US20120237507A1 - Monovalent Antigen Binding Proteins - Google Patents
Monovalent Antigen Binding Proteins Download PDFInfo
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- US20120237507A1 US20120237507A1 US13/406,503 US201213406503A US2012237507A1 US 20120237507 A1 US20120237507 A1 US 20120237507A1 US 201213406503 A US201213406503 A US 201213406503A US 2012237507 A1 US2012237507 A1 US 2012237507A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/66—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a swap of domains, e.g. CH3-CH2, VH-CL or VL-CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
Definitions
- sequence listing comprising SEQ ID NOS: 1-12 is attached hereto. Each sequence provided in the sequence listing is incorporated herein by reference, in its entirety, for all purposes.
- the present invention relates to monovalent antigen binding proteins with a CH1-CL domain exchange, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
- c-Met monovalent antibodies have different properties such as lack of agonistic function or reduced receptor internalization upon antibody binding than their corresponding bivalent forms and therefore represent attractive formats for therapeutic use.
- E.g. WO 2005/063816 refers to monovalent antibody fragments as therapeutics.
- US 2004/0033561 describes a method for the generation of monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain; however, a disadvantage of this method is the formation of a binding inactive homodimer of VL-CL-CH2-CH3 chains as depicted in FIG. 2 . Due the similar molecular weight such homodimeric by-products are the difficult to separate.
- WO 2007/048037 also refers to monovalent antibodies based on the co-expression of a VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain, but having a tagging moiety attached to the heavy chain for easier purification of the heterodimer from the difficult-to-separate homodimeric by-product.
- WO 2009/089004 describes another possibility to generate a heterodimeric monovalent antibody using electrostatic steering effects.
- WO 2010/145792 relates tetravalent bispecific antibodies, wherein mismatched byproducts of similar weight are reduced resulting in higher yields of the desiered bispecific antibody.
- the invention comprises a monovalent antigen binding protein comprising
- the monovalent antigen binding protein according to the invention is characterized in that is of human IgG isotype.
- the monovalent antigen binding protein according to the invention is characterized in that the modified heavy chains of a) and b) are of IgG1 isotype, and the antigen binding protein is afucosylated with an the amount of fucose of 80% or less (preferably of 65% to 5%) of the total amount of oligosaccharides (sugars) at Asn297.
- the invention further comprises a method for the preparation of a monovalent antigen binding protein according to the invention
- the invention further comprises nucleic acid encoding the monovalent antigen binding protein according to the invention.
- the invention further comprises vectors comprising nucleic acid encoding the monovalent antigen binding protein according to the invention.
- the invention further comprises host cell comprising said vectors.
- the invention further comprises composition, preferably a pharmaceutical or a diagnostic composition of a monovalent antigen binding protein according to the invention.
- the invention further comprises pharmaceutical composition comprising a monovalent antigen binding protein according to the invention and at least one pharmaceutically acceptable excipient.
- the invention further comprises method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of a monovalent antigen binding protein according to the invention.
- the antigen binding proteins according to the invention are based on the principle that a VL-CH1-CH2-CH3 and VH-CL-CH2-CH3 chain only forms heterodimers and cannot form a difficult-to-separate homodimeric by-product of similar structure and molecular weight.
- the effect of this modification lays not primarily in a reduction of by-products, but in that the only by-product which is formed is changed from a homodimeric by-product of similar size into a High-Molecular weight tetramer ( FIG. 1D ). This High-Molecular weight tetramer then can be easily removed with SEC or other MW separation techniques.
- the formed dimeric byproduct ( FIG. 1D ) can be easily separated due to the different molecular weight (the molecular weight is approximately doubled) and structure. Therefore the purification without the introduction of further modifications (like e.g. genetic introductions of tags) is possible.
- the monovalent antigen binding proteins according to the invention have valuable characteristics such as biological or pharmacological activities (as e.g. ADCC, or antagonistic biological activity as well as lack of agonistic activities). They can be used e.g. for the treatment of diseases such as cancer.
- the monovalent antigen binding proteins have furthermore highly valuable pharmacokinetic properties (like e.g. halftime (term t1 ⁇ 2) or AUC).
- FIG. 1 A) Scheme of the monovalent antigen binding protein according to the invention with CH1-CL domain exchange based on VL-CH1-CH2-CH3 and VH-CL-CH2-CH3 chains (abbreviated as MoAb). B) Scheme of a MoAb according to the invention including knobs-into-holes in the CH3 domains. C) Scheme of the dimeric bivalent antigen binding protein (MoAb-Dimer that is formed as a byproduct which can be easily separated due to different structure and molecular weight).
- FIG. 2 Scheme of A) a monovalent antibody of VL-CL-CH2-CH3 and of VH-CH1-CH2-CH3 chains (described e.g in US 2004/0033561) and B) the binding inactive difficult-to-separate homodimer byproduct of VL-CL-CH2-CH3 chains (described e.g in WO 2007/048037).
- FIG. 3 Biochemical characterization of MoAb c-Met (c-Met 5D5 MoAb (“wt”)).
- A Protein A purified antigen binding protein was separated on a Superdex 200 26/60 column. Individual peaks correspond to MoAb (3), MoAb Dimer (2) and an aggregate fraction (1).
- B Peak fractions (1, 2, 3) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- FIG. 4 Biochemical characterization of monovalent MoAb IGF1R (IGF1R AK18 MoAb (“wt”)).
- A Protein A purified antigen binding protein was separated on an Superdex 200 26/60 column. Individual peaks correspond to MoAb (2) and MoAb Dimer (1).
- B Peak fractions (1, 2) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- C The molecular mass of the peaks fractions 1 and 2 was investigated by SEC-MALLS. Peak 2 was identified as monovalent antigen binding protein MoAb IGF1R.
- FIG. 5 Biochemical characterization of MoAb Her3 (Her3 205 MoAb (“wt”)).
- A Protein A purified antibody was separated on an Superdex 200 26/60 column. Individual peaks correspond to MoAb (3), MoAb Dimer (2) and an aggregate fraction (1).
- B Peak fractions (1, 2, 3) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- FIG. 6 Biochemical characterization of MoAb Her3 with KiH mutations (Her3 205 MoAb KiH).
- A Protein A purified antigen binding protein was separated on an Superdex 200 26/60 column.
- B Peak fraction was pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- FIG. 7 Biochemical characterization of MoAb IGF1R with KiH mutations (IGF1R AK18 MoAb KiH).
