Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
  • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1089—Design, preparation, screening or analysis of libraries using computer algorithms
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B10/00—Directed molecular evolution of macromolecules, e.g. RNA, DNA or proteins
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/567—Framework region [FR]
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)

Definitions

• the present invention relates to methods of generating antibody libraries, antibody libraries produced using such methods, and variant antibodies.
• Antibody affinity is the measure of the strength of interaction between the antibody and protein that it specifically binds to, as a ratio of the association rate and dissociation rate. There are many reasons why optimisation of this ratio is desirable. Increased affinity may mean that an antibody therapeutic is more effective at a given dose, or it may mean that less of the drug is required per dose, and diagnostic tests could improve sensitivity.
• Reducing affinity may also be beneficial for some drugs that require tissue penetration and therefore a faster dissociation. It has also been shown that bispecific antibodies require intricate tuning of affinity of each binding site in order to maximise efficacy.
• the present invention addresses many of the problems of the prior art.
• the inventors have surprisingly shown that by limiting mutations to a nucleotide sequence encoding a given antibody sequence to sites which correspond to DNA motifs which are targeted by enzymes involved in somatic hypermutation, a library of variants of a given antibody sequence may be created, the library, when compared to those prepared by many existing technologies, being relatively small but comprising a relatively high proportion of variants having increased affinity, or aggregation, or melting point, or expression level in CHO cells or combinations of the same.
• the inventors have exemplified the invention utilising two unrelated antibodies, an anti-CathepsinS antibody, Fsn0503h (Fusion Antibodies Ltd), and the anti-HER2 antibody trastuzumab, (Herceptin ® , Roche).
• a first aspect of the present invention provides a library of antibody molecules, wherein each antibody molecule is a variant of a reference antibody, wherein the amino acid sequence of each antibody molecule differs from the amino acid sequence of the reference antibody at one or more amino acid residues, wherein each of said amino acid residues are independently encoded from a DNA segment of the DNA sequence encoding the variant, wherein said DNA segment of the variant differs from that of the corresponding DNA sequence encoding the reference antibody by a point mutation in a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.
• a second aspect of the invention provides a method of generating/producing a library of variant antibody molecules, wherein said variant antibody molecules are variants of a reference antibody, said method comprising the steps: a) providing a nucleotide sequence encoding the reference antibody, b) in said nucleotide sequence, identifying one or more DNA motifs susceptible to deamination by a somatic hypermutation inducing enzyme; c) for one or more of said DNA motifs, selecting at least one variant nucleotide residue to substitute for a residue of said DNA motif, wherein said substitution will result in a variant nucleotide sequence which encodes a variant antibody molecule having, relative to the reference antibody, a change in the amino acid sequence being encoded by said DNA motif; and d) repeating steps (b) and (c); such that a library containing a plurality of variants of said reference antibody is generated.
• the reference antibody may be any antibody molecule of which variants are desired or required.
• said reference antibody is a humanised antibody molecule.
• a third aspect of the invention provides a method of generating a variant antibody molecule, wherein said variant antibody molecule is a variant of a reference antibody, said method comprising the steps: a) providing a nucleotide sequence encoding the reference antibody, b) in said nucleotide sequence, identifying one or more DNA motifs susceptible to deamination by a somatic hypermutation inducing enzyme; c) for one or more of said DNA motifs, selecting at least one variant nucleotide residue to substitute for a residue of said DNA motif, wherein said substitution will result in a variant nucleotide sequence which encoding a variant antibody molecule having, relative to the reference antibody, a change in the amino acid sequence being encoded by said DNA motif.
• said somatic hypermutation inducing enzyme is Activation-Induced Deaminase (AID).
• said DNA motif is DGYW or WRCH, for example RGYW or WRCY, where D is adenine, guanine or thymine, R is adenine or guanine, G is guanine, C is cytosine, H is adenine or cytosine or thymine, W is adenine or thymine, and Y is cytosine or thymine.
• Said DNA motif may be on either strand of the DNA. Where there are more than one of said DNA motifs, the motifs may overlap.
• the inventors have shown that the antibody library of the invention or produced according to a method of the invention may be refined by further limiting the members of the library to those variants resulting from mutations at the DNA motif targeted by a somatic hypermutation inducing enzyme which do not result in either a STOP codon or indeed a other motifs in the variant antibody molecule which relative to the reference antibody would be considered to be potentially undesirable, for example in terms of stability or binding.
• the DNA sequence of each variant does not comprise (or encode) a deamination site, isomerisation site, N-linked glycosylation site or oxidation site which originates from said point mutation in said DNA motif.
• one or more of said DNA motifs are in DNA sequences which encode CDRs of said antibody molecule.
• the inventors have shown that using the method with some reference antibodies, the variants generated may not have any mutations in some CDRs compared to the corresponding CDRs of the reference antibody.
• the variants have no changes in one or more of its CDRs compared to the corresponding CDRs of the reference sequence.
• the variants have no changes in Light chain CDR1 compared to the corresponding CDR of the reference sequence.
• the variants have no changes in Light chain CDR2 compared to the corresponding CDR of the reference sequence.
• the variants have no changes in Light chain CDR3 compared to the corresponding CDR of the reference sequence.
• the variants have no changes in heavy chain CDR1 compared to the corresponding CDR of the reference sequence.
• the variants have no changes in heavy chain CDR2 compared to the corresponding CDR of the reference sequence.
