder(s) as disclosed herein.
  • a vector which comprises the nucleic acid disclosed herein.
  • nucleic acid or the vector as disclosed herein can e.g. be used in gene therapy.
  • the invention further encompasses a host cell comprising the vector and/or the nucleic acid disclosed herein.
  • a composition comprising the immunobinder acceptor framework as disclosed herein, the immunobinder as disclosed herein, the isolated nucleic acid as disclosed herein or the vector as disclosed herein.
  • sequences disclosed herein are the following (X residues are CDR insertion sites and contain at least 3 and up to 50 amino acids):
  • SEQ ID NO. 2 variable light chain framework KI27
  • the invention provides methods for the humanization of non-human antibodies by grafting CDRs of non-human donor antibodies onto stable and soluble antibody frameworks.
  • the CDRs stem from rabbit antibodies and the frameworks are those described above.
  • a general method for grafting CDRs into human acceptor frameworks has been disclosed by Winter in US Patent No. 5,225,539 and by Queen et al. in WO9007861A1, which are hereby incorporated by reference in their entirety.
  • the general strategy for grafting CDRs from rabbit monoclonal antibodies onto selected frameworks is related to that of Winter et al. and Queen et al., but diverges in certain key respects.
  • the methods of the invention diverge from the typical Winter and Queen methodology known in the art in that the human antibody frameworks as disclosed herein are particularly suitable as acceptors for human or non-human donor antibodies.
  • the framework sequence used for the humanization methods of the invention is not necessarily the framework sequence which exhibits the greatest sequence similarity to the sequence of the non-human (e.g., rabbit) antibody from which the donor CDRs are derived.
  • framework residue grafting from the donor sequence to support CDR conformation is not required. At most, antigen binding amino acids located in the framework or other mutations that occurred during somatic hypermutation may be introduced.
  • the amino acid sequence of the CDR donor antibody is first identified and the sequences aligned using conventional sequence alignment tools (e.g., Needleman-Wunsch algorithm and Blossum matrices).
  • sequence alignment tools e.g., Needleman-Wunsch algorithm and Blossum matrices.
  • the introduction of gaps and nomenclature of residue positions may be done using a conventional antibody numbering system.
  • the AHo numbering system for immunoglobulin variable domains may be used.
  • the Kabat numbering scheme may also be applied since it is the most widely adopted standard for numbering the residues in an antibody. Kabat numbering may e.g. be assigned assigned using the SUBIM program. This program analyses variable regions of an antibody sequence and numbers the sequence according to the system established by Kabat and co-workers (Deret et al 1995).
  • framework and CDR regions are generally done following the Kabat definition which is based on sequence variability and is the most commonly used.
  • the designation is preferably a combination of the definitions of Rabat's, mean contact data generated by analysis of contacts between antibody and antigen of a subset of 3D complex structures (MacCallum et al., 1996) and Chotia's which is based on the location of the structural loop regions (see also Fig. 1).
  • Conversion tables for the two different numbering systems used to identify amino acid residue positions in antibody heavy and light chain variable regions are provided in A. Honegger, J.Mol.Biol. 309 (2001) 657-670.
  • the Kabat numbering system is described further in Kabat et al.
  • variable domains of the rabbit monoclonal antibodies may e.g. be classified into corresponding human sub-groups using e.g. an EXCEL implementation of sequence analysis algorithms and classification methods based on analysis of the human antibody repertoire (Knappik et al., 2000, J Mol Biol. Feb 1 l;296(l):57-86).
  • CDR conformations may be assigned to the donor antigen binding regions, subsequently residue positions required to maintain the different canonical structures can also be identified.
  • the CDR canonical structures for five of the six antibody hypervariable regions of rabbit antibodies (LI, L2, L3, HI and H2) are determined using Chothia's (1989) definition.
  • the antibodies of the invention may be further optimized to show enhanced functional properties, e.g., enhanced solubility and/or stability.
  • the antibodies of the invention are optimized according to the "functional consensus" methodology disclosed in PCT Application Serial No. PCT/EP2008/001958, entitled “Sequence Based Engineering and Optimization of Single Chain Antibodies", filed on March 12, 2008, which is incorporated herein by reference.