- A Protein A purified antibody was separated on an Superdex 200 26/60 column. Individual peaks correspond to MoAb (2) and MoAb Dimer (1).
- B Peak fractions (1, 2) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- FIG. 8 Biochemical characterization of MoAb c-Met with KiH mutations (c-Met 5D5 MoAb KiH).
- A Protein A purified antibody was separated on an Superdex 200 26/60 column.
- B Peak fraction was pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. Polyacrylamide gels were stained with Coomassie Blue dye.
- FIG. 9 c-Met receptor phosphorylation assay in A549 cells.
- A549 cells were stimulated with HGF in the absence or presence of c-Met binding antibodies or c-Met 5D5 MoAb (“wt”)).
- Total cell lysates were subjected to immunoblot analysis.
- Asterisk marks phospho-c-Met band in between two unspecific bands.
- FIG. 10 Cellular binding of MoAb c-Met (c-Met 5D5 MoAb (“wt”))) to A549 cells with flow cytrometric analysis.
- A549 cells were incubated with a dilution series of the indicated antibodies. Bound antibodies were visualized with an Fc-binding secondary fluorophor coupled antibody.
- FIG. 11 Schematic picture of the surface plasmon resonance assay applied to analyze the binding affinity of the monovalent antigen binding protein IGF1R AK18 MoAb (“wt”).
- FIG. 12 Cellular binding of MoAb IGF-1R (IGF1R AK18 MoAb (“wt”)) to A549 cells with flow cytometric analysis. A549 cells were incubated with a dilution series of the indicated antibodies. Bound antibodies were visualized with an Fc-binding secondary fluorophor coupled antibody.
- MoAb IGF-1R IGF1R AK18 MoAb (“wt”)
- FIG. 13 ADCC Assay with parent non-glycoengineered (non-ge) IGF Mab and parent glycoengineered (ge) IGF Mab and non-glycoengineered monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)).
- FIG. 14 Internalization of IGF-1R was assessed following incubation with parent IGF-1R IgG1 antibody and monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)), the data show that internalization of IGF-1R is reduced in terms of potency and absolute internalization when the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) is bound.
- FIG. 15 IGF-1 induced autophosphorylation of IGF-1R was assessed following incubation with IGF-1R IgG1 antibody and monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)), the data show that IGF-1 induced autophoshorylation of IGF-1R is reduced in terms of potency when the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) is bound.
- FIG. 16 Aggregation tendency of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)) was assessed by a DLS timecourse experiment. Over a period of five days, no measurable increase in the hydrodynamic radius (Rh) of the isolated monomer fraction (see FIG. 4 ) could be detected.
- IGF1R MoAb IGF1R AK18 MoAb
- FIG. 17 ESI-MS spectrum of the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) after deglycosylation and under non-reducing conditions.
- IGF MoAb IGF1R AK18 MoAb
- FIG. 18 ESI-MS spectrum of the IGF-1R monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)) after deglycosylation and reduction.
- IGF1R AK18 MoAb IGF1R AK18 MoAb
- the invention comprises a monovalent antigen binding protein comprising
- the CH3 domains of said monovalent antigen binding protein according to the invention can be altered by the “knobs-into-holes” (KiH) technology which is described in detail with several examples in e.g. WO 96/027011, Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; and Merchant, A. M., et al., Nat Biotechnol 16 (1998) 677-681.
- the interaction surfaces of the two CH3 domains are altered to increase the heterodimerisation of both heavy chains containing these two CH3 domains.
- Each of the two CH3 domains (of the two heavy chains) can be the “knob”, while the other is the “hole”. The effect of this modification is that the High-Molecular weight tetramer by-product, is reduced significantly.
- amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
- amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).
- both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain such that a disulfide bridge between both CH3 domains can be formed.
- C cysteine
- said monovalent antigen binding protein comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain”.
- An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M., et al., Nature Biotech 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs chain” and a E356C mutation or a S354C mutation into the CH3 domain of the “hole chain”.
- said monovalent antigen binding protein comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or said monovalent antigen binding protein comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain forming a interchain disulfide bridge) (numbering always according to EU index of Kabat).
- knobs-in-holes technologies as described by EP 1 870 459 A1, can be used alternatively or additionally.
- a preferred example for said monovalent antigen binding protein are R409D; K370E mutations in the CH3 domain of the “knobs chain” and D399K; E357K mutations in the CH3 domain of the “hole chain” (numbering always according to EU index of Kabat).
- said monovalent antigen binding protein comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain” and additionally R409D; K370E mutations in the CH3 domain of the “knobs chain” and D399K; E357K mutations in the CH3 domain of the “hole chain”.
- said monovalent antigen binding protein comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains or said monovalent antigen binding protein comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally R409D; K370E mutations in the CH3 domain of the “knobs chain” and D399K; E357K mutations in the CH3 domain of the “hole chain”.
- antibody denotes a full length antibody consisting of two antibody heavy chains and two antibody light chains (see FIG. 1 ).
- a heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody heavy chain variable domain (VH), an antibody constant heavy chain domain 1 (CH1), an antibody hinge region (HR), an antibody heavy chain constant domain 2 (CH2), and an antibody heavy chain constant domain 3 (CH3), abbreviated as VH-CH1-HR-CH2-CH3; and optionally an antibody heavy chain constant domain 4 (CH4) in case of an antibody of the subclass IgE.
- VH antibody heavy chain variable domain
- CH1 antibody constant heavy chain domain 1
- HR antibody hinge region
- CH2 antibody heavy chain constant domain 2
- CH3 antibody heavy chain constant domain 3
- the heavy chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of VH, CH1, HR, CH2 and CH3.
- the light chain of full length antibody is a polypeptide consisting in N-terminal to C-terminal direction of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL.
- the antibody light chain constant domain (CL) can be ⁇ (kappa) or ⁇ (lambda).
- the antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and the CH1 domain (i.e. between the light and heavy chain) and between the hinge regions of the full length antibody heavy chains.
- full length antibodies are natural antibodies like IgG (e.g. IgG 1 and IgG2), IgM, IgA, IgD, and IgE.)
- the antibodies according to the invention can be from a single species e.g. human, or they can be chimerized or humanized antibodies.
- the full length antibodies according to the invention comprise two antigen binding sites each formed by a pair of VH and VL, which both specifically bind to the same (first) antigen.
