WO2014072741A1 - Anticorps humanisé et son procédé de production - Google Patents

Anticorps humanisé et son procédé de production Download PDF

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Publication number
WO2014072741A1
WO2014072741A1 PCT/GB2013/052965 GB2013052965W WO2014072741A1 WO 2014072741 A1 WO2014072741 A1 WO 2014072741A1 GB 2013052965 W GB2013052965 W GB 2013052965W WO 2014072741 A1 WO2014072741 A1 WO 2014072741A1
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amino acid
seq
antibody molecule
humanised
molecule according
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PCT/GB2013/052965
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English (en)
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Richard John BUICK
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Fusion Antibodies Limited
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Publication of WO2014072741A1 publication Critical patent/WO2014072741A1/fr

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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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/464—Igs containing CDR-residues from one specie grafted between FR-residues from another
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • 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
    • 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

Definitions

  • the application relates to the field of antibodies, in particular to humanised antibodies and methods of making such humanised antibodies.
  • the invention also extends to the use of such humanised antibodies in therapeutic uses, such as for the treatment of medical conditions such as cancer.
  • rodent monoclonal antibodies such as murine monoclonal antibodies
  • HAMA human anti-mouse antibody
  • Composite Human Antibodies are entirely human in origin and comprise multiple segments of human variable region sequence from different human antibodies. Such technology may thus be used to produce a fully humanized monoclonal antibody which displays a single binding specificity and which has variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. This contrasts with humanised antibodies produced by "CDR grafting", wherein the complementarity determining regions (CDRs] from a murine antibody are grafted into a framework acceptor sequence, provided by regions of human antibody light and heavy chain variable domains. This results in the production of an antibody which retains the binding specificity of the murine antibody, but where the only non-human components are the grafted murine CDR regions.
  • the therapeutic effectiveness of the resulting humanised antibody can be impaired.
  • simple transplantation of the CDR regions often results in a reduced therapeutic efficacy of the antibody due to the binding affinity of the antibody being diminished.
  • back mutations are often employed, back mutations being the replacement of an amino acid residue at a specific position of the framework sequence (outside of the CDR sequence] so as to improve the binding specificity of the humanised antibody.
  • the CDRs of a donor antibody are selected using one of the Rabat, IMGT or Chothia methods and, typically, it is necessary to back-mutate certain residues outside the CDR sequences to restore satisfactory affinity.
  • the present inventors have surprisingly shown that, when using the CDR grafting technique, humanised antibodies of high affinity may be readily obtained by using CDRs defined by a method which combines the IMGT and Rabat methods for defining CDR sequences. Accordingly, in a first aspect of the present invention, there is provided a humanised antibody molecule having a heavy chain, wherein the complementarity determining regions (CDRs] are derived from a non-human donor antibody, wherein
  • the first amino acid residue and the final amino acid residue of CDRH1 of the humanised antibody molecule are the first amino acid residue of CDRH1 of the donor antibody as defined by the IMGT method and the final amino acid residue as defined by the Rabat method respectively and the CDRH1 comprises the intervening amino acid residues of said donor CDRH1 between said first and final amino acid residues;
  • the first amino acid residue and the final amino acid residue of CDRH3 of the humanised antibody molecule are the first amino acid residue of CDRH3 of the donor antibody as defined by the IMGT method and the final amino acid residue as defined by the Rabat method respectively and the CDRH3 comprises the intervening amino acid residues of said donor CDRH3 between said first and final amino acid residues;
  • CDRH2 of the humanised antibody molecule corresponds to CDRH2 of the donor antibody as defined by the Rabat method.
  • a humanised antibody molecule is a chimeric antibody molecule which comprises a variable domain at least part of which is derived from a human source.
  • the term "derived" is intended to not only encompass the source of material as being the physical source but also to define material which is structurally identical to the material but which does not originate from the reference source.
  • the CDRs derived from a non-human donor antibody need not necessarily be purified or isolated from the donor antibody or antibody framework.
  • the humanised antibody molecule comprises a light chain.
  • the CDRL1, CDRL2, and CDRL3 of the light chain correspond to CDRL1, CDRL2, and CDRL3 of the donor antibody as defined by the Rabat method.
  • the humanised antibody of the first aspect of the invention to maintain a predetermined affinity equivalent to the corresponding non-human donor antibody comprises fewer back mutations in the VH sequence of the human acceptor sequence than the number of back mutations required by a corresponding humanised antibody in which the CDRs are as defined by the Rabat method only to maintain said predetermined level of affinity.
  • the humanised antibody of the invention comprises 3 or fewer, such as 2 or fewer back mutations in the VH sequence of the human acceptor sequence.
  • the humanised antibody of the invention comprises no back mutations in the VH sequence of the human acceptor sequence.
  • the present inventors have humanised a number of distinct antibodies against different targets using the novel method for humanisation. They have demonstrated that, by using this method, antibodies with excellent binding efficacy may be readily obtained.
  • the humanised antibody of the first aspect of the invention is an antibody which is cross-specific to two EGF molecules.
  • the humanised antibody of the invention may have binding specificity for both HBEGF and AREG.
  • the humanised antibody has binding specificity for the antigenic fragment of AREG having the amino acid sequence shown as SEQ. ID NO: 1 and the antigenic fragment of HBEGF having the amino acid sequence shown as SEQ. ID NO: 2:
  • humanised antibody molecules according to the present invention based on a murine antibody raised against an antigenic fragment from this region were found to have specificity for both AREG and HB-EGF.
  • a humanised antibody molecule of the invention with binding specificity for both HBEGF and AREG is an antibody molecule which comprises:
  • CDRH1 SEQ ID NO: 3
  • CDRs complementarity determining regions
  • CDRL1 SEQ ID NO: 6,
  • CDRL2 SEQ ID NO: 7,
  • CDRL3 SEQ ID NO: 8.