• the variants have no changes in heavy chain CDR3 compared to the corresponding CDR of the parent sequence.
• many of the mutations characterising the variants may be in the framework regions of the variant antibody molecules.
• one or more of said DNA motifs are in DNA sequences which encode framework regions of said antibody molecule.
• all of said DNA motifs are in DNA sequences which encode framework regions of said antibody molecule.
• greater than 20% for example greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% of the nucleotide residues of each variant antibody which differ from nucleotide residues in corresponding positions in the reference antibody are residues at a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.
• the nucleotide sequence encoding each of said antibody molecules does not differ from that of the reference antibody at any residue other than residues of said DNA motif.
• An antibody library of the invention and/or produced using the method of the invention may be further refined to enhance the proportion of variants of high affinity and/or stability.
• An antibody library of the invention and/or produced using the method of the invention may be further refined to enhance the proportion of variants with differential aggregation, i.e. less able to aggregate to each other.
• An antibody library of the invention and/or produced using the method of the invention may be further refined to enhance the proportion of variants with particular melting point characteristics.
• An antibody library of the invention and/or produced using the method of the invention may be further refined to enhance the proportion of variants which show favourable or desired expression level in CHO cells.
• An antibody library of the invention and/or produced using the method of the invention may be further refined to enhance the proportion of variants with high affinity, stability, desired aggregation characteristics, desired melting point characteristics, desired expression level characteristics or combinations of the same.
• the method may further comprise determining the affinity and/or stability of binding of said variant antibody molecule to the binding target of the reference antibody, melting point in relation to a reference antibody, aggregation in relation to a reference antibody, expression level in relation to a reference antibody or combinations of the same. Accordingly in one embodiment of the method of the invention, said method further comprises screening said library of variants to determine binding to an epitope to which the reference antibody binds. Suitably, the method of the invention, may further comprises screening said library of variants to determine a melting point, expression level, aggregation level or stability of a variant with respect to the reference antibody.
• Those variants determined to bind to said epitope with an affinity and/or stability/ or have a melting characteristic or aggregation characteristic or expression characteristic less or greater than a predetermined value relative to the reference antibody may be used to generate an optimised library of variant antibody molecules.
• Said screening method may be via conventional in vitro techniques. Such techniques may include an affinity ELISA assay, a BIAcore assay, a kinetic method or an equilibrium/solution method.
• said screening may be via an in silico technique, using, for example computer implemented molecular docking software to model the binding of the variant to the epitope of the reference antibody.
• variants can be ranked by predicted affinity and stability allowing the selection of a small library of variants for DNA synthesis and expression.
• the inventors have demonstrated that within such a small library a very high number of the variants have increased affinity compared to the number which would be expected to be identified in a library generated by existing technologies such as error-prone PCR and phage display.
• Molecular docking software products are commercially available. Any suitable software or tool suitable for modelling antibody binding to an epitope may be used in the present invention.
• suitable software includes Bioluminate software from Schrodinger but others are available.
• the antibody library comprises greater than 1%, for example greater than 5%, 10%, 20%, 30% , 40% or 50% of variants having increased affinity to the epitope bound by the reference antibody compared to the affinity of the reference antibody to said epitope.
• the method is a computer implemented method.
• a fourth aspect of the invention provides a computer readable storage media comprising instructions to perform a method for generating a library of variant antibody molecules according to the second aspect of the invention.
• the method may further comprise the step of synthesising the variant antibody molecules.
• the method of the invention may further comprise in vitro screening of said library of variants to determine binding to an epitope to which the reference antibody binds.
• a fifth aspect of the present invention is a variant of a trastuzumab antibody, wherein said variant has (i) at least two amino acid changes in the light chain sequence when compared to the light chain amino acid sequence of a reference antibody, wherein said reference antibody is trastuzumab, (ii) at least two amino acid changes in the heavy chain sequence when compared to the heavy chain amino acid sequence of trastuzumab, or (iii) at least one amino acid change in the light chain sequence when compared to the light chain amino acid sequence of trastuzumab and at least one amino acid change in the heavy chain sequence when compared to the heavy chain amino acid sequence of trastuzumab; wherein each of said amino acid changes are at amino acid residues independently encoded from a DNA segment of the variant DNA sequence, wherein said DNA segment of the variant differs from that of the corresponding DNA sequence encoding the reference antibody by a point mutation in a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.
• said amino acid changes are selected from the group consisting of lc9N, lc9T, Ic9l, lc9R, lc9K, lc25G, lc25V, lc25D, lc31N, lc31S, Ic31l, lc32D, lc32G, lc32V, lc32T, lc32N, lc32S, Ic32l, lc32P, lc32L, lc32F, lc33L, Ic33l, lc34G, lc34V, lc34D, lc38E, lc38K, lc40A, lc40S, lc40T, lc43G, lc43V, lc43T, lc43N, lc43S, Ic43l, lc43P, lc43L, lc
• lc9N refers to an asparagine at position 9 of the variant trastuzumab light chain.
• said amino acid changes are selected from the group consisting of lc9T, Ic9l, lc9R, lc9K, lc43F, lc47V, lc51P, lc51T, IclOlD, hc2L, hc3H, hcl4S, hcl6V, hc24P, hc26A, hc26V, hc48l, hc58S, hc61V, hc79V, hc85R, hc88G, hc92V, hc92D, hcl03A, hcl03V, hcl06V, hcll4S, hcll4N, and hcll4l.