- the monovalent antigen binding proteins of the invention are derived by modifying: a) the first heavy chain of said antibody by replacing the VH domain by the VL domain of said antibody; and by modifying b) the second heavy chain of said antibody by replacing the CH1 domain by the CL domain of said antibody.
- the resulting monovalent antigen binding protein comprise two modified heavy chains and no light chains.
- the C-terminus of the heavy or light chain of said full length antibody denotes the last amino acid at the C-terminus of said heavy or light chain.
- binding site or “antigen-binding site” as used herein denotes the region(s) of antigen binding protein according to the invention to which a ligand (e.g the antigen or antigen fragment of it) actually binds and which is derived from antibody molecule or a fragment thereof (e.g. a Fab fragment).
- the antigen-binding site according to the invention comprise an antibody heavy chain variable domains (VH) and an antibody light chain variable domains (VL).
- the antigen-binding sites i. the pairs of VH/VL that specifically bind to the desired antigen can be derived a) from known antibodies to the antigen or b) from new antibodies or antibody fragments obtained by de novo immunization methods using inter alia either the antigen protein or nucleic acid or fragments thereof or by phage display.
- An antigen-binding site of a monovalent antigen binding protein of the invention contains six complementarity determining regions (CDRs) which contribute in varying degrees to the affinity of the binding site for antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL3). The extent of CDR and framework regions (FRs) is determined by comparison to a compiled database of amino acid sequences in which those regions have been defined according to variability among the sequences.
- Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. Bispecific antibodies are antibodies which have two different antigen-binding specificities. The monovalent antigen binding proteins according to the invention are “monospecific” and specifically bind to an epitope of the respective antigen.
- valent as used within the current application denotes the presence of a specified number of binding sites in an antibody molecule.
- a natural antibody for example has two binding sites and is bivalent.
- monovalent antigen binding protein denotes the a polypeptide containing only one antigen binding site.
- the full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes.
- Immunoglobulin classes include IgG, IgM, IgA, IgD, and IgE class (or isotypes) and, in the case of IgG and IgA, their subclasses (or subtypes).
- an full length antibody of the invention and thus a monovalent antigen binding protein of the invention has a constant domain structure of an IgG class antibody.
- monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition.
- chimeric antibody refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as “class-switched antibodies”.
- Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
- humanized antibody refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
- CDR complementarity determining regions
- a murine CDR is grafted into the framework region of a human antibody to prepare the “humanized antibody.” See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270.
- Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies.
- humanized antibodies encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
- FcR Fc receptor
- human antibody is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences.
- Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).
- Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production.
- Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.
- human antibody as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to C1q binding and/or FcR binding, e.g. by “class switching” i.e. change or mutation of Fc parts (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutation).
- recombinant human antibody is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell.
- recombinant human antibodies have variable and constant regions in a rearranged form.
- the recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation.
- the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
- variable domain (variable domain of a light chain (VL), variable region of a heavy chain (VH) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen.
- the domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three “hypervariable regions” (or complementarity determining regions, CDRs).
- the framework regions adopt a ⁇ -sheet conformation and the CDRs may form loops connecting the ⁇ -sheet structure.
- the CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site.
- the antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention.
- hypervariable region or “antigen-binding portion of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding.
- the hypervariable region comprises amino acid residues from the “complementarity determining regions” or “CDRs”.
- “Framework” or “FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding.
- CDR and FR regions are determined according to the standard definition of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
- binding refers to the binding of the monovalent antigen binding protein to an epitope of the antigen in an in vitro assay, preferably in an plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) with purified wild-type antigen.
- the affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), k D (dissociation constant), and K D (k D /ka).
- Binding or specifically binding means a binding affinity (K D ) of 10 ⁇ 8 mol/l or less, preferably 10 ⁇ 9 M to 10 ⁇ 13 mol/l.
- a monovalent antigen binding protein according to the invention is specifically binding to each antigen for which it is specific with a binding affinity (K D ) of 10 ⁇ 8 mol/l or less, preferably 10 ⁇ 9 M to 10 ⁇ 13 mol/l.
- Binding of the monovalent antigen binding protein to the Fc ⁇ RIII can be investigated by a BIAcore assay (GE-Healthcare Uppsala, Sweden).
- the affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), k D (dissociation constant), and K D (k D /ka).
- epitope includes any polypeptide determinant capable of specific binding to a monovalent antigen binding proteins.
- epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics.
- An epitope is a region of an antigen that is bound by a monovalent antigen binding protein.
- an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
- the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of human IgG1 subclass, or of human IgG1 subclass with the mutations L234A and L235A.
- the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of human IgG2 subclass.
- the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of human IgG3 subclass.
- the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of human IgG4 subclass or, of human IgG4 subclass with the additional mutations S228P and L235E (also named IgG4 SPLE).
- constant region denotes the sum of the domains of an antibody other than the variable region.
- the constant region is not involved directly in binding of an antigen, but exhibit various effector functions.
- antibodies are divided in the classes (also named isotypes): IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (also named isotypes), such as IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2.
- the heavy chain constant regions that correspond to the different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the light chain constant regions (CL) which can be found in all five antibody classes are called ⁇ (kappa) and ⁇ (lambda).
- constant region derived from human origin denotes a constant heavy chain region of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region.
- constant regions are well known in the state of the art and e.g. described by Kabat, E. A., (see e.g. Johnson, G. and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E. A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788).
- an antibody according to the invention has a reduced FcR binding compared to an IgG1 antibody and the full length parent antibody is in regard to FcR binding of IgG4 subclass or of IgG1 or IgG2 subclass with a mutation in 5228, L234, L235 and/or D265, and/or contains the PVA236 mutation.
- the mutations in the full length parent antibody are S228P, L234A, L235A, L235E and/or PVA236.
- the mutations in the full length parent antibody are in IgG4 S228P and L235E and in IgG1 L234A and L235A.
- ADCC antibody-dependent cell-mediated cytotoxicity
- CDC complement-dependent cytotoxicity
- C1q complement factor C1q
- binding site is known in the state of the art and described e.g. by Lukas, T. J., et al., J. Immunol. 127 (1981) 2555-2560; Bunkhouse, R. and Cobra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, D.
- Such constant region binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
- ADCC antibody-dependent cellular cytotoxicity
- an antigen binding protein according to the invention show improved ADCC properties compared to its parent full length antibody. These improved ADCC effects achieved without further modification of the Fc part like glycoengineering.