  • SEQ. ID NOS: 3 to 8 are as follows:
  • SEQ ID NO: 3 GFNIKDSYIH
  • SEQ ID NO: 5 VSASYRYGFSY
  • SEQ ID NO: 6 RSNKSLLHTNGNTYLY
  • SEQ ID NO: 8 MQHLEYPYT
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 9, SEQ. ID NO: 10, or SEQ. ID NO: 11 and/or a VL domain having an amino acid sequence shown as SEQ. ID NO: 12, SEQ. ID NO: 13, or SEQ. ID NO: 14.
  • SEQ. ID NOS: 9 to 14 are as follows: SEQ. ID NO: 9:
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 9 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 12.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 9 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 13.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 9 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 14.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 10 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 13.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 10 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 14.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 11 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 12.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 11 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 13.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 11 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 14.
  • the humanised antibody molecule has binding specificity for CD20.
  • the antibody molecule comprises:
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • CDRL1 SEQ ID NO: 18,
  • CDRL2 SEQ ID NO: 19, and
  • CDRL3 SEQ ID NO: 20.
  • SEQ. ID NOS: 15 to 20 are as follows: SEQ. ID NO: 15: GYTFTSYNMH
  • SEQ. ID NO: 16 AIYPGNGDTSYNQKFKG
  • SEQ. ID NO: 17 ARSTYYGGDWYFNV
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO:21, SEQ. ID NO: 22, or SEQ. ID NO: 23 and/or a VL domain having an amino acid sequence shown as SEQ. ID NO: 24, SEQ. ID NO: 25, or SEQ. ID NO: 26.
  • SEQ. ID NOS: 21 to 23 correspond to the VH sequences shown as VHl to VH3 of Figure 9.
  • SEQ. ID NOS: 24 to 26 correspond to the VL sequences shown as VL1 to VL3 of Figure 10.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO:21. In a particular embodiment, the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 21 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 24. In another embodiment, the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 21 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 25. In another embodiment, the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 22 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 24.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 22 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 25.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 22 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 26.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 23 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 24.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 23 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 25.
  • the humanised antibody molecule of the invention comprises a VH domain having an amino acid sequence shown as SEQ. ID NO: 23 and a VL domain having an amino acid sequence shown as SEQ. ID NO: 26.
  • the invention provides a nucleic acid molecule which encodes a humanised antibody molecule of the first aspect of the invention.
  • the present invention provides a method of humanising an antibody molecule, said method comprising producing an antibody molecule in which the heavy chain CDRs of a non-human donor antibody are grafted onto a first human acceptor framework, and optionally the light chain CDRs of a non-human donor antibody are grafted onto a second human acceptor framework, wherein
  • the first amino acid residue and the final amino acid residue of CDRHl of the humanised antibody molecule are the first amino acid residue of CDRHl of the donor antibody as defined by the IMGT method and the final amino acid residue as defined by the Rabat method and the CDRHl comprises the intervening amino acid residues of said donor CDRHl between said first and final amino acid residues;
  • the first amino acid residue and the final amino acid residue of CDRH3 of the humanised antibody molecule are the first amino acid residue of CDRH3 of the donor antibody as defined by the IMGT method and the final amino acid residue as defined by the Rabat method and the CDRH3 comprises the intervening amino acid residues of said donor CDRH3 between said first and final amino acid residues;
  • CDRH2 of the humanised antibody molecule corresponds to CDRH2 of the donor antibody as defined by the Rabat method.
  • the method encompasses any method of producing an antibody having heavy chain CDRs derived from a non-human donor antibody within a human acceptor framework.
  • the method thus encompasses any method of producing an antibody molecule according to the first aspect of the invention and will thus include methods involving expression of nucleic acid
  • a fourth aspect of the invention is an antibody molecule obtained by the method of the third aspect of the invention.
  • a further aspect of the invention provides a humanised antibody molecule of the first or fourth aspect of the invention or a nucleic acid molecule according to the second aspect of the invention for use in medicine.
  • a humanised antibody molecule of the first or fourth aspect of the invention or a nucleic acid molecule according to the second aspect of the invention for use in the treatment of a disease or disorder associated with angiogenesis.
  • neoplastic disease in a subject, said method comprising administration to said subject of an effective amount of humanised antibody molecule of the first or fourth aspect of the invention or a nucleic acid molecule of the second aspect of the invention.
  • a method of treating a disease or disorder associated with angiogenesis in a subject comprising administration to said subject of an effective amount of humanised antibody molecule of the first or fourth aspect of the invention or a nucleic acid molecule of the second aspect of the invention.
  • the invention also extends to a pharmaceutical composition
  • a pharmaceutical composition comprising a humanised antibody molecule of the first or fourth aspect of the invention or a nucleic acid molecule of the second aspect of the invention.
  • the pharmaceutical composition further comprises a chemotherapeutic agent and/or an angiogenesis inhibitor.
  • kits comprising:
  • a chemotherapeutic agent and/or an angiogenesis inhibitor for simultaneous, sequential or separate administration with said humanised antibody molecule or nucleic acid molecule.
  • Figure 1 illustrates the VH and VL sequences of the murine Fsnl006 antibody which has binding specificity for both HBEGF and AREG; the CDRs as defined by the Rabat method, the IMGT method and the Chothia method are shown in Table 1.
  • FIG 2 illustrates VH acceptor frameworks with the Fsnl006 "Fusion CDR” residue sequences and VL acceptor frameworks with the Fsnl006 "Fusion CDR” residue sequences as employed in humanized Fsn 1006 variants; the CDRs are underlined.
  • Figure 3 illustrates a sequence alignment of the murine Fsnl006 VH sequence with three humanised VH variants.
  • Figure 4 illustrates a sequence alignment of the murine Fsnl006 VL sequence with three humanised VL variants.
  • Figure 5 illustrates the results of analysis of binding of the nine humanised antibody constructs to HB-EGF in comparison to the murine antibody (2F7] and the chimeric antibody (H0LO).
  • Figure 6 illustrates a Western blot of HCTtll6 cel ls treated with HBEGF a nd FSN 1006 HC2LC2.
  • Figure 7 illustrates the results of an MTT assay performed on HCT116 cells after 72 hours incubation with three humanised antibody variants of the invention.