• the variant light chain and heavy chain sequences do not differ from that of the reference antibody at any residue other than by the amino acid changes recited above in relation to the fifth aspect.
• a sixth aspect of the invention provides a variant of a Cathepsin S antibody, wherein said variant has (i) at least two amino acid changes in the light chain sequence when compared to the light chain amino acid sequence of a reference antibody, wherein said reference antibody is Fsn503h, (ii) at least two amino acid changes in the heavy chain sequence when compared to the heavy chain amino acid sequence of Fsn503h, or (iii) at least one amino acid change in the light chain sequence when compared to the light chain amino acid sequence of Fsn503h and at least one amino acid change in the heavy chain sequence when compared to the heavy chain amino acid sequence of Fsn503h; wherein each of said amino acid changes are at amino acid residues independently encoded from a DNA segment of the variant DNA sequence, wherein said DNA segment of the variant differs from that of the corresponding DNA sequence encoding the reference antibody by a point mutation in a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.
• said amino acid changes are selected from the group consisting of lcl2A, lcl2S, lcl2T, lcl9V, lc28R, lc32T, Ic32l, lc45A, lc45S, lc45T, lc50H, lc51V, lc51F, Ic51l, lc56L, lc56F, Ic56l, lc58K, lc66S, lc69A, lc69V, lc81T, Ic81l, lc81N, lc85P, lc85S, lc85T, lc90L, lc90F, Ic96l, lc96S, Ic96l, lc96N, lcl08N, hc3H, hc4V, hc4M, hclOA, hclOV
• said amino acid changes are selected from the group consisting of group consisting of lcl2A, lcl2S, lcl2T, lcl9V, lc45S, lc45T, lc50H, lc51V, Ic56l, Ic81l, Ic96l, lc96S, Ic96l, lc96N, lcl08N, hclOA, hclOV, hcl4S, hc30l, hc31R, hc37L, hc37F, hc40P, hc40S, hc52l, and hc92G.
• the variant light chain and heavy chain sequences do not differ from that of the reference antibody at any residue other than by the amino acid changes recited above in relation to the sixth aspect.
• said variant antibody molecule has the combination of amino acid mutations as shown for any of the antibodies listed in Tables 2 to 7. In another embodiment of the fourth aspect, said variant antibody molecule has the combination of amino acid mutations as shown for any of the antibodies listed in Table 8. In one such embodiment, said variant antibody has the combination of amino acid mutations as shown for any one of the antibodies listed in Table 8 and does not have any amino acid mutations relative to the Trastuzumab light chain and heavy chain sequences other than those shown for said antibody listed in Table 8.
• said variant antibody has the mutation lc43F.
• said variant antibody has the combination of amino acid mutations as shown for any of variant antibodies 19, 5 or 6 in Table 8. In one such embodiment, said variant does not have any amino acid mutations relative to the Trastuzumab light chain and heavy chain sequences other than those shown for said variant antibody in Table 8.
• the variant antibody is a variant of a trastuzumab antibody comprising relative to trastuzumab, the following amino acid changes: lc9K, lc43F, and hcl06V.
• the variant antibody is variant antibody 19 as listed in Table 8.
• the variant antibody is a variant of a trastuzumab antibody comprising relative to trastuzumab, the following amino acid changes: lc9R, lc43F, and hcll4S.
• the variant antibody is variant antibody 5 as listed in Table 8.
• the variant antibody is a variant of a trastuzumab antibody comprising relative to trastuzumab, the following amino acid changes: Ic9l, lc43F,lcl01D, and hc79V.
• the variant antibody is variant antibody 6 as listed in Table 8.
• said variant antibody molecule has the combination of amino acid mutations as shown for any of the antibodies listed in Table 1.
• said variant light chain and heavy chain sequences comprise in total at least three, for example at least 4, at least 5 or at least 6 amino acid changes compared to the amino acid sequence of the reference antibody.
• one or more of said amino acid changes are in framework regions of said variant antibody.
• all of said amino acid changes are in framework regions of said variant antibody.
• one or more of said amino acid changes are in CDRs of said variant antibody.
• the change of affinity of said variant antibody molecule relative to the reference antibody is greater than -2 and the change of stability of said variant antibody molecule relative to reference antibody is greater than -2. In certain embodiments of the fifth or sixth aspects of the invention, the change of affinity of said variant antibody molecule relative to the reference antibody is greater than -10, for example greater than - 15, for example, greater than -20, such as greater than -25.
• the change of stability of said variant antibody molecule relative to the reference antibody is greater than -10, for example greater than - 30, for example, greater than -50, such as greater than -60.
• the aggregation characteristic, melting point characteristic or expression level may be at least 2 fold, three fold, 10 fold different to the reference antbody.
• an antibody with an affinity value of -10 is considered to have a greater affinity value of an antibody with an affinity value of -5.
• the greater the negative stability value the greater the stability.
• an antibody with a stability value of -10 is considered to have a greater stability value of an antibody with a stability value of -5.
• Affinity and stability may be assessed by any suitable method.
• the inventors used the residue scanning affinity maturation tool as part of Schrodinger's Biologies tool Maestro. Values were relative to the parental antibody with the minimum value for improved affinity taken to be -2 keal/mol for both affinity and stability.
• DNA segment refers to a section of a DNA sequence.
• the DNA segment may be a section of DNA residues, said section being part of a longer DNA sequence which encodes an antibody molecule.