- the term “complement-dependent cytotoxicity (CDC)” denotes a process initiated by binding of complement factor C1q to the Fc part of most IgG antibody subclasses. Binding of C1q to an antibody is caused by defined protein-protein interactions at the so called binding site. Such Fc part binding sites are known in the state of the art (see above).
- Fc part binding sites are, e.g., characterized by the amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat).
- Antibodies of subclass IgG1, IgG2, and IgG3 usually show complement activation including C1q and C3 binding, whereas IgG4 does not activate the complement system and does not bind C1q and/or C3.
- IgG1 type antibodies the most commonly used therapeutic antibodies, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain.
- the two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, forming extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody dependent cellular cytotoxicity (ADCC) (Lifely, M.
- ADCC antibody dependent cellular cytotoxicity
- the monovalent antigen binding protein according to the invention is characterized in that the modified heavy chains of a) and b) are of IgG1 isotype, and the antigen binding protein is afucosylated with an the amount of fucose of 80% or less of the total amount of oligosaccharides (sugars) at Asn297.
- the antigen binding protein is afucosylated with an the amount of fucose of 65% to 5% of the total amount of oligosaccharides (sugars) at Asn297.
- afucosylated antigen binding protein refers to an antigen binding proteins of IgG1 or IgG3 isotype (preferably of IgG1 isotype) with an altered pattern of glycosylation in the Fc region at Asn297 having a reduced level of fucose residues.
- Glycosylation of human IgG1 or IgG3 occurs at Asn297 as core fucosylated bianntennary complex oligosaccharide glycosylation terminated with up to 2 Gal residues.
- CHO type glycosylation of antibody Fc parts is e.g. described by Routier, F. H., Glycoconjugate J. 14 (1997) 201-207.
- Antibodies which are recombinantely expressed in non glycomodified CHO host cells usually are fucosylated at Asn297 in an amount of at least 85%.
- an afucosylated antibody as used herein includes an antibody having no fucose in its glycosylation pattern. It is commonly known that typical glycosylated residue position in an antibody is the asparagine at position 297 according to the EU numbering system (“Asn297”).
- an afucosylated antigen binding protein means an antibody of IgG1 or IgG3 isotype (preferably of IgG1 isotype) wherein the amount of fucose is 80% or less (e.g. of 80% to 1%) of the total amount of oligosaccharides (sugars) at Asn297 (which means that at least 20% or more of the oligosaccharides of the Fc region at Asn297 are afucosylated). In one embodiment the amount of fucose is 65% or less (e.g.
- amount of fucose means the amount of said oligosaccharide (fucose) within the oligosaccharide (sugar) chain at Asn297, related to the sum of all oligosaccharides (sugars) attached to Asn 297 (e.g. complex, hybrid and high mannose structures) measured by MALDI-TOF mass spectrometry and calculated as average value (for a detailed procedure to determine the amount of fucose, see e.g. WO 2008/077546).
- the oligosaccharides of the Fc region are bisected.
- the afucosylated antibody according to the invention can be expressed in a glycomodified host cell engineered to express at least one nucleic acid encoding a polypeptide having GnTIII activity in an amount sufficient to partially fucosylate the oligosaccharides in the Fc region.
- the polypeptide having GnTIII activity is a fusion polypeptide.
- ⁇ -1,6-fucosyltransferase activity of the host cell can be decreased or eliminated according to U.S. Pat. No. 6,946,292 to generate glycomodified host cells.
- the amount of antibody fucosylation can be predetermined e.g. either by fermentation conditions (e.g.
- afucosylated antigen binding proteins and respective glycoengineering methods are described in WO 2005/044859, WO 2004/065540, WO 2007/031875, Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180, WO 99/154342, WO 2005/018572, WO 2006/116260, WO 2006/114700, WO 2005/011735, WO 2005/027966, WO 97/028267, US 2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739.
- glycoengineered antigen binding proteins according to the invention have an increased ADCC (compared to the parent antigen binding proteins).
- Other glycoengineering methods yielding afucosylated antigen binding proteins according to the invention are described e.g. in Niwa, R. et al., J. Immunol. Methods 306 (2005) 151-160; Shinkawa, T., et al., J. Biol. Chem., 278 (2003) 3466-3473; WO 03/055993 or US 2005/0249722.
- one aspect of the invention is an afucosylated antigen binding protein according to the invention which of IgG1 isotype or IgG3 isotype (preferably of IgG1 isotype) with an amount of fucose of 60% or less (e.g. of 60% to 1%) of the total amount of oligosaccharides (sugars) at Asn297, for the treatment of cancer in.
- an afucosylated anti-CD20 antibody of IgG1 or IgG3 isotype specifically binding to CD20 with an amount of fucose of 60% or less of the total amount of oligosaccharides (sugars) at Asn297, for the manufacture of a medicament for the treatment of cancer.
- the amount of fucose is between 60% and 20% of the total amount of oligosaccharides (sugars) at Asn297.
- the amount of fucose is between 60% and 40% of the total amount of oligosaccharides (sugars) at Asn297.
- the amount of fucose is between 0% of the total amount of oligosaccharides (sugars) at Asn297.
- EU index (according to Kabat) is generally used when referring to a residue or position in an immunoglobulin heavy chain constant region (e.g., the EU index is reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference).
- the sugar chains show characteristics of N-linked glycans attached to Asn297 of an antibody recombinantly expressed in a CHO cell” denotes that the sugar chain at Asn297 of the full length parent antibody according to the invention has the same structure and sugar residue sequence except for the fucose residue as those of the same antibody expressed in unmodified CHO cells, e.g. as those reported in WO 2006/103100.
- NGNA as used within this application denotes the sugar residue N-glycolylneuraminic acid.
- the antibody according to the invention is produced by recombinant means.
- one aspect of the current invention is a nucleic acid encoding the antibody according to the invention and a further aspect is a cell comprising said nucleic acid encoding an antibody according to the invention.
- Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity.
- nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods.
- the monovalent antigen binding proteins according to the invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures.
- the hybridoma cells can serve as a source of such DNA and RNA.
- the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells.
- Amino acid sequence variants (or mutants) of the monovalent antigen binding protein are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
- host cell denotes any kind of cellular system which can be engineered to generate the antibodies according to the current invention.
- HEK293 cells and CHO cells are used as host cells.
- the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
- the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included.