  • Figure 8 illustrates the VH and VL sequences of rituximab; the CDRs as defined by the Rabat method, the IMGT method and the Chothia method are shown in Table 2.
  • Figure 9 illustrates a sequence alignment of the rituximab VH sequence with three humanised VH variants.
  • Figure 10 illustrates a sequence alignment of the rituximab VL sequence with three humanised VL variants.
  • Figure 11 illustrates the results of a flow cytometry experiment in which the binding ability of humanised rituximab antibodies (panels c, d, e, f and g] to CD20 is compared with that of a negative control (panel a], and, as a positive control, rituximab (panel b).
  • the present invention provides humanised antibody molecules produced by a modified CDR grafting technique in which the heavy chain CDR sequences as employed in the humanised antibody molecule comprise a combination of the CDR residues as defined by the IMGT method and a combination of the CDR residues as defined by the Rabat method.
  • the Rabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain].
  • the Rabat definition is based on sequence variability.
  • the Rabat residue designations do not always correspond directly with the linear numbering of the amino acid residues of the heavy and light chain variable regions of the present invention.
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Rabat numbering corresponding to a shortening of, or insertion into, a structural component, whether a framework region or complementarity determining region (CDR], of the basic variable domain structure of the heavy or light chain.
  • CDR complementarity determining region
  • Rabat numbering of residues may be determined for any given antibody by alignment of residues in the sequence of the antibody with a standard sequence to which the Rabat numbering has been applied.
  • Alternative numbering systems include the IMGT system and the Chothia system.
  • the Chothia definition is based on the location of the structural loop regions.
  • the IMGT definition is based on sequence variability, structure and evolutionary data.
  • an "antibody molecule” is an immunoglobulin, whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide, protein or peptides having a binding domain that is, or is homologous to, the binding domain of the humanised antibody molecules of the invention.
  • Immunoglobulins typically have a heterotetrameric structure comprising two identical heavy chains and two identical light chains, linked together by disulphide bonds.
  • Each heavy and light chain comprises a variable domain which confers the binding specificity of the antigen, with these domains being known as VH and VL domains for the heavy and light chains respectively.
  • Each chain also comprises at least one constant domain, with the light chain having at a single constant domain, designated the CL domain, while the heavy domain comprises three constant domains, CHI, CH2 and CH3.
  • Some antibody isotypes additionally include a further constant domain referred to as the CH4 domain.
  • the acceptor sequence of the humanised antibodies of the invention is an IgG.
  • the IgG is of subclass IgGl.
  • the Fc domain of an antibody typically comprises the last 2 heavy chain constant region domains of each chain. These dimerise to form the Fc domain which is responsible for mediating the effector functions of the antibody, such as ADCC (antibody -dependent ceil -mediated cytotoxicity ) and complement fixation.
  • ADCC antibody -dependent ceil -mediated cytotoxicity
  • complement fixation such as ADCC (antibody -dependent ceil -mediated cytotoxicity ) and complement fixation.
  • the Fc region of the antibody also has a role in the circulatory half-life of the antibody. Modifications can be made to the Fc domain to modulate antibody function.
  • a binding member may comprise a VH domain comprising, in sequence, VH CDR1 (or CDRH1], CDR2 (CDRH2] and CDR3 (CDRH3] regions along with a plurality of associated framework regions.
  • a binding member may additionally or alternatively comprise a VL domain comprising VL CDRl(or CDRL1], CDR2(CDRL2] and CDR3 (CDRL3] regions along with associated framework regions.
  • the VH and VL domains typically each comprise four framework regions, FR1, FR2, FR3 and FR4, interspersed between the 3 complementarity determining regions in the following arrangement: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the complementarity determining regions which may also be known as hypervariable regions, have a role in conferring the binding specificity of the antibody, or binding fragment.
  • fragments of a whole antibody can perform the function of binding antigens.
  • binding fragments include, but are not limited to: (i] Fab fragments consisting of the VL, VH, CL and CHI domains of a heterotetrameric antibody, (ii] F(ab']2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region, (iii] Fab' fragment, a Fab fragment with part of the hinge region, (iv] Fd fragments consisting of the VH and CHI domains of a
  • antibody molecule should be construed as extending to fragments of humanised antibodies of the invention. The invention therefore extends to a fragment of the humanized antibody molecule of the present invention, e.g.
  • the heavy only or, for example, the variable domain of the heavy chain.
  • the CDRs of the light chain associate with the CDRs of the heavy chain to confer the binding specificity of an antibody, or antibody binding fragment, in instances where both sets of CDRs are present It is known that the contribution made by the light chain variable region to the energetics of binding is small relative to the associated heavy chain variable region. Accordingly, isolated heavy chain regions, which comprise, in sequence, the 3 complementarity determining regions (CDRH1, CDRH2, and CDRH3], are known to have an antigen binding capability and are commonly referred to as single domain antibodies. Such antibody fragments of the humanized antibody molecules of the invention are provided by the present invention.
  • the antibody molecule may be selected from the group comprising, but not limited to; a Fab fragment, a Fab' fragment, a scFv (single chain variable fragment], a peptidomimetic, a diabody, or a related multivalent derivative.
  • antibody fragments can be derived from full length antibodies by proteolytic digestion according to the method of Morimoto (Morimoto et al., "Single- step purification of F(ab' ]2 fragments of mouse monoclonal antibodies
  • Antibody fragments can also be produced directly by host cells (see Carter et al., Bio/Technology 10: 163-167 (1992]].
  • the invention extends to single binding domains which are based on the VH region of the humanised antibodies of the antibodies of the invention.
  • a binding member comprising, consisting or consisting essentially of a single binding domain derived from a humanised antibody of the invention.
  • the single binding domain is derived from the amino acid sequence of the VH (heavy chain variable domain], for example as defined in SEQ ID NO:10, of a humanized antibody molecule of the invention.
  • Such a binding domain may be used as a targeting agent to AREG and HBEGF, as it is known that immunoglobulin VH domains are capable of binding to target antigens in a specific manner.