• the DNA segment may consist of the DNA residues which form a DNA motif, said DNA motif being a sequence- specific binding site for a somatic hypermutation inducing enzyme
• an “epitope” refers to a plurality of amino acid residues which are capable of being recognised by, and bound to by, an antibody molecule.
• Epitopes are generally comprised of chemically active surface groups and have specific three-dimensional structural characteristics, as well as specific charge characteristics which contribute to the three-dimensional structure of the epitope.
• Antibody molecules of or for use in the invention may bind to a non-contiguous epitope.
• a "non-contiguous epitope” is an epitope that is comprised of a series of amino acid residues that are non-linear in alignment, such that the residues are spaced or grouped in a non- continuous manner along the length of a polypeptide sequence.
• polypeptide and “protein” are used herein interchangeably to describe a series of at least two amino acids covalently linked by peptide bonds or modified peptide bonds such as isosteres. No limitation is placed on the maximum number of amino acids which may comprise a peptide or protein.
• polypeptide extends to fragments, analogues and derivatives of a peptide, wherein said fragment, analogue or derivative retains the same biological functional activity as the peptide from which the fragment, derivative or analogue is derived.
• an “antibody” is an immunoglobulin, whether natural or partly or wholly synthetically produced.
• the term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.
• Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb or Fd, and a bi-specific antibody.
• antibody and antibody molecule should be construed as covering any binding member or substance having a binding domain with the required specificity.
• An antibody molecule of and for use in the invention may be a monoclonal antibody, or a fragment, derivative, functional equivalent or homologue thereof.
• the term includes any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.
• the constant region of the antibody may be of any suitable immunoglobulin subtype.
• the subtype of the antibody may be of the class IgA, IgM, IgD and IgE where a human immunoglobulin molecule is used.
• Such an antibody may further belong to any subclass, e.g. IgGl, lgG2a, lgG2b, lgG3 and lgG4.
• Fragments of a whole antibody can perform the function of antigen binding.
• binding fragments are a Fab fragment comprising or consisting of the VL, VH, CL and CHI antibody domains; an Fv fragment consisting of the VL and VH domains of a single antibody; a F(ab')2 fragment; a bivalent fragment comprising two linked Fab fragments; a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; and a bi-specific antibody, which may be multivalent or multispecific fragments constructed by gene fusion.
• humanized antibodies may be used.
• a humanised antibody may be a modified antibody having the hypervariable region of non-human antibody and the constant region of a human antibody.
• the binding member may comprise a human constant region.
• the variable region other than the hypervariable region may also be derived from the variable region of a human antibody and/or may also be derived from a non-human antibody. In other cases, the entire variable region may be derived from a non human antibody and the antibody is said to be chimerised.
• An antibody may be selected from the group consisting of a human antibody, a humanised antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a camelid antibody, a shark antibody and an in-vitro antibody.
• an antigen binding fragment may be used.
• the antigen binding fragment may be derived from any of the aforementioned antibodies.
• the antigen binding fragment is selected from the group consisting of a Fab fragment, a scFv fragment, a Fv fragment and a dAb fragment.
• the antibody comprises two complete heavy chains and two complete light chains, or an antigen binding fragment thereof.
• the antibody is of the isotype IgG, IgA, IgE or IgM, or an antigen binding fragment thereof. In certain embodiments where the antibody is of the isotype IgG, the antibody may be of the subtype IgGl, lgG2 or lgG3, or an antigen binding fragment thereof. In certain embodiments the antibody is of the subtype lgG4, or an antigen binding fragment thereof.
• Antibodies may be provided by a number of techniques. For example, a combinatorial screening technique such as a phage display-based biopanning assay may be used in order to identify amino acid sequences which have binding specificity to an antigen.
• a combinatorial screening technique such as a phage display-based biopanning assay may be used in order to identify amino acid sequences which have binding specificity to an antigen.
• phage display biopanning techniques involve the use of phage display libraries, which are utilised in methods which identify suitable epitope binding ligands in a procedure which mimics immune selection, through the display of antibody binding fragments on the surface of filamentous bacteria. Phage with specific binding activity are selected. The selected phage can thereafter be used in the production of chimeric, CDR-grafted, humanised or human antibodies.
• Antibodies can be tested for their ability to bind to an antigen using methods known in the art.
• Antibodies or antigen fragments for use in the present invention may also be generated wholly or partly by chemical synthesis.
• the antibodies can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available and are well known by the person skilled in the art. Further, they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry.
• Another convenient way of producing antibodies or antibody fragments suitable for use in the present invention is to express nucleic acid encoding them by use of nucleic acid in an expression system.
• Antibodies may be generated by mutagenesis of antibody genes to produce artificial repertoires of antibodies. This technique allows the preparation of antibody libraries.
• Artificial repertoires of immunoglobulins such as artificial scFv repertoires, may be employed as an immunoglobulin source in order to identify binding molecules which have specificity for a specific epitope.
• Any suitable means of generating antibody libraries may be used in conjunction with the present invention.
• Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
• Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in-vitro selection and amplification of specific antibody fragments that bind a target antigen.
• the nucleotide sequences encoding the VH and VL regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
• phage-based display systems are that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straight forward.
• a method for producing polypeptides may comprise culturing host cells transformed with a recombinant expression vector encoding a polypeptide under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture.