- NS0 cells Expression in NS0 cells is described by, e.g., Barnes, L. M., et al., Cytotechnology 32 (2000) 109-123; Barnes, L. M., et al., Biotech. Bioeng. 73 (2001) 261-270.
- Transient expression is described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9.
- Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci.
- HEK 293 A preferred transient expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199.
- control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
- Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.
- a nucleic acid is “operably linked” when it is placed in a functional relationship with another nucleic acid sequence.
- DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
- Purification of monovalent antigen binding proteins is performed in order to eliminate cellular components or other contaminants, e.g. other cellular nucleic acids or proteins (e.g. byproducts) by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art (see Ausubel, F., et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)).
- Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g.
- cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).
- An example of a purification is described in Example 1 and the corresponding FIGS. 3 to 8 .
- One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention.
- Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition.
- a further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising an antibody according to the invention.
- the present invention provides a composition, e.g. a pharmaceutical composition, containing an antibody according to the present invention, formulated together with a pharmaceutical carrier.
- One embodiment of the invention is the monovalent antigen binding protein according to the invention for the treatment of cancer.
- Another aspect of the invention is said pharmaceutical composition for the treatment of cancer.
- One embodiment of the invention is the monovalent antigen binding protein according to the invention for use in the treatment of cancer.
- Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.
- Another aspect of the invention is method of treatment of patient suffering from cancer by administering an antibody according to the invention to a patient in the need of such treatment.
- “pharmaceutical carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
- the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or infusion).
- a composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
- the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.
- Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
- Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.
- parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
- cancer refers to proliferative diseases, such as lymphomas, lymphocytic leukemias, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ure
- compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
- the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
- Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
- the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
- composition must be sterile and fluid to the extent that the composition is deliverable by syringe.
- carrier preferably is an isotonic buffered saline solution.
- Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
- isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
- the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
- the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
- transfection refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without daunting cell wall barriers are used as host cells, transfection is carried out e.g. by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 546. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride as described by Cohen, F. N, et al., PNAS. 69 (1972) 7110.
- expression refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins.
- the transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.
- a “vector” is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells.
- the term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.
- an “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide.
- An “expression system” usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
- DNA sequences were determined by double strand sequencing performed at SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).
- Desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis.
- the gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids.
- the plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy.
- the DNA sequence of subcloned gene fragments was confirmed by DNA sequencing.
- DNA sequences encoding for the two antibody chains (VH-CL-CH2-CH3 and VL-CH1-CH2-CH3) were prepared as whole fragments by gene synthesis with flanking 5′HpaI and 3′NaeI restriction sites.
- a Roche expression vector was used for the construction of all antibody chains.
- the vector is composed of the following elements:
- the immunoglobulin genes in the order of VH-CL-CH2-CH3 and VL-CH1-CH2-CH3 as well as “knobs-into-hole” constructs were prepared by gene synthesis and cloned into pGA18 (ampR) plasmids as described.
- the pG18 (ampR) plasmids carrying the synthesized DNA segments and the Roche expression vector were digested either with HpaI and NaeI or with BclI and NaeI restriction enzymes (Roche Molecular Biochemicals) and subjected to agarose gel electrophoresis.
- Recombinant immunoglobulin variants were expressed by transient transfection of human embryonic kidney 293-F cells using the FreeStyleTM 293 Expression System according to the manufacturer's instruction (Invitrogen, USA). Briefly, suspension FreeStyleTM 293-F cells were cultivated in FreeStyleTM 293 Expression medium at 37° C./8% CO 2 . Cells were seeded in fresh medium at a density of 1-2 ⁇ 10 6 viable cells/ml on the day of transfection.
- DNA-293fectinTM complexes were prepared in Opti-MEM® I medium (Invitrogen, USA) using 325 ⁇ l of 293fectinTM (Invitrogen, Germany) and 250 ⁇ g of each plasmid DNA in a 1:1 molar ratio for a 250 ml final transfection volume.
- Antibody containing cell culture supernatants were harvested 7 days after transfection by centrifugation at 14000 g for 30 minutes and filtered through a sterile filter (0.22 ⁇ m). Supernatants were stored at ⁇ 20° C. until purification.
- antibodies were generated by transient transfection in HEK293-EBNA cells.
- Antibodies were expressed by transient co-transfection of the respective expression plasmids in adherently growing HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr-Virus nuclear antigen; American type culture collection deposit number ATCC # CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mM L-Glutamine (Gibco), and 250 ⁇ g/ml Geneticin (Gibco).
- FuGENETM 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a ratio of FuGENETM reagent ( ⁇ l) to DNA ( ⁇ g) of 4:1 (ranging from 3:1 to 6:1). Proteins were expressed from the respective plasmids using an equimolar ratio of plasmids. Cells were feeded at day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco]. Bispecific antibody containing cell culture supernatants were harvested from day 5 to 11 after transfection by centrifugation and stored at ⁇ 200 C. General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.
- Antibodies were purified from cell culture supernatants by affinity chromatography using Protein A-SepharoseTM (GE Healthcare, Sweden) and Superdex200 size exclusion chromatography. Briefly, sterile filtered cell culture supernatants were applied on a HiTrap ProteinA HP (5 ml) column equilibrated with PBS buffer (10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound proteins were washed out with equilibration buffer.
- PBS buffer 10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4
- Antibody and antibody variants were eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing fractions were neutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted protein fractions were pooled, concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded on a Superdex200 HiLoad 120 ml 16/60 or 26/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractions containing purified antibodies with less than 5% high molecular weight aggregates were pooled and stored as 1.0 mg/ml aliquots at ⁇ 80° C.
- MWCO Amicon Ultra centrifugal filter device
- the protein concentration of purified protein samples was determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.
- Purity and molecular weight of antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiotreitol) and staining with Coomassie brilliant blue.
- the NuPAGE® Pre-Cast gel system (Invitrogen, USA) was used according to the manufacturer's instruction (4-12% Tris-Glycine gels).
- the aggregate content of antibody samples was analyzed by high-performance SEC using a Superdex 200 analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM KH 2 PO 4 , 250 mM KCl, pH 7.0 running buffer at 25° C. 25 ⁇ g protein were injected on the column at a flow rate of 0.5 ml/min and eluted isocratic over 50 minutes.
- concentrations of 1 mg/ml of purified proteins were incubated at 4° C. and 40° C. for 7 days and then evaluated by high-performance SEC (e.g. HP SEC Analysis (Purified Protein).