  • the amino acid sequences of the VH or VL domains may comprise at least one back mutation, said back mutation being the replacement of an amino acid residue at a specific position of the sequence so as to improve the binding specificity of the humanised antibody or fragment thereof to its target and/or to enhance the therapeutic efficacy of the humanised antibody.
  • Typically such modification can be made to the framework residues within the light and heavy chain variable regions so as to decrease the immunogenicity of the antibody.
  • back mutations may not be necessary.
  • further engineering techniques can be used to modify the antibodies of the present invention, for example by including modifications of the Fc region which can alter serum half life, complement fixation, Fc receptor binding and/or antigen dependent cellular cytotoxicity.
  • the antibodies or antibody fragments can be produced which have altered glycosylation patterns.
  • an antibody molecule of the invention is altered to increase or decrease the extent to which the antibody molecule is glycosylated.
  • Glycosylation of polypeptides is typically either relinked or O-linked.
  • N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X -threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • the antibodies can be PEGylated by reacting the antibody with a plyethylene glycol (PEG] derivative.
  • PEG plyethylene glycol
  • the antibody is defucosylated and therefore lacks fucose residues.
  • modifications in the biological properties of an antibody may be accomplished by selecting substitutions that affect (a] the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b] the charge or hydrophobicity of the molecule at the target site, or (c] the bulk of the side chain.
  • Amino acids may be grouped according to similarities in of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.
  • Naturally occurring residues may be divided into groups based on common side-chain properties: (1] hydrophobic: Norleucine, Met, 5 Ala, Val, Leu, He; (2] neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3] acidic: Asp, GIu; (4] basic: His, Lys, Arg; (5] residues that influence chain orientation: Gly, Pro; (6] aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Such substituted residues also may be introduced into the
  • amino acid substitutions may be made in one or more CDRs.
  • the humanized antibody molecule of the invention may comprise 3 or fewer, for example 2, 1 or zero amino acid substitutions in the intervening amino acid residues between the first and the final amino acid residues of one or more of CDRH1,
  • the humanized antibody molecule of the invention comprises one or more amino acid substitutions in said CDRs, optionally the antibody molecule maintains affinity of a magnitude at least equivalent to that of the corresponding antibody molecule having no such amino acid substitutions.
  • the humanized antibody molecule of the invention comprises zero amino acid substitutions in the intervening amino acid residues between the first and the final amino acid residues of each of CDRH1, CDRH2, CDRH3, CDRLl, CDRL2, or CDRL3, i.e. the intervening amino acids correspond to those of the CDRs of the donor antibody as defined above.
  • the present invention extends to a multivalent
  • monospecific antigen binding protein comprising two, three, four or more of the antibody molecules as defined in the foregoing aspects of the invention, or to fragments thereof, wherein said antibody molecules are bound to each other by a connecting structure.
  • the invention also extends to bispecific antibodies.
  • bispecific antibodies may include conventional bispecific antibodies which may be prepared by chemical conjugation means, or by hybrid hybridoma cell lines.
  • the bispecific antibodies may be derived from bispecific antibody fragments, such as scFv dimers or diabodies.
  • scFv dimers may be used, rather than whole antibodies.
  • diabodies can be constructed using only variable domains and therefore are provided without an Fc region, such a structure reducing the possible occurrence of a HAMA or anti-idiotypic immune response.
  • the antibody or antibody fragments of the invention may be subject to one or more techniques which may further be used to ensure that an immunogenic response is not mounted against the antibody when administered to a subject
  • techniques will be well known to the person skilled in the art and include, but are not limited to, de-immunisation, gene shuffling and PEGylation.
  • Deimmunisation techniques may involve the introduction of mutations to remove T-cell epitopes without significantly reducing the binding affinity of an antibody.
  • such "deimmunised" antibodies are created with human constant domains.
  • optimisation techniques include, but are not limited to, modifying the following attributes of the antibody: potency, affinity, binding specificity, K on (association rate constant], K off (dissociation rate constant], thermodynamic stability, solubility, serum half-life, expression, folding kinetics, protease susceptibility, Fc region effector function and drug recycling.
  • the biological function or therapeutic activity which may be modulated includes, but is not limited to: enhancement of efficacy, improved pharmacokinetic profile, enhanced patient convenience, improved safety profile, reduced immunogenicity and extended shelf-life.
  • the antibody molecules of the invention may be produced wholly or partly by chemical synthesis.
  • the antibody molecules of the invention can be prepared by techniques which are well known to the person skilled in the art, such as standard liquid peptide synthesis, or by solid-phase peptide synthesis methods.
  • the antibody molecules may be prepared in solution using liquid phase peptide synthesis techniques, or further by a combination of solid-phase, liquid phase and solution chemistry.
  • the present invention further extends to the production of the antibody molecules of the invention by expression in a suitable expression system of a nucleic acid which encodes at least one amino acid sequence which, alone or in combination with one or more other amino acid sequences, comprises an antibody molecule of the invention, such that a desired peptide or polypeptide can be encoded.
  • a nucleic acid encoding the amino acid sequence of the light chain and a second nucleic acid encoding an amino acid of a heavy chain can be expressed to provide an antibody molecule of the present invention.
  • nucleic acid encoding an amino acid sequence which forms an antibody molecule of the present invention.
  • nucleic acids encoding the amino acid sequences which form antibody molecules of the present invention can be provided in an isolated or purified form, or provided in a form which is substantially free of material which can be naturally associated with it, with the exception of one or more regulatory sequences.
  • Nucleic acid which expresses an antibody molecule of the invention may be wholly or partially synthetic and may include, but is not limited to, DNA, cDNA and RNA
  • Nucleic acid sequences encoding the antibody molecules of the invention can be readily prepared by the skilled person using techniques which are well known to those skilled in the art, such as those described in Sambrook et al. "Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Volumes 1-3, 2001 (ISBN-0879695773), and Ausubel et al. "Short Protocols in Molecular Biology", 4 th Edition, 1999, John Wiley and Sons, (ISBN - 0471250929]. Said techniques include (i] the use of the polymerase chain reaction (PCR] to amplify samples of nucleic acid, (ii] chemical synthesis, or (iii] preparation of cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding antibody molecules of the invention may be generated and used in any suitable way known to those skilled in the art, including taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA.