• the person skilled in the art will recognise that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is intracellular, membrane-bound or a soluble form that is secreted from the host cell.
• Vectors may include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, avian, microbial, viral, bacterial, or insect gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence.
• An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
• a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
• a DNA sequence for a signal peptide may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide.
• a signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, but allows secretion of polypeptide from the cell.
• Suitable host cells for expression of polypeptides include higher eukaryotic cells and yeast. Prokaryotic systems are also suitable.
• Mammalian cells, and in particular CHO cells are particularly preferred for use as host cells.
• CHO cells are used widely in the production of proteins due to their ease of culture and transfectability.
• ExpiCHO-S cells are a suspension cell line that can grow to very high densities and allows for high expression of proteins.
• the plasmid DNA of interest can be transfected into ExpiCHO cells by complexing with ExpiFectamine (a cationic lipid-based transfection reagent) to allow the DNA to condense. This condensed DNA then enters the ExpiCHO cells by endocytosis and is expressed in the nucleus.
• the expressed proteins are present in the cell culture supernatant and harvested after an appropriate number of days have passed.
• Nucleic acid for use in accordance with the present invention may comprise DNA or RNA and may be wholly or partially synthetic.
• nucleic acid for use in the invention codes for antibodies or antibody fragments of the invention as defined above.
• the skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide an antibody molecule of or for use in the present invention.
• Nucleic acid sequences encoding antibodies or antibody fragments for use with the present invention can be readily prepared by the skilled person. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
• PCR polymerase chain reaction
• DNA encoding antibody fragments may be generated and used in any suitable way known to those of skill in the art, including by 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 portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
• the nucleic acid may be comprised as constructs in the form of a plasmid, vector, transcription or expression cassette which comprises at least one nucleic acid as described above.
• 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 the nucleic acid. Following production by expression the antibody or antibody fragments may be isolated and/or purified using any suitable technique, then used as appropriate.
• Suitable host cells 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, NSO mouse myeloma cells.
• a common, preferred 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. General techniques for the production of antibodies are well known to the person skilled in the field.
• nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies.
• nucleic acids comprise coding 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.
• Figure 1 shows amino acid sequences of the Fsn0503h antibody light chain and heavy chain showing potential mutations above each chain (SEQ ID Nos 1 & 2). Doubly circled amino acids were not permitted in the library due to their uncommon usage at that position. Doubly circled asterisks (*)represent STOP codons that also were not permitted. Singly circled amino acid regions are CDRs.
• Figure 2 is a schematic model showing predicted protein-protein interaction between Fsn0503h and Cathepsin S.
• Figure 3 shows Table 1 which lists affinity and stability predictions of amino acid mutations in a combinatorial fashion for 1 to 6 mutations. Groups of mutations highlighted in blue represent variants with both improved stability and affinity where the change is greater than -2.
• Figure 4 illustrates an ELISA comparison of each of the expressed variants with the parental Fsn0503h antibody.
• Figure 5 shows affinity (KD) measurements of each variant alongside the parental Fsn0503h antibody, as measured by BLI with an Octet instrument (Pall).
• Figure 6 illustrates an affinity on-off rate map of the variants of Fsn0503h.
• the parental antibody is shown as the hollow dot.
• the 4.45 nM line represents equal affinity to the parental antibody, and any variants with an increased affinity ( ⁇ 4.45 nM) are within the area to the left of the 4.45 nM line.
• Figure 7 shows amino acid sequences of the trastuzumab antibody light chain (SEQ ID NO: 3) showing in bold the amino acid residue at each position of the wild type antibody light chain and, where applicable, in the boxes to the right of each residue, potential mutations. Amino acids shown with two lines were not permitted in the library due to their uncommon usage at that position. Asterisks (*) in two lines represent STOP codons that also were not permitted. Complementarity determining regions CDRs and framework regions (FR) are indicated.
• Figure 8 shows amino acid sequences of the trastuzumab antibody heavy chain (SEQ ID NO: 4) showing in bold the amino acid residue at each position of the wild type antibody antibody chain and, where applicable, in the boxes to the right of each residue, potential mutations. Amino acids shown with two lines were not permitted in the library due to their uncommon usage at that position. Asterisks (*) in two lines represent STOP codons that also were not permitted. Complementarity determining regions CDRs and framework regions (FR) are indicated.
• Figure 9 schematically illustrates how somatic hypermutations may be used to cause alterations to the DNA sequence of antibodies which results in amino acid changes in the protein structure of the antibody leading to enhanced affinity.
• Figure 10 illustrates the fully resolved crystal structure of trastuzumab in complex with human HER2 which was used to perform residue scanning. Domain IV of the extracellular domain of HER2 was found to bind to trastuzumab.
• Figure 11a shows Table 2 which lists trastuzumab variants having one mutation relative to the reference trastuzumab antibody.
• L indicates light chain, while H indicates heavy chain.
• D Affinity is predicted free energy change (AAG) in kcal/mol relative to trastuzumab affinity.
• D Stability is predicted free energy change (AAG) relative to trastuzumab stability. Negative D Affinity and negative D Stability values are considered to represent improved affinity and improved stability relative to trastuzumab.
• Figure lib shows Table 3 which lists trastuzumab variants having two mutations relative to the reference trastuzumab antibody.
• L indicates light chain
• H indicates heavy chain.
• Figure 11c shows Table 4 which lists trastuzumab variants having three mutations relative to the reference trastuzumab antibody.