  • Exemplary framework residue positions for substitution and exemplary framework substitutions are described in PCT Application No. PCT/ CH2008/000285, entitled “Methods of Modifying Antibodies, and Modified Antibodies with Improved Functional Properties", filed on June 25, 2008, and PCT Application No. PCT/ CH2008/000284, entitled “Sequence Based Engineering and Optimization of Single Chain Antibodies", filed on June 25, 2008.
  • the immunobinders of the invention comprise one or more of the stability enhancing mutations described in U.S. Provisional Application Serial No. 61/075,692, entitled “Solubility Optimization of Immunobinders", filed on June 25, 2008.
  • the immunobinder comprises a solubility enhancing mutation at an amino acid position selected from the group of heavy chain amino acid positions consisting of 12, 103 and 144 (AHo Numbering convention).
  • the immunobinder comprises one or more substitutions selected from the group consisting of: (a) Serine (S) at heavy chain amino acid position 12; (b) Serine (S) or Threonine (T) at heavy chain amino acid position 103; and (c) Serine (S) or Threonine (T) at heavy chain amino acid position 144.
  • the immunobinder comprises the following substitutions: (a) Serine (S) at heavy chain amino acid position 12; (b) Serine (S) or Threonine (T) at heavy chain amino acid position 103; and (c) Serine (S) or Threonine (T) at heavy chain amino acid position 144.
  • the immunobinder comprises stability enhancing mutations at a framework residue of the light chain acceptor framework in at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the light chain variable region according to the AHo numbering system.
  • the light chain acceptor framework comprises one or more substitutions selected from the group consisting of (a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c) leucine (L) at position 4; (d) Serine (S) at position 10; (e) Arginine (R) at position 47; (e) Serine (S) at position 57; (f) phenylalanine (F) at position 91 ; and (g) Valine (V) at position 103.
  • the present invention provides an immunobinder humanized according to the method described herein.
  • the target antigen of said immunobinder is VEGF or TNFa.
  • polypeptides described in the present invention or generated by a method of the present invention can, for example, be synthesized using techniques known in the art.
  • nucleic acid molecules encoding the desired variable regions can be synthesized and the polypeptides produced by recombinant methods.
  • variable region or a polypeptide comprising it can be made by techniques well known in the art of molecular biology. More specifically, recombinant DNA techniques can be used to produce a wide range of polypeptides by transforming a host cell with a nucleic acid sequence (e.g. , a DNA sequence that encodes the desired variable region (e.g. , a modified heavy or light chain; the variable domains thereof, or other antigen-binding fragments thereof)).
  • a nucleic acid sequence e.g. , a DNA sequence that encodes the desired variable region (e.g. , a modified heavy or light chain; the variable domains thereof, or other antigen-binding fragments thereof)
  • a cell line e.g. , an immortalized mammalian cell line
  • variable regions comprising donor CDRs and acceptor FR amino acid sequences can be made and then changes introduced into the nucleic acid molecules to effect the CDR amino acid substitution.
  • Exemplary art recognized methods for making a nucleic acid molecule encoding an amino acid sequence variant of a polypeptide include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide.
  • Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in carrying out site-directed mutagenesis of DNA, the parent DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such parent DNA.
  • a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the parent DNA as a template.
  • the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.
  • PCR mutagenesis is also suitable for making amino acid sequence variants of polypeptides. See Higuchi, in PCR Protocols, pp.177- 183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • the starting material is the plasmid (or other vector) comprising the DNA to be mutated.
  • the codon(s) in the parent DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the DNA encoding the polypeptide.
  • the plasmid DNA 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, wherein the two strands of the oligonucleotide 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 5' and 3' 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.
  • variable region generated by the methods of the invention can be remodeled and further altered to further increase antigen binding.
  • steps described above can be preceded or followed by additional steps, including, e.g. affinity maturation.
  • empirical binding data can be used for further optimization.
  • the present invention contemplates other modifications, e.g., to Fc region amino acid sequences in order to generate an Fc region variant with altered effector function.
  • One may, for example, delete one or more amino acid residues of the Fc region in order to reduce or enhance binding to an FcR.
  • one or more of the Fc region residues can be modified in order to generate such an Fc region variant.
  • no more than one to about ten Fc region residues will be deleted according to this embodiment of the invention.