  • the excised portion may then be operably linked to a suitable promoter and expressed in a suitable expression system, such as a commercially available expression system.
  • a suitable expression system such as a commercially available expression system.
  • the relevant portions of DNA can be amplified by using suitable PCR primers. Modifications to the DNA sequences can be made by using site directed mutagenesis.
  • Nucleic acid sequences encoding the antibody molecules of the invention may be provided as constructs in the form of a plasmid, vector, transcription or expression cassette which comprises at least one nucleic acid as described above.
  • constructs form a further aspect of the present invention.
  • the construct may be comprised within a recombinant host cell which comprises one or more constructs as above. Expression may conveniently be achieved by culturing, under appropriate conditions, recombinant host cells containing suitable nucleic acid sequences. Following expression, the antibody or antibody fragments may be isolated and/or purified using any suitable technique, then used as appropriate.
  • Suitable host cells may include bacteria, mammalian cells, yeast, insect and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO] cells, HeLa cells, baby hamster kidney cells and NSO mouse myeloma cells.
  • a commonly employed bacterial host is E. coli.
  • the expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a binding member.
  • the invention extends to a host cell comprising a nucleic acid or antibody molecule of the invention.
  • nucleic acids encode single stranded nucleic acids, double stranded nucleic acids consisting of said coding nucleic acids and of complementary nucleic acids thereto, or these complementary (single stranded] nucleic acids themselves.
  • nucleic acids encoding a heavy chain variable domain and/or a light chain variable domain of antibodies can be enzymatically or chemically synthesised nucleic acids having the authentic sequence coding for a naturally-occurring heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
  • An antibody of the invention may be produced by recombinant means, not only directly, but also as a fusion polypeptide with a heterologous polypeptide, for example a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide for example a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected may be one that is recognized and processed (i.e., cleaved by a signal peptidase] by the host cell.
  • the signal sequence may be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Lpp, or heat-stable enterotoxin II leaders.
  • Antibody molecules and nucleic acids may be formulated with diluents or adjuvants and still, for practical purposes, be considered as being provided in an isolated form.
  • the antibody molecules can be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy.
  • the antibody molecules may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NSO cells], or they may be (for example if produced by expression in a prokaryotic cell] unglycosylated.
  • Heterogeneous preparations comprising humanised antibody molecules also form part of the invention.
  • such preparations may be mixtures of antibodies with full-length heavy chains and heavy chains lacking the C-terminal lysine, with various degrees of glycosylation and/or with derivatized amino acids, such as cyclization of an N-terminal glutamic acid to form a pyroglutamic acid residue.
  • Administration may be mixtures of antibodies with full-length heavy chains and heavy chains lacking the C-terminal lysine, with various degrees of glycosylation and/or with derivatized amino acids, such as cyclization of an N-terminal glutamic acid to form a pyroglutamic acid residue.
  • the antibody molecules of the present invention may be administered alone but will preferably be administered as a pharmaceutical composition which will generally comprise a suitable pharmaceutically acceptable excipient, diluent or carrier selected depending on the intended route of administration.
  • a suitable pharmaceutically acceptable excipient diluent or carrier selected depending on the intended route of administration.
  • pharmaceutical carriers include water, glycerol, ethanol and the like.
  • the antibody molecules of the present invention may be administered to a patient in need of treatment via any suitable route.
  • the composition can be
  • parenteral administration examples include, but are not limited to intravenous, intracardial, intraarterial, intraperitoneal, intramuscular, intracavity, subcutaneous, transmucosal, inhalation or transdermal. Routes of administration may further include topical and enteral, for example, mucosal (including pulmonary], oral, nasal, rectal.
  • the active ingredient can be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as sodium chloride injection, Ringer's injection or, Lactated Ringer's injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.
  • a composition of the invention may be administered to a subject in a "therapeutically effective amount" this being an amount sufficient to show benefit to the subject to whom the composition is administered.
  • the actual dose administered, and rate and time-course of administration will depend on, and can be determined with due reference to, the nature and severity of the condition which is being treated, as well as factors such as the age, sex and weight of the subject being treated, as well as the route of administration. Further due consideration should be given to the properties of the composition, for example, its binding activity and in-vivo plasma life, the concentration of the antibody or binding member in the formulation, as well as the route, site and rate of delivery.
  • Dosage regimens can include a single administration of the composition, or multiple administrative doses of the composition.
  • the compositions can further be administered sequentially or separately with other therapeutics and medicaments which are used for the treatment of the condition for which the antibody or binding member of the present invention is being administered to treat
  • Examples of dosage regimens which can be administered to a subject can be selected from the group comprising, but not limited to; l ⁇ g/kg/day through to 20mg/kg/day, l ⁇ g/kg/day through to lOmg/kg/day, and l ⁇ g/kg/day through to lmg/kg/day.
  • the dosage will be such that a plasma concentration of from l ⁇ g/ml to 100 ⁇ g/ml of the antibody is obtained.
  • the actual dose of the composition administered, and rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g.
  • the formulation is a liquid formulation, a lyophilized formulation, a lyophilized formulation that is reconstituted as a liquid, or an aerosol formulation.
  • the antibody in the formulation is at a
  • the formulation further comprises a buffer.
  • the pH of the formulation is from about pH 5.5 to about pH 6.5.
  • the buffer may comprise from about 4 mM to about 60 mM histidine buffer, about 5 mM to about 25 mM succinate buffer, or about 5 mM to 25 mM acetate buffer.
  • the buffer comprises sodium chloride at a concentration of from about lOmM to 300mM, for example in the range 100 to 150mM, such as about 125mM concentration and sodium citrate at a concentration of from about 5mM to 50mM, for example about 25mM.
  • the formulation can further comprise a surfactant at a concentration of about 0% to about 0.2%.
  • the surfactant is selected from the group consisting of, but not limited to: polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, and combinations thereof.
  • the surfactant is polysorbate-20.