• L indicates light chain
• H indicates heavy chain.
• Figure lid shows Table 5 which lists trastuzumab variants having four mutations relative to the reference trastuzumab antibody.
• L indicates light chain
• H indicates heavy chain.
• Figure lie shows Table 6 which lists trastuzumab variants having five mutations relative to the reference trastuzumab antibody.
• L indicates light chain, while H indicates heavy chain.
• Figure Ilf shows Table 7 which lists trastuzumab variants having six mutations relative to the reference trastuzumab antibody.
• L indicates light chain, while H indicates heavy chain.
• Figure 12 shows Table 8 which lists the 100 trastuzumab variants from tables 1 to 7 having both improved affinity and improved stability, ranked according to improved affinity.
• Figure 13 schematically illustrates the top 5 trastuzumab variants scored based on affinity using the predicted free energy change (AAG) in kcal/mol.
• AAG predicted free energy change
• Figure 14 illustrates sensorgrams from 20 samples showing report points used for ranking. Assay performed at 25°C.
• Figure 15 shows a graph illustrating off-rate ranking of Trastuzumab variants binding to HER2 and shows stability_early vs stabilityjate for identification of stable binder (best binders circled in blue). In total, 89 samples were analyzed and ranked with respect to binding stability, however for clarity only 29 are shown.
• Trastuzumab is commercially available antibody material. Wild-Type is antibody having sequence of trastuzumab and transiently expressed in parallel with all Trastuzumab variants/mutants. Mutant is a Trastuzumab variant. Mutants 5, 6 and 19 are trastuzumab variants listed as numbers 5, 6 and 19 in Table 8. Assay performed at 25°C. Figure 16.
• Trastuzumab shows a graph illustrating off-rate ranking of Trastuzumab variants binding to HER2 and shows Stability_early vs stabilityjate for identification of stable binders from a small sub-set of the top binders shown in Figure 15 (those binders closest the 100% left line).
• Trastuzumab is commercially available antibody. Wild-Type is material transiently expressed in parallel with all Trastuzumab variants/mutants. Mutant; is a Trastuzumab variant.
• Mutants 5, 6 and 19 are trastuzumab variants listed as numbers 5, 6 and 19 in Table 8. Assay performed at 37°C.
• Figure 17 shows reference corrected BLI binding curves (black), monitored on a surface of non-covalently immobilised Trastuzumab (a), wild-type (b), mutant 5 (c) and (d) mutant 19 antibodies, for various HER2 concentrations (serial diluted two-fold; highest concentration shown on curve) at 37°C in running buffer.
• Figure 18 illustrates schematically the correlation between affinity prediction and measurement for trastuzumab variants.
• Figure 19 shows blank corrected BLI binding curves (black), monitored on a surface of non- covalently immobilised HER2 at a range of concentrations of wild-type (a), mutant 5 (b) and (c) mutant 19 antibodies, at 37°C in running buffer.
• Figure 20 shows double-reference corrected BLI binding curves (black), monitored on a surface of non-covalently immobilised HER2 at coated at a range of concentrations, presented to a concentration of wild-type (a), mutant 5 (b) and (c) mutant 19 antibodies, at 37°C in running buffer.
• Figure 21 displays the Expression yield of Fsn0503 variants expressed as mg of purified IgG by Octet immunoassay obtained per ml of transfected CHO supernatant. Wild-Type shown in orange.
• Figure 22 displays the melting point determination values (TM1 and TM2) of Fsn0503 variants in degrees centrigrade. Wild-Type is in position 1.
• Figure 23 Shows the measurement of proportion of monodisperse (non-aggregated) molecule of Fsn0503 variants by size-exclusion chromatography. Wild-Type shown in orange.
• An antibody library was generated for each of (i) a humanised anti-Cathepsin S antibody, Fsn0503h (Fusion Antibodies, Harbor) (Kwok et al. Molecular Cancer 2011, 10:147) and (ii) trastuzumab (Roche) by mimicking the natural somatic hypermutation in humans, searching for specific DNA sequence motifs RGYW in the 3' to 5' strand and WRCY in the 5' to 3' strand, recognised by the AID enzyme, responsible for making mutations within antibodies in the human body.
• the DNA sequence has a point mutation introduced at the guanine in position 2 of the DNA sequence motif RGYW or the cytosine in position 3 of the DNA sequence motif WRCY, where the nucleotide is mutated to be any other nucleotide.
• the AID enzyme in humans introduces natural mutations at these positions. This can cause a possible change to the amino acid at a single point. All these possible mutations were identified throughout the antibody DNA sequence in both directions in the 3' to 5' and 5' to 3' strands to generate all the amino acids that would be possible naturally throughout the antibody sequence to form the initial library.
• Each library's size was then curated by removing any recognised sequence liabilities, such as deamidation sites, isomerization sites, n-linked glycosylation sites and oxidation sites from the newly generated amino acid sequences.
• the antibody prediction tool in Maestro 11.7 was used to perform homology modelling, to generate an antibody model based on the amino acid sequences for the heavy and light variable chain regions.
• the antigen was imported from PDB.In thecase of Fsn0503h, the inventors imported the crystal structure of human
• Cathepsin S (Cat S) with a C25S mutation with a bound drug (PDB Code: 3MPE ).
• PDB Code: 3MPE the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB Code: 1N8Z) was used.