  • the Fc region herein comprising one or more amino acid deletions will preferably retain at least about 80%, and preferably at least about 90%, and most preferably at least about 95%, of the starting Fc region or of a native sequence human Fc region.
  • the polypeptides described in the present invention or generated by a method of the present invention may be produced by recombinant methods.
  • a polynucleotide sequence encoding a polypeptide can be inserted in a suitable expression vector for recombinant expression.
  • polypeptide is an antibody
  • polynucleotides encoding additional light and heavy chain variable regions, optionally linked to constant regions may be inserted into the same or different expression vector.
  • An affinity tag sequence e.g. a His (6) tag
  • the DNA segments encoding immunoglobulin chains are the operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.
  • Expression control sequences include, but are not limited to, promoters (e.g., naturally- associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the polypeptide.
  • expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin- resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Patent No. 4,704,362).
  • E. coli is one prokaryotic host particularly useful for cloning the polynucleotides (e.g., DNA sequences) of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • yeast Other microbes, such as yeast, are also useful for expression.
  • Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.
  • E. coli and S. cerevisiae are preferred host cells.
  • mammalian tissue culture may also be used to express and produce the polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, and include CHO cell lines, various Cos cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B- cells, and hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148: 1149 (1992).
  • the vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989).
  • transgenic animals can be microinjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes.
  • the subject polypeptide can also be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression, e.g., in the milk of a transgenic animal (see, e.g., Deboer et al. 5,741,957; Rosen 5,304,489; and Meade 5,849,992.
  • Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.
  • Polypeptides can be expressed using a single vector or two vectors.
  • antibody heavy and light chains may be cloned on separate expression vectors and co-transfected into cells.
  • signal sequences may be used to facilitate expression of polypeptides of the invention.
  • the polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns (e.g., protein A or protein G), column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer- Verlag, N.Y., (1982)).
  • Either the humanized Ig variable regions or polypeptides comprising them can be expressed by host cells or cell lines in culture. They can also be expressed in cells in vivo.
  • the cell line that is transformed (e.g. , transfected) to produce the altered antibody can be an immortalized mammalian cell line, such as those of lymphoid origin (e.g. , a myeloma, hybridoma, trioma or quadroma cell line).
  • the cell line can also include normal lymphoid cells, such as B-cells, that have been immortalized by transformation with a virus (e.g. , the Epstein-Barr virus).
  • the cell line used to produce the polypeptide is a mammalian cell line
  • cell lines from other sources such as bacteria and yeast
  • E. co/i-derived bacterial strains can be used, especially, e.g. , phage display.
  • Some immortalized lymphoid cell lines such as myeloma cell lines, in their normal state, secrete isolated Ig light or heavy chains. If such a cell line is transformed with a vector that expresses an altered antibody, prepared during the process of the invention, it will not be necessary to carry out the remaining steps of the process, provided that the normally secreted chain is complementary to the variable domain of the Ig chain encoded by the vector prepared earlier.
  • the immortalized cell line does not secrete or does not secrete a complementary chain, it will be necessary to introduce into the cells a vector that encodes the appropriate complementary chain or fragment thereof.
  • the transformed cell line may be produced for example by transforming a suitable bacterial cell with the vector and then fusing the bacterial cell with the immortalized cell line (e.g. , by spheroplast fusion).
  • the DNA may be directly introduced into the immortalized cell line by electroporation.
  • a humanized Ig variable region as described in the present invention or generated by a method of the present invention can be present in an antigen-binding fragment of any antibody.
  • the fragments can be recombinantly produced and engineered, synthesized, or produced by digesting an antibody with a proteolytic enzyme.
  • the fragment can be a Fab fragment; digestion with papain breaks the antibody at the region, before the inter-chain (i.e., V H -V H ) disulphide bond, that joins the two heavy chains. This results in the formation of two identical fragments that contain the light chain and the V H and C H I domains of the heavy chain.
  • the fragment can be an F(ab') 2 fragment.
  • Single-chain Fv (scFv) fragments can be constructed in a variety of ways. For example, the C-terminus of V H can be linked to the N-terminus of V L - Typically, a linker (e.g., (GGGGS) 4 ; SEQ ID NO: 4) is placed between V H and V L .