  • the formulation further comprises about 0.001% to about 0.05% Tween and may further comprise sodium chloride, e.g. in the range 100 to 150mM such as at a concentration of about 125mM and sodium citrate, e.g. at a concentration in the range 5mM to 50mM, such as about 25mM. Therapeutic Uses
  • An antibody molecule of the invention may be used in, for example, in vitro, ex vivo, and in vivo therapeutic methods.
  • the invention extends to the use of the antibody molecules and nucleic acids of the invention for the treatment of neoplastic disease.
  • treatment means the reduction of the progression, severity and/or duration of a disease or condition or at least one symptom thereof, wherein said reduction or amelioration results from the administration of a binding compound which has specificity for the target binding epitope.
  • the term 'treatment' therefore refers to any regimen that can benefit a subject
  • the treatment may be in respect of an existing condition or may be prophylactic (preventative treatment].
  • Treatment may include curative, alleviative or prophylactic effects.
  • references herein to "therapeutic” and “prophylactic” treatments are to be considered in their broadest context The term “therapeutic” does not necessarily imply that a subject is treated until total recovery. Similarly, “prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition.
  • antibody molecules, nucleic acids or compositions of the invention may be used in the treatment of neoplastic diseases or cancers.
  • Treatment of neoplastic disease or cancer includes treatment of conditions caused by cancerous growth and/or vascularisation and includes the treatment of neoplastic growths or tumours.
  • tumours examples include, for instance, sarcomas, including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , kidney, cervical and ovarian carcinoma, non-small cell lung cancer, hepatocellular carcinoma, lymphomas, including Hodgkin and non-Hodgkin lymphomas,
  • sarcomas including osteogenic and soft tissue sarcomas
  • carcinomas e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , kidney, cervical and ovarian carcinoma
  • non-small cell lung cancer hepatocellular carcinoma
  • lymphomas including Hodgkin and non-Hodgkin lymphomas
  • neuroblastoma neuroblastoma, melanoma, myeloma, Wilms tumor, and leukemias, including acute lymphoblastic leukaemia and acute myeloblasts leukaemia, astrocytomas, gliomas and retinoblastomas.
  • Certain embodiments of the invention may be particularly useful in the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.
  • antibody molecules, nucleic acids and compositions of the invention may be used to treat other conditions, for example other conditions or disorders mediated by or associated with angiogenesis.
  • conditions include, various inflammatory disorders, tumours, various autoimmune disorders, some hereditary disorders, and ocular disorders.
  • the antibody molecules and methods of the invention may be used in the treatment of angiogenesis associated inflammation, including various forms of arthritis, such as rheumatoid arthritis and osteoarthritis, chronic inflammatory conditions including ulcerative colitis, Crohn's disease, bartonellosis, and
  • angiogenesis-mediated disorders for which the invention may find use include hemangioma, solid tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, retrolental fibroplasia, arthritis, diabetic neovascularization, peptic ulcer, Helicobacter related diseases, fractures, keloids, and vasculogenesis.
  • Ocular Disorders mediated by angiogenesis for which some embodiments of the invention may be used include macular degeneration, ocular neovascular disease corneal graft rejection, neovascularization following injury or infection, rubeosis, diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma, corneal diseases and macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, diseases associated with corneal neovascularization including, but are not limited to, epidemic
  • keratoconjunctivitis Vitamin A deficiency, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, and periphigoid radial keratotomy, diseases associated with retinal/choroidal neovascularization including, but not limited to, macular
  • the antibody molecules may be used in combination with chemotherapeutic agents.
  • chemotherapeutic agents for example
  • chemotherapeutic agents which may be used include antimetabolites, including thymidylate synthase inhibitors, nucleoside analogs, platinum cytotoxic agents, topoisomerase inhibitors or antimicrotubule agents.
  • thymidylate synthase inhibitors which may be used in the invention include 5-FU, MTA and raltitrexed (TDX].
  • platinum cytotoxic agents which may be used include cisplatin and oxaliplatin.
  • chemotherapeutic agents which may be used in the present invention, in addition or instead of the specific agents recited above, may include alkylating agents; alkyl sulfonates; aziridines; ethylenimines; methylamelamines; nitrogen mustards; nitrosureas; anti-metabolites; folic acid analogues; purine analogs; pyrimidine analogs; androgens; anti-adrenals; folic acid replenishers; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;
  • defofamine demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid;
  • gallium nitrate gallium nitrate; hydroxyurea; lentinan; ionidamine; mitoguazone; and mitoxantrone.
  • the chemotherapeutic agent is a
  • the topoisomerase inhibitor is a topoisomerase I inhibitor, for example a camptothecin.
  • a suitable topoisomerase I inhibitor which may be used in the present invention is irinotecan (CPT-11] or its active metabolite SN-38.
  • CPT-11 specifically acts in the S phase of the cell cycle by stabilizing a reversible covalent reaction intermediate, referred to as a cleavage or cleavage complex, and may also induce G2-M cell cycle arrest.
  • the chemotherapeutic agent is a fluoropyrimidine e.g. 5-FU.
  • fluoropyrimidine e.g. 5-FU.
  • binding specificity refers to the ability of the antibody molecules of the invention to bind to a target epitope with a greater affinity than that which results when bound to a non-target epitope.
  • specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target epitope. In certain embodiments, this affinity is determined by an affinity ELISA assay.
  • affinity can be determined by a BIAcore assay.
  • affinity can be determined by a kinetic method. In certain embodiments, affinity can be determined by an equilibrium /solution method.
  • the present inventors have previously developed an antibody with cross-specificity for AREG and HBEGF; this antibody is described in WO2009/127881.
  • the VH and VL sequences of this murine antibody, designated Fsnl006, are shown in Figure 1.
  • the CDRs as defined by the Rabat method, the IMGT method and the Chothia method are shown in Table 1 below the sequences.