• the protein preparation wizard (Bioluminate, Schrodinger) was used to assign bond orders (using Chemical Component Dictionary (CCD) database), add hydrogens, create zero-order bonds to metals, create disulphide bonds, convert selenomethionines to methionines, fill in missing side chains and loops using Prime, and generating het states using Epik, for both the antibody and antigen models.
• the structures were further refined using ProtAssign
• the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB Code: 1N8Z) was docked to the surface of the antibody model using the protein-protein docking tool, Prime. Only the CDR regions of the antibody were considered for molecular docking. Non-CDR regions were masked. A suitable docked pose was selected based on rank and from our inspection of shape complementarity and surface interactions (using the protein interaction analysis tool).
• Residue scanning was performed on the docked pose of the antibody-antigen complex. Informed mutations were made to the antibody, avoiding highly conserved residues, to increase the affinity of the antibody for the antigen and to enhance stability.
• Residue scanning was first done by generating models with a single amino acid variation from the original structure, and repeated (with the same mutations) for up to 6
• the residue mutation tool calculated the stability and affinity of the mutants relative to the original wild type antibody-antigen complex.
• the variants were then sorted by difference in affinity and difference in stability relative to the wild type. Results that scored below a threshold of -2 for difference in stability and difference in affinity were selected and ranked based on the combination of the two scores (prioritising difference in affinity).
• Suspension adapted ExpiCHO cells were routinely cultivated at 4-6 xlO 6 cells/ml at 130 rpm, 37°C, 8% C0 2 , in ExpiCHO Expression Medium in 500ml vented Erlenmeyer flasks.
• lpg/ml of DNA was diluted in 4% (v/v) OptiPRO SFM in a centrifuge tube.
• 0.32% (v/v) ExpiFectamine was diluted in 3.7% OptiPRO SFM.
• the ExpiFectamine/OptiPRO mix was then added to the DNA/OptiPRO mix and incubated at room temperature for 3 minutes before adding to 25ml ExpiCHO cells at a final density of 6 x 10 6 cells/ml in 125ml vented Erlenmeyer flasks. Each transfected culture was cultivated at 37°C, 8% CO2 and 130 rpm overnight. Twenty hours post transfection, cells were supplemented with 0.6% (v/v) ExpiCHO enhancer and 24% (v/v) ExpiCHO feed. Cultures were then transferred to incubators at 32°C, 5% CO2 and 130 rpm. Cultures were harvested by centrifugation at 4000 rpm for 40 minutes at 18°C.
• Two-step Fsn0503h WT and variant antibody purifications were performed using a Tricorn 5/50 column (GE) packed with 1 ml of MabSelectTM PrismA (GE) followed by a 10 ml (2x5ml) Hitrap Desalting (Desalt) column (GE).
• the MabSelectTM PrismA affinity medium was chosen for its high mAb binding and specificity properties and its alkali tolerance for efficient Cleaning-in-place (CIP). All steps were performed at room temperature, using a flow-rate of 4 ml/min, unless otherwise stated.
• the protein A column was washed (in reverse flow mode) with 10 column volumes (CV) of PBS followed by a one-step elution (in reverse flow mode) with 100 mM glycine, pH 3.0.
• the protein A eluate was collected in a 2 ml loop when the absorbance was above 120 mAU at 280 nm (AKTA equipped with a 10 mm flow cell) and injected immediately onto the pre equilibrated Desalt column.
• the Desalt peak elution was collected in a 96-well-2 ml block at 2-8°C when the eluate had an absorbance above 100 mAU.
• the automated process also included a CIP of both the affinity and desalt columns. CIP was performed between each sample, for all contact pathways, using 0.2M NaOH (reverse flow mode was used for column cleaning). The level of expression was determined as the total yield of material, following purification per ml of culture media.
• trastuzumab variants were synthesised using similar techniques.
• the plates were washed with PBS-T three times and dried. 100mI of anti-his-HRP at 5pg/ml was added to each well and left for lhr 30mins shaking at 150rpm, RT. The plates were washed three time PBS-T, once with PBS and dried. 100mI of TMB was added to each well and incubated at 37 °C for lOmins, which was followed by 50mI of 1M HCL and the absorbance of the plates was measured at 450nm.
• Affinity ranking assays were performed by first capturing IgG using anti-human Octet biosensors (ForteBio part no. 18-5060) followed by a baseline step of 2 minutes in HBS-EBT buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 1 mg/ml BSA, and 0.05% Tween-20, pH 7.4). The mAb capture biosensors were then submerged in wells containing 200 ng/ml of recombinant Cathepsin S antigen for 10 minutes (association step), followed by a 10 min dissociation step in running buffer.
• IgG-captured sensors were dipped into wells containing only buffer and blank sensors were also dipped into wells containing the antigen.
• This referencing provided a means of compensating for both the natural dissociation of the capture IgG and also non-specific binding of the antigen to the sensor surface. All steps were performed at 25°C in HBS-EBT buffer at a constant flow-rate of 1000 rpm. New sensors were used for each sample. Dissociation rate constants (koff) were calculated using the ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.
• IgG containing cell supernatants 200 pL aliquots of antibody standards (spanning 0.06 to 512 pg/ml) and IgG containing cell supernatants (diluted within the measurable range of calibration curve) were prepared in duplicate using lx HBS-EBT buffer (10 mM HEPES, 150 mM NaCI, 3 mM EDTA, 1 mg/ml BSA, and 0.05% Tween-20, pH 7.4) and placed in the wells of a 96-well black microtiter plate (Greiner Bio-One part no. 655209). All samples and standards were measured in duplicate using protein A Biosensors (Fortebio PN 18-5010).