- the total deglycosylated mass of antibodies was determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 ⁇ g purified antibodies were deglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH2PO4/K2HPO4, pH 7 at 37° C. for 12-24 h at a protein concentration of up to 2 mg/ml and subsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare). The mass of the respective heavy and light chains was determined by ESI-MS after deglycosylation and reduction.
- PNGaseF N-Glycosidase F
- SEC-MALLS size-exclusion chromatography with multi-angle laser light scattering
- MALLS allows molecular weight estimation of macromolecules irrespective of their molecular shape or other presumptions.
- SEC-MALLS is based on a separation of proteins according to their size (hydrodynamic radius) via SEC chromatography, followed by concentration- and scattered light-sensitive detectors.
- SEC-MALLS typically gives rise to molecular weight estimates with an accuracy that allows clear discrimination between monomers, dimers, trimers etc., provided the SEC separation is sufficient.
- 5 ⁇ 10e5 A549 cells were seeded per well of a 6-well plate the day prior HGF stimulation in RPMI with 0.5% FCS (fetal calf serum). The next day, growth medium was replaced for one hour with RPMI containing 0.2% BSA (bovine serum albumine). 12.5 ⁇ g/mL of the bispecific antibody was then added to the medium and cells were incubated for 15 minutes upon which HGF (R&D, 294-HGN) was added for further 10 minutes in a final concentration of 25 ng/mL.
- FCS fetal calf serum
- BSA bovine serum albumine
- Cells were washed once with ice cold PBS containing 1 mM sodium vanadate upon which they were placed on ice and lysed in the cell culture plate with 100 ⁇ L lysis buffer (50 mM Tris-Cl pH7.5, 150 mM NaCl, 1% NP40, 0.5% DOC, aprotinine, 0.5 mM PMSF, 1 mM sodium-vanadate). Cell lysates were transferred to eppendorf tubes and lysis was allowed to proceed for 30 minutes on ice. Protein concentration was determined using the BCA method (Pierce).
- 2 ⁇ 10e5 MCF7 cells were seeded per well of a 12-well plate in complete growth medium (RPMI 1640, 10% FCS). Cells were allowed to grow to 90% confluency within two days. Medium was then replaced with starvation medium containing 0.5% FCS. The next day the respective antibodies were supplemented at the indicated concentrations 1 hour prior addition of 500 ng/mL Heregulin (R&D). Upon addition of Heregulin cells were cultivated further 10 minutes before the cells were harvested and lysed. Protein concentration was determined using the BCA method (Pierce). 30-50 ⁇ g of the lysate was separated on a 4-12% Bis-Tris NuPage gel (Invitrogen) and proteins on the gel were transferred to a nitrocellulose membrane. Membranes were blocked for one hour with TBS-T containing 5% BSA and developed with a phospho-specific Her3/ErbB3 antibody specifically recognizing Tyr1289 (4791, Cell Signaling).
- A549 were detached and counted. 1.5 ⁇ 10e5 cells were seeded per well of a conical 96-well plate. Cells were spun down (1500 rpm, 4° C., 5 min) and incubated for 30 min on ice in 50 ⁇ L of a dilution series of the respective bispecific antibody in PBS with 2% FCS (fetal calf serum). Cells were again spun down and washed once with 200 ⁇ L PBS containing 2% FCS followed by a second incubation of 30 min with 5 ⁇ g/mL of Alexa488-coupled antibody directed against human Fc which was diluted in PBS containing 2% FCS (Jackson Immunoresearch, 109116098).
- mfi mean fluorescence intensity of the cells was determined by flow cytometry (FACS Canto, BD). Mfi was determined at least in duplicates of two independent stainings Flow cytometry spectra were further processed using the FlowJo software (TreeStar). Half-maximal binding was determined using XLFit 4.0 (IDBS) and the dose response one site model 205.
- the binding properties of monovalent anti-IGF-1R antibodies were analyzed by surface plasmon resonance (SPR) technology using a Biacore instrument (Biacore, GE-Healthcare, Uppsala).
- SPR surface plasmon resonance
- Biacore Biacore instrument
- This system is well established for the study of molecule interactions. It allows a continuous real-time monitoring of ligand/analyte bindings and thus the determination of association rate constants (ka), dissociation rate constants (kd), and equilibrium constants (KD) in various assay settings.
- SPR-technology is based on the measurement of the refractive index close to the surface of a gold coated biosensor chip. Changes in the refractive index indicate mass changes on the surface caused by the interaction of immobilized ligand with analyte injected in solution.
- FIG. 17 depicts the applied assay format. A low loading density with capturing antibody density and IGF-1R antibody was chosen to enforce monovalent binding.
- human FcgIIIa was immobilized to a CM-5 sensor chip by capturing the His-tagged receptor to an anti-His antibody (Penta-His, Qiagen) which was coupled to the surface by standard amine-coupling and blocking chemistry on a SPR instrument (Biacore T100). After FcgRIIIa capturing, 50 nM IGF1R antibodies were injected at 25° C. at a flow rate of 5 ⁇ L/min. The chip was afterwards regenerated with a 60s pulse of 10 mM glycine-HCl, pH 2.0 solution.
- ADCC Antibody-Dependent Cellular Cytotoxicity Assay
- ADCC antibody-dependent cell cytotoxicity
- cell numbers were determined and cells diluted to 1 ⁇ 10e5 cells/ml in RPMI-FM/PenStrep medium from the pellet afterwards.
- the cells were plated 5,000 per well in a round bottom plate, in a volume of 50 ⁇ l.
- HuMAb antibodies were added at a final concentration ranging from 25-0.1 ⁇ g/ml in a volume of 50 ⁇ l cell culture medium to 50 ⁇ l cell suspension.
- 50 ⁇ l of effector cells, freshly isolated PBMC were added at an E:T ratio of 25:1.
- the plates were centrifuged for 1 minutes at 200 ⁇ g, followed by an incubation step of 2 hours at 37° C.
- the binding of antibodies and antigen binding protein according the invention to the IGF-1R results in internalization and degradation of the receptor. This process can be monitored by incubating IGF-1R expressing HT29 CRC cells with IGF-1R targeting antibodies followed by a quantification of remaining IGF-1R protein levels in cell lysates by ELISA.
- HT29 cells at 1.5 ⁇ 104 cells/well were incubated in a 96 well MTP in RPMI with 10% FCS over night at 37° C. and 5% CO2 in order to allow attachment of the cells.