  • the inventors selected the CDR sequences by employing a combined IMGT and Rabat CDR sequence in which (i] the CDRH1 comprises the first amino acid residue of the CDRH1 of the donor antibody as defined by the IMGT method, the final amino acid residue of the CDRH1 as defined by the Rabat method, and therebetween the intervening amino acid residues of said donor CDR between said first and final amino acid residues; (ii] the CDRH3 comprises the first amino acid residue of the CDRH3 of the donor antibody as defined by the IMGT method, the final amino acid residue of the CDRH3 as defined by the Rabat method and therebetween the intervening amino acid residues of said donor CDR between said first and final amino acid residues; and (iii] the CDRH2, CDRL1, CDRL2, and CDRL3 sequences of the humanised antibody molecule correspond to the CDRH2, CDRL1, CDRL2, and CDRL3 respectively of the donor antibody as defined by the Rabat method.
  • VH2 ABM67208, (VH2) ABI50723, (VH3) AAR32431, (VL1) ABC66929, (VL2) ABI74076, and (VL3) CAA51099.
  • FIG 4 the murine Fsnl006 VL sequence has been aligned with the three humanized VL variants.
  • Fsnl006 VL is the murine sequence and VL1-3 are the humanized variants.
  • the Fusion CDRs are underlined. Rey residues important for the VH/VL interface and canonical loop structure are asterisked (*]. As far as possible these have been maintained in the humanized variants and have not been mutated.
  • Each of the VH domains was synthesised in-frame with a human IgGl isotype constant domain sequence.
  • the entire heavy chain sequence was codon optimised (GeneArt, Germany] and the DNA sequence verified.
  • the amino acid sequence of the IgGl constant domain is ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMI SRTPE VTC VWD VSHED PE VKFN WYVD GVE VHN AKTKPRE E QYN STYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQGNVFSC
  • VL domains were synthesised in-frame with a human IgK isotype constant domain sequence.
  • the entire light chain sequence was codon optimised (LifeTech,
  • the humanised variants were expressed in CHO cells. There is one chimeric antibody (VH0-VL0], having the murine variable domains and the human IgG constant domains, and 9 humanised variants having humanised variable domains and human IgG constant domains (VH1-VL1 to VH3-VL3]. N.B. VH1, VH2 etc and VL1, VL2 etc are alternative nomenclature for HC1, HC2 etc and HL1, HL2 etc respectively.
  • Suspension adapted CHO Kl cells (CHO-S cells] (Invitrogen, UK] were routinely cultivated at 2 - 3 X 10 5 cells/ml at 150rpm, 8% C0 2 , 37°C in Pro CHO 4 serum free medium (Lonza, UK] plus 8mM L-glutamine (PAA, Analab, UK] and HT (Invitrogen, UK] in 500ml vented Erlenmeyer flasks (Corning, Netherlands]. Before transfection (24hours and 48 hours] cells were passed at 3X10 5 cells/ml.
  • the cells were then left standing for 30 mins at 37°C and then placed on a shaker at 150 rpm, 8% CO2 and 37°C for 4 hours. An equal volume of Pro CHO 5 medium was then added (final cell density was lX10 6 cells/ml]. After 7 days incubation at 150 rpm, 8% CO2, 37°C, the supernatant was harvested by centrifugation at 4,000 rpm for 40 mins at 4°C for purification.
  • the Murine 2F7 antibody was humanized and nine variants of this FSN1006 human antibody was produced in CHO-S cells by transient transfection and purified using in house standard operating procedures as described above. The subsequent humanized variants were sent for Biacore analysis (Biaffin, Kassel, Germany). The top three variants were then chosen based on these results according to antibody binding affinities and kinetics. Subsequent in house experiments were carried out to ascertain the best lead candidate in terms of binding affinity and activity.
  • Heavy Chain 1 Light Chain 1 (HC1:LC1)
  • Heavy Chain 2 Light Chain 1 (HC2:LC1
  • Heavy Chain 1 Light Chain 2 (HC1:LC2
  • Heavy Chain 2 Light Chain 2 (HC2:LC2)
  • Heavy Chain 3 Light Chain 2 (HC3:LC2)
  • Heavy Chain 1 Light Chain 3 (HC1:LC3
  • Heavy Chain 2 Light Chain 3 (HC2:LC3)
  • Heavy Chain 3 Light Chain 3 (HC3:LC3)
  • Heavy Chain 0 Light Chain 0 Chimeric Antibody
  • Biacore analysis was performed as a single cycle kinetics experiment by immobilizing the antibody to a a-human Fc on CM5 sensor chips and subsequently injecting five concentrations of the antigen without regeneration of the surface in order to monitor association and dissociation rates providing sufficiently accurate values of kinetic constants [kass, Miss] and affinities [KD) for a kinetic ranking.
  • the Biacore results are summarised in Tables 4 and 5.).
  • the binding of the generated humanised antibodies was tested via competition ELISA. Briefly, 96-well Maxisorp plate (Nunc] were coated with lOOng/well of AREG or HB EGF antigen ( ⁇ g/ml stock] in coating buffer and incubated overnight at 4°C. Coating solution was removed and 200 ⁇ /well of blocking solution (3.0% w/v semi skimmed milk in IX PBS] was added. Plates were agitated at room temperature for 2 hours.
  • Blocking solution was removed and plates were washed IX PBS Tween 20 (0.1%] (PBS- T].
  • Murine or humanized Fsnl006 antibodies were diluted to a starting concentration of ⁇ g/ml and serial 1:1 dilutions then made down to 0.03 ⁇ g/ml in PBS. Each dilution (100 ⁇ /well] was added in triplicate with a PBS negative control. Plates were incubated with agitation at room temperature for 2 hours. Primary antibody was removed and plates were washed 4 x PBS-T.
  • Figure 5 illustrates the results of analysis of binding of the nine humanised antibody constructs to HB-EGF in comparison to the murine antibody (2F7] and the chimeric antibody (H0LO].
  • the ELISA binding data confirmed that the variants H2L1(HC2:LC1], H2L2(HC2:LC2], and H2L3(HC2:LC3] had very similar binding characteristics to the murine antibody.
  • binding of the variants having the HI or H3 chains was not as strong, binding nevertheless occurred to the H1L1(HC1:LC1], H1L2(HC1:LC2], H3L1(HC3:LC1] and H3L2(HC3:LC2] antibodies.