  • a linker e.g., (GGGGS) 4 ; SEQ ID NO: 4
  • tags that facilitate detection or purification e.g., Myc-, His-, or FLAG-tags
  • tags such as these can be appended to any antibody or antibody fragment of the invention; their use is not restricted to scFv.
  • tagged antibodies are within the scope of the present invention.
  • the antibodies described herein, or generated by the methods described herein can be heavy chain dimers or light chain dimers.
  • an antibody light or heavy chain, or portions thereof, for example, a single domain antibody (DAb) can be used.
  • a humanized Ig variable region as described in the present invention or generated by a method of the present invention is present in a single chain antibody (ScFv) or a minibody (see e.g., US Pat No. 5,837,821 or WO 94/09817A1).
  • Minibodies are dimeric molecules made up of two polypeptide chains each comprising an ScFv molecule (a single polypeptide comprising one or more antigen binding sites, e.g., a V L domain linked by a flexible linker to a V H domain fused to a CH3 domain via a connecting peptide).
  • ScFv molecules can be constructed in a V H -linker-V L orientation or V L -linker-V H orientation.
  • the flexible hinge that links the V L and V H domains that make up the antigen binding site preferably comprises from about 10 to about 50 amino acid residues.
  • An exemplary connecting peptide for this purpose is (Gly4Ser)3 (Huston et al . (1988). PNAS, 85:5879). Other connecting peptides are known in the art.
  • a minibody of the invention comprises a connecting peptide.
  • the connecting peptide comprises a Gly/Ser linker, e.g., GGGSSGGGSGG (SEQ ID NO: 6).
  • a tetravalent minibody in another embodiment, can be constructed.
  • Tetravalent minibodies can be constructed in the same manner as minibodies, except that two ScFv molecules are linked using a flexible linker, e.g., having an amino acid sequence (G 4 S) 4 G 3 AS (SEQ ID NO: 7).
  • a humanized variable region as described in the present invention or generated by a method of the present invention can be present in a diabody.
  • Diabodies are similar to scFv molecules, but usually have a short (less than 10 and preferably 1-5) amino acid residue linker connecting both variable domains, such that the V L and V H domains on the same polypeptide chain can not interact. Instead, the V L and V H domain of one polypeptide chain interact with the V H and V L domain (respectively) on a second polypeptide chain (WO 02/02781).
  • a humanized variable region of the invention can be present in an immunoreactive fragment or portion of an antibody ⁇ e.g. an scFv molecule, a minibody, a tetravalent minibody, or a diabody) operably linked to an FcR binding portion.
  • an antibody e.g. an scFv molecule, a minibody, a tetravalent minibody, or a diabody
  • the FcR binding portion is a complete Fc region.
  • the humanization methods described herein result in Ig variable regions in which the affinity for antigen is not substantially changed compared to the donor antibody.
  • polypeptides comprising the variable domains of the instant invention bind to antigens with a binding affinity greater than (or equal to) an association constant Ka of about 10 5 M _1 , 10 6 M _1 , 10 7 M _1 , 10 8 M _1 , 10 9 M _1 , 10 10 M _1 , 10 11 M "1 , or 10 12 M _1 , (including affinities intermediate of these values).
  • Affinity, avidity, and/or specificity can be measured in a variety of ways.
  • the methods of the invention improve antibody affinity when they generate an antibody that is superior in any aspect of its clinical application to the antibody (or antibodies) from which it was made (for example, the methods of the invention are considered effective or successful when a modified antibody can be administered at a lower dose or less frequently or by a more convenient route of administration than an antibody (or antibodies) from which it was made).
  • the affinity between an antibody and an antigen to which it binds can be measured by various assays, including, e.g. , an ELISA assay, a BiaCore assay or the KinExATM 3000 assay (available from Sapidyne Instruments (Boise, ID)).
  • an ELISA assay e.g. , a BiaCore assay or the KinExATM 3000 assay (available from Sapidyne Instruments (Boise, ID)).
  • sepharose beads are coated with antigen (the antigen used in the methods of the invention can be any antigen of interest (e.g. , a cancer antigen; a cell surface protein or secreted protein; an antigen of a pathogen (e.g.
  • a bacterial or viral antigen e.g., an HIV antigen, an influenza antigen, or a hepatitis antigen
  • an allergen by covalent attachment.