- the medium was aspirated and 100 ⁇ l anti IGF-1R antibody diluted in RPMI+10% FCS was added in concentrations from 10 nM to 2 pM in 1:3 dilution steps.
- the cells were incubated with antibody for 18 hours at 37° C. Afterwards, the medium was again removed and 120 ⁇ l MES lysis buffer (25 mM MES pH 6.5+ Complete) were added.
- 96-Well streptavidin coated polystyrene plates (Nunc) were loaded with 100 ⁇ l MAK ⁇ hu IGF-1R ⁇ >hu-1a-IgG-Bi (Ch.10) diluted 1:200 in 3% BSA/PBST (final concentration 2.4 ⁇ g/ml) and incubated under constant agitation for 1 hour at room temperature. Afterwards, the well content was removed and each well was washed three times with 200 ⁇ l PBST. 100 ⁇ l of the cell lysate solution were added per well, again incubated for 1 hour at room temperature on a plate shaker, and washed three times with 200 ⁇ l PBST.
- IGF-1R IGF-1 receptor
- 3T3-IGF-1R cells a murine fibroblast cell line overexpressing human IGF-1R, were treated for 10 minutes with 10 nM recombinant human IGF-1 in the presence of different concentrations of monovalent and bivalent IGF-1R antibody. After lysis of the cells, the levels of phosphorylated IGF-1R protein were determined by a phospho-IGF-1R specific ELISA, combining a human IGF-1R specific capture antibody and a phospho-Tyrosine specific detection antibody.
- NMRI mice female, fed, 23-32 g body weight at the time point of compound administration.
- mice were allocated to 3 groups with 2-3 animals each. Blood samples are taken from group 1 at 0.5, 168 and 672 hours, from group 2 at 24 and 336 hours and from group 3 at 48 and 504 hours after dosing.
- Blood samples of about 100 ⁇ L were obtained by retrobulbar puncture. Serum samples of at least 40 ⁇ l were obtained from blood after 1 hour at room temperature by centrifugation (9300 ⁇ g) at room temperature for 2.5 min. Serum samples were frozen directly after centrifugation and stored frozen at ⁇ 20° C. until analysis.
- mice serum The concentrations of the human antibodies in mice serum were determined with an enzyme linked immunosorbent assay (ELISA) using 1% mouse serum.
- Biotinylated monoclonal antibody against human Fc ⁇ (mAb ⁇ hFc ⁇ PAN >IgG-Bi) was bound to streptavidin coated microtiterplates in the first step.
- serum samples in various dilutions
- reference standards respectively, were added and bound to the immobilized mAb ⁇ hFc ⁇ PAN >IgG-Bi.
- digoxigenylated monoclonal antibody against human Fc ⁇ mAb ⁇ hFc ⁇ PAN >IgG-Dig was added.
- the human antibodies were detected via anti-Dig-horseradish-peroxidase antibody-conjugate.
- ABTS-solution was used as the substrate for horseradish-peroxidase.
- the specificity of the used capture and detection antibody which does not cross react with mouse IgG, enables quantitative determination of human antibodies in mouse serum samples.
- the pharmacokinetic parameters were calculated by non-compartmental analysis, using the pharmacokinetic evaluation program WinNonlinTM, version 5.2.1.
- FIG. 3-5 depict the chromatograms of the size exclusion chromatography of the three different monovalent antigen binding proteins without knobs-into-holes as well as the corresponding SDS-PAGE under non-reducing and reducing conditions.
- FIG. 6-5 depict the chromatograms of the size exclusion chromatography of the three different monovalent antigen binding proteins with knobs-into-holes as well as the corresponding SDS-PAGE under non-reducing and reducing conditions.
- c-Met has been described as oncogenic receptor tyrosine kinase which upon deregulation fosters cellular transformation.
- Antibodies targeting c-Met have been described in the past.
- MetMAb/OA-5D5 (Genentech) is one such antibody inhibiting ligand-dependent activation of c-Met.
- c-Met 5D5 MoAb monovalent antigen binding protein
- c-Met MoAb c-Met 5D5 MoAb (“wt”)
- wt c-Met 5D5 MoAb
- c-Met MoAb monovalent antigen binding protein c-Met MoAb
- a cell suspension was incubated with a threefold dilution series (100-0.0003 ⁇ g/mL) of the indicated antibodies. Bound antibodies were visualized with a secondary Alexa488-coupled antibody binding to the constant region of human immunoglobulin. Fluorescence intensity of single cells was measured on a FACS Canto (BD Biosciences) flow cytometer. No differences in binding of c-Met MoAb and OA-5D5 are observable indicating that the c-Met MoAb (c-Met 5D5 MoAb (“wt”)) efficiently binds to cell surface c-Met.
- FIG. 17 depicts the scheme of the SPR assay to determine the monovalent affinity. The analysis (double determination) showed that the IGF-1R binding affinity is retained in the monovalent antibody.
- IGF1R MoAb monovalent antigen binding protein IGF1R MoAb
- the data show that there is a difference in halfmaximal binding to cells due to the fact that the IGF-1R IgG1 antibody can bind with two arms to IGF-1R on cells and exhibits an avidity effect whereas the monovalent antibody can only bind with one arm.
- IGF-1R 150 kDa: 0.76 nM
- IGF-1R MoAb 100 kDa: 5.65 nM
- Donor-derived peripheral blood mononuclear cells can be used to measure effector cell recruitment by non-glycoengineered and glycoengineered antibodies to cancer cells. Lysis of cancer cells correlates with NK cell mediated cytotoxicity and is proportional to the antibody's ability to recruit NK cells.
- DU145 prostate cancer cells were incubated in a 1:25 ratio (DU145:PBMC) ratio with PBMC in the absence or presence of the respective antibodies. After 2 hours cellular lysis was determined using the BATDA/Europium system as described above. The magnitude of cell lysis by ADCC is expressed as % of the maximum release of TDA fluorescence enhancer from the target cells lysed by detergent corrected for spontaneous release of TDA from the respective target cells.
- IGF1R AK18 MoAb Monovalent IGF-1R antigen binding proteins (IGF1R AK18 MoAb (“wt”)) that mediate reduced IGF-1R internalization and enhanced ADCC due to reduced internalization (see below) and double the amount of Fc-parts to engage FcRIIIa receptors on effector cells may thus represent a promising approach to target IGF-1R on cancer cells; as non-glycoengineered or as glycoengineered antibodies.