  • Table 6 The results are summarised in Table 6 below
  • the ability of the FSN1006 antibody to inhibit phosphorylation of the Epidermal Growth Factor Receptor (EGFR] was determined using western blots.
  • the MTT assay was used to determine the cytotoxicity and anti-proliferative activities of FSN1006.
  • HBEGF Inhibition of EGFR phosphorylation HBEGF (30ng/ml) and FSN 1006 antibodies (50nM-l,000nM) were pre- incubated on ice for 30 minutes before treating HCT 116 cells for 20 minutes to determine if the presence of FSN 1006 constructs could block the phosphorylation of EGFR by HBEGF. Lysates were prepared form the treated cells and separated by SDS PAGE and western blotted. The blots were then probed with anti- phospho EGFR antibody. The presence of a heavy band indicated that phopsorylated EGFR was detected and was being activated by HBEGF.
  • Lane 1 PBS treated HCT116 cells neg control
  • Lane 2 ⁇ , ⁇ HC2LC2
  • Lane 3 ⁇ , ⁇ HC2LC2 + 30ng/ml HBEGF
  • Lane 7 50nM HC2LC2 + 30ng/ml HBEGF
  • the humanised antibodies were tested for antiproliferative activities on a number of cell lines using the MTT (3-(4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide] assay. Briefly, 5000 cells were seeded per well on 96 well plate. After 24 hours cells were treated with the humanised antibody of interest at different concentrations.
  • MTT 3-(4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • FIG. 7 illustrates the results of an MTT assay using the humanised antibody variants having heavy chain 2.
  • the HC2LC3 variant appeared to be the most effective of the three constructs with a cell kill of 30-40% observed at concentrations of 100-150 ⁇ g/ml.
  • the HC2LC2 antibody proved the next best effective with cell kill at 25-30% whilst HC2LC1 showed cell death from 15-32% observed at concentrations of 100-150 ⁇ g/ml.
  • Example 1 Using the method as employed in Example 1, the inventors have also designed and generated a humanised anti-CD20 antibody, specifically a humanised version of rituximab.
  • VH and VL sequences of rituximab are shown in Figure 8.
  • the CDRs as defined by the Rabat method, the IMGT method and the Chothia method are shown in the Table below the sequences. Humanised versions of this antibody were prepared by CDR grafting.
  • the inventors selected the CDR sequences by employing a combined IMGT and Rabat CDR sequence in which (i] the CDRH1 comprises the first amino acid residue of the CDRH1 of the donor antibody as defined by the IMGT method, the final amino acid residue of the CDRH1 as defined by the Rabat method and therebetween the intervening amino acid residues of said donor CDR between said first and final amino acid residues; (ii] the CDRH3 comprises the first amino acid residue of the CDRH3 of the donor antibody as defined by the IMGT method, the final amino acid residue of the CDRH3 as defined by the Rabat method and therebetween the intervening amino acid residues of said donor CDR between said first and final amino acid residues; and (iii] the CDRH2, CDRL1, CDRL2, and CDRL3 sequences of the humanised antibody molecule correspond to the CDRH2, CDRL1, CDRL2, and CDRL3 of the donor antibody as defined by the Rabat method.
  • the CDRH1 comprises the first amino acid residue of the C
  • FIG 9 the murine rituximab VH sequence has been aligned with the three humanized VH variants.
  • VHO is the murine sequence and VH1-3 are the humanized variants.
  • the CDRs are marked by boxes.
  • VLO murine rituximab VL sequence
  • Fsnl006 VLO is the murine sequence
  • VL1-3 are the humanized variants.
  • the "Fusion CDRs" are indicated by the boxes.
  • Each of the VH domains was synthesised in-frame with a human IgGl isotype constant domain sequence.
  • the entire heavy chain sequence was codon optimised (GeneArt, Germany] and the DNA sequence verified.
  • Each of the VL domains was synthesised in- frame with a human IgK isotype constant domain sequence.
  • the entire light chain sequence was codon optimised (LifeTech, Germany] and the DNA sequence verified.
  • the humanised variants were expressed in CHO cells.
  • the humanised heavy chains and humanised light chains were recombined to 9 humanised variants.
  • Binding of the nine humanized rituximab variants to Raji cells was assessed to compare their functional activity in binding to CD20.
  • Raji cells were harvested in the log phase of growth and washed x3 in PBS. Cells were blocked with BSA, and then incubated with primary antibody (rituximab positive control, humanized rituximab variants, PBS control, human IgG negative control] for lhr at room temp. Cells were washed x 3 with PBS and then incubated with secondary goat-anti-human-FITC antibody for lhr at room temperature. Cells were washed x3 with PBS and then analysed by flow cytometry for positive binding.

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Abstract

La présente invention concerne un procédé permettant d'humaniser des molécules d'anticorps, ledit procédé consistant à utiliser des CDR définis par un procédé qui combine les méthodes IMGT et de Kabat pour définir les séquences CDR. L'invention concerne spécifiquement un anticorps humanisé doté d'une spécificité de liaison pour AREG et HBEGF et un anticorps anti-CD20 humanisé.
PCT/GB2013/052965 2012-11-09 2013-11-11 Anticorps humanisé et son procédé de production WO2014072741A1 (fr)

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WO2020232165A1 (fr) * 2019-05-14 2020-11-19 Qlb Biotherapeutics Anticorps anti-cd3 x anti-cd20 bispécifiques et leurs utilisations
WO2021190437A1 (fr) * 2020-03-27 2021-09-30 National Institute Of Biological Sciences, Beijing Anticorps contre areg et leur utilisation
JP2022506430A (ja) * 2018-10-31 2022-01-17 ジョイント・ストック・カンパニー “バイオキャド” Ｃｄ２０に特異的に結合するモノクローナル抗体
US11492394B1 (en) 2021-10-29 2022-11-08 Nascent Biotech, Inc. Kits and containers for treating vimentin expressing tumors
WO2024189340A1 (fr) 2023-03-10 2024-09-19 Fusion Antibodies Plc Anticorps et leurs utilisations

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