  • Dilutions of antibody to be tested are prepared and each dilution is added to the designated wells on a plate.
  • a detection antibody e.g. goat anti- human IgG -HRP conjugate
  • a chromagenic substrate e.g. HRP
  • determining affinity is not always as simple as looking at a single figure. Since antibodies have two arms, their apparent affinity is usually much higher than the intrinsic affinity between the variable region and the antigen (this is believed to be due to avidity). Intrinsic affinity can be measured using scFv or Fab fragments.
  • the present invention features a humanized rabbit antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g. , an immunosuppressant) or a radiotoxin.
  • a therapeutic moiety such as a cytotoxin, a drug (e.g. , an immunosuppressant) or a radiotoxin.
  • conjugates are referred to herein as "immunoconjugates”.
  • the antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon- ⁇ ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-l”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-l interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • the invention provides pharmaceutical formulations comprising humanized rabbit antibodies for the treatment disease.
  • pharmaceutical formulation refers to preparations which are in such form as to permit the biological activity of the antibody or antibody derivative to be unequivocally effective, and which contain no additional components which are toxic to the subjects to which the formulation would be administered.
  • “Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

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Abstract

La présente invention concerne une charpente d'anticorps accepteur et des procédés permettant la greffe d'anticorps non humains, par ex. des anticorps de lapin, à l'aide d'une charpente d'anticorps accepteur. Les anticorps produits par les procédés de l'invention sont utiles dans une variété d'applications diagnostiques et thérapeutiques.
EP13705169.4A 2013-02-20 2013-02-20 Charpente d'anticorps pour greffe de cdr Withdrawn EP2958939A1 (fr)

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Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4704362A (en) 1977-11-08 1987-11-03 Genentech, Inc. Recombinant cloning vehicle microbial polypeptide expression
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
EP0832981A1 (fr) 1987-02-17 1998-04-01 Pharming B.V. Séquences d'ADN pour diriger des protéines vers les glandes mammaires, afin d'être secrétées efficacement
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
IL162181A (en) 1988-12-28 2006-04-10 Pdl Biopharma Inc A method of producing humanized immunoglubulin, and polynucleotides encoding the same
US5633076A (en) 1989-12-01 1997-05-27 Pharming Bv Method of producing a transgenic bovine or transgenic bovine embryo
US5837821A (en) 1992-11-04 1998-11-17 City Of Hope Antibody construct
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US6905668B1 (en) 1998-09-25 2005-06-14 Tokyo Gas Company Limited Diagnostic agents for pancreatic exocrine function
US7258986B2 (en) 1999-12-28 2007-08-21 Esbatech Ag Intrabodies with defined framework that is stable in a reducing environment and applications thereof
CA2410551A1 (fr) 2000-06-30 2002-01-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw (Vib) Proteines de fusion heterodimeres
CN105175535A (zh) * 2002-05-22 2015-12-23 艾斯巴技术-诺华有限责任公司 在胞内环境中表现出增强的稳定性的免疫球蛋白构架及其鉴定方法
AU2003264009A1 (en) 2002-08-15 2004-03-03 Epitomics, Inc. Humanized rabbit antibodies
AU2003259718A1 (en) 2003-08-07 2005-03-07 Epitomics, Inc. Methods for humanizing rabbit monoclonal antibodies
US7429487B2 (en) 2005-07-05 2008-09-30 Epitomics, Inc. Fusion partner for production of monoclonal rabbit antibodies
WO2008144753A2 (fr) * 2007-05-21 2008-11-27 Alder Biopharmaceuticals, Inc. Anticorps anti-tnf alpha et leur utilisation
EP2162469A4 (fr) 2007-05-21 2012-08-01 Alderbio Holdings Llc Nouveaux procedes d'humanisation d'anticorps de lapin et anticorps de lapin humanises
CN104004094B (zh) * 2008-06-25 2017-09-22 艾斯巴技术-诺华有限责任公司 使用通用抗体构架进行的兔抗体的人源化
LT3241843T (lt) * 2008-06-25 2021-09-27 Novartis Ag Imuninių rišiklių tirpumo optimizavimas
US8399624B1 (en) * 2009-06-25 2013-03-19 Esbatech, An Alcon Biomedical Research Unit Llc Acceptor framework for CDR grafting

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2014127811A1 *

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