- IGF-1R AK18 MoAb monovalent antigen binding protein IGF MoAb
- FIG. 14 show that internalization of IGF-1R is reduced in terms of potency and absolute internalization when the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) is bound.
- the targeting IGF-1R on tumor cells by bivalent IGF-1R antibodies results in internalization and lysosomal degradation of IGF-1R.
- IGF1R AK18 MoAb monovalent antigen binding protein IGF MoAb
- HT29 colon cancer cells were treated for 18 hours with different concentrations of monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)) and bivalent parent IGF-1R antibody. After lysis of the cells, the remaining levels of IGF-1R protein were determined by IGF-1R specific ELISA.
- IGF-1R Autophosphorylation (IGF-1 Stimulation) (FIG. 15)
- IGF-1R AK18 MoAb a murine fibroblast cell line overexpressing human IGF-1R, were treated for 10 minutes with 10 nM recombinant human IGF-1 in the presence of different concentrations of monovalent antigen binding protein IGF1R MoAb (IGF1R AK18 MoAb (“wt”)) and bivalent parent IGF-1R antibody. After lysis of the cells, the levels of phosphorylated IGF-1R protein were determined by a phospho-IGF-1R specific ELISA, combining a human IGF-1R specific capture antibody and a phospho-Tyrosine specific detection antibody.
- the data in FIG. 15 show that the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) can inhibit IGF-1 induced autophosphorylation although at a higher concentration due to monovalent binding on cells (lack of avidity effect due to bivalent binding).
- the concentration required for halfmax inhibition increased from 1.44 nM (IgG1) to 27.9 nM (MoAb). Since the difference in IC50 values of monovalent and bivalent antibodies is slightly less pronounced in IGF-1R autophosphorylation (19 fold) compared to IGF-1R downregulation (59 fold), the reduced impact of monovalent binding on downregulation cannot solely explained by reduced affinity to the IGF-1R.
- the stability of the monovalent antigen binding protein IGF1R MoAb was studied by dynamic light scattering as described above. Briefly, aggregation tendency of the monovalent antigen binding protein IGF1R MoAb was assessed by a DLS timecourse experiment at 40° C. Over a period of five days, no measurable increase in the hydrodynamic radius (Rh) of the isolated monomer fraction (c.f. FIG. 10 ) could be detected ( FIG. 22 ).
- Pharmacokinetic properties of the monovalent antibodies according to the invention were determined in NMRI mice, female, fed, 23-32 g body weight at the time point of compound administration mice in a single dose PK study, as described above (in the methods sections).
- the PK properties are given in the subsequent table and indicate that the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) has improved PK properties compared to the parental ⁇ IGF-1R>IgG1 antibody.
- IGF1R AK18 MoAb monovalent antigen binding protein
- the monovalent antigen binding protein IGF MoAb (IGF1R AK18 MoAb (“wt”)) was transiently expressed and purified via Protein A affinity and size exclusion chromatography. After preparative SEC the antibody eluted within two separate peaks (peak 1 and peak 2), which were collected. Analytical SEC from the fraction 2 (peak 2) corresponds to a molecular weight of 100 kDa indicating a defined monomer. SEC-MALS confirmed the initial SEC result and shows for the fraction 2 (monomer,) a MW of 99.5 kDa. SDS-PAGE analysis of this fraction under denaturing and reducing conditions shows one major band with an apparent molecular weight of 50-60 kDa. Under non reducing conditions fraction 2 (monomer) shows a major band around a MW of 100 kDa.
- ESI-MS spectra of deglycosylated MoAbs from fraction 2 show one peak series corresponding to a monomer with a mass of 98151 Da.
- MS measurements under reducing conditions of fraction 2 show the correct sequence and expression of the construct.
- the MS data from fraction 2 show two different heavy chains with a molecular weight of 47959 Da and 50211 Da in approximately equal amounts.
- HEK-EBNA cells are transfected, using the calcium phosphate method, with four plasmids. Two encoding the antibody chains, one for a fusion GnTIII polypeptide expression (a GnT-III expression vector), and one for mannosidase II expression (a Golgi mannosidase II expression vector) at a ratio of 4:4:1:1, respectively. Cells are grown as adherent monolayer cultures in T flasks using DMEM culture medium supplemented with 10% FCS, and are transfected when they are between 50 and 80% confluent.
- a solution of DNA, CaCl 2 and water is prepared by mixing 94 ⁇ g total plasmid vector DNA divided equally between the light and heavy chain expression vectors, water to a final volume of 469 ⁇ l and 469 ⁇ l of a 1M CaCl 2 solution.
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2012
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- 2012-02-24 AR ARP120100617A patent/AR085403A1/es not_active Application Discontinuation
- 2012-02-24 CA CA2824824A patent/CA2824824A1/en not_active Abandoned
- 2012-02-24 KR KR1020137022150A patent/KR101572338B1/ko not_active IP Right Cessation
- 2012-02-24 CN CN201280010809.1A patent/CN103403025B/zh not_active Expired - Fee Related
- 2012-02-24 RU RU2013141078/10A patent/RU2013141078A/ru not_active Application Discontinuation
- 2012-02-24 WO PCT/EP2012/053119 patent/WO2012116927A1/en active Application Filing
- 2012-02-24 BR BR112013020338A patent/BR112013020338A2/pt not_active IP Right Cessation
- 2012-02-24 JP JP2013555826A patent/JP5768147B2/ja not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
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AR085403A1 (es) | 2013-09-25 |
JP2014509199A (ja) | 2014-04-17 |
WO2012116927A1 (en) | 2012-09-07 |
CN103403025B (zh) | 2016-10-12 |
US10611825B2 (en) | 2020-04-07 |
MX342034B (es) | 2016-09-12 |
EP2681240A1 (en) | 2014-01-08 |
KR20130135896A (ko) | 2013-12-11 |
RU2013141078A (ru) | 2015-04-10 |
CA2824824A1 (en) | 2012-09-07 |
JP5768147B2 (ja) | 2015-08-26 |
EP2681240B1 (en) | 2017-08-16 |
BR112013020338A2 (pt) | 2016-10-18 |
MX2013009781A (es) | 2013-09-26 |
US20180037633A1 (en) | 2018-02-08 |
KR101572338B1 (ko) | 2015-11-26 |
CN103403025A (zh) | 2013-11-20 |
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