• the plate was placed in the Octet and allowed to equilibrate to 25° C in the thermostatted chamber.
• the run was initiated by placing the sensors in the wells and measuring the change in layer thick-ness (in nanometers, nm) with time, all under computer control. Data were taken for each set of eight samples at a time (one plate column is measured simultaneously) for 180-600 sec at a flow rate of 400-1000 rpm (orbital flow). Data were processed automatically using the Octet User Software version 3.1. The measurement time and flow rate were altered according to the sensitivity required.
• Affinity ranking assays were performed by first capturing *lgG using anti-human Octet biosensors (ForteBio part no. 18-5060) followed by a baseline step of 2 minutes in HBS-P+ buffer (10 mM HEPES, 150 mM NaCI, 1 mg/ml BSA, and 0.05% Tween-20, pH 7.4). The mAb capture biosensors were then submerged in wells containing 5 nM of recombinant HER2 (Aero Biosystems; P/N. H5225) antigen for 15 minutes (association step), followed by a 20 min dissociation step in running buffer.
• IgG-captured sensors were dipped into wells containing only buffer and blank sensors were also dipped into wells containing the antigen.
• This referencing provided a means of compensating for both the natural dissociation of the capture IgG and also non-specific binding of the antigen to the sensor surface. Steps were performed at either 25°C or 37°C in HBS-EBT buffer at a constant flow-rate of 1000 rpm. New sensors were used for each sample. Dissociation rate constants (koff) were calculated using the ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.
• Kinetic assays were performed by first capturing IgG using anti-human Fc Octet biosensors followed by two baseline steps of 2 minutes each in in HBS-P+ buffer running buffer. The mAb capture biosensors were then submerged in wells containing various concentrations of HER2 for 15 mins followed by 20 mins of dissociation time in running buffer. To allow for double reference correction, IgG-captured sensors were dipped into wells containing only buffer and blank sensors were also dipped into wells containing the antigen concentration series. This referencing providing a means of compensating for both the natural dissociation of the capture IgG and also non-specific binding of the antigen to the sensor surface. All steps were performed at 37°C in kinetics buffer at a constant flow-rate of 1000 rpm.
• Antibodies commonly display 2 measurable melting points in analysis, designated TM1 and TM2 as a result of thermal denaturing of different parts of the assembled molecule. These values were determined by Thermal Shift Assay. Solutions containing 5 pi of Sypro Orange ([diluted 1/200 in PBS, pH 7.4); Molecular Probes) and 45 mI of 0.3 mg ml -1 antibody were added to low profile PCR tubes (Bio-Rad; TLS0851). Tubes were sealed with optical ultra- clear sealing caps (Bio-Rad; TCS0803) and heated in an i-Cycler iQ5 real-time PCR detection system (Bio-Rad) from 20 to 90 °C in increments of 1 °C. Fluorescence changes in the wells of the plate were monitored simultaneously with a charge-coupled (CCD) camera. The wavelengths for excitation and emission were 485 and 575 nm, respectively.
• CCD charge-coupled
• T m temperature midpoint for the protein unfolding transition
• the level of monodispersity, defined as free individual molecules of immunoglobulin within an antibody preparation, of each variant was also shown to differ from the wild type molecule. This measurement is commonly used as an indicator of aggregation propensity for antibody molecules. Aggregation is the tendency of protein molecules to associate into multimeric complexes, diminishing the solubility and activity of an antibody preparation over time and is a key attribute contributing to the stability of an antibody molecule in solution.
• variable domains of the Fsn0503h antibody light and heavy light chains were analysed for motifs susceptible to mutation during somatic hypermutation, and the corresponding potential amino acid results of these mutation plotted above the parental sequence, as shown in Figure 1. Any undesirable amino acids or STOP codons generated as a result of the DNA mutations were identified.
• the 66 variants expressed in CHO as described above also demonstrated a range of level of expression relative to the wild type.
• IgG level was determined by a quantitative human IgG immunoassay on the BLI Octet instrument following purification and expressed the total amount relative to the supernatant volume purified.
• the 66 variants also demonstrated a range of variation in stability characteristics associated with antibody molecules. This included changes in melting temperature profile at 2 melting points commonly observable in immunoglobulin molecules. The results of melting point determination are shown in Figure 22. It is noteable that some variants such as Mut 6 and Mut 49 appear to have lost their distinct 2-phase melting pattern, with only a single melting temperature being observable.
• a library of antibody variants was produced for trastuzumab based on naturally occurring somatic hypermutation in humans. Using the method described above somatic
• trastuzumab in complex with the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB Code: 1N8Z) is illustrated in figure 10.
• the library of trastuzumab mutations illustrated in Figures 7 and 8 was used for mutant scanning. Mutants were scanned consecutively whereby a single amino acid mutation was first analysed, followed by two mutations up until six total mutations in the heavy and light chains and subsequently variants were ranked based on affinity and stability.
• the Schrodinger molecular docking software was used as described in the Methods.
• R 2 values indicate how well the fit and experimental data correlate and above 0.95 are considered a good fit; X 2 is the sum of the squared deviation should be generally below 3 X 2 is the measure of error between the experimental data and the fitted line. The smaller the X 2 indicates a better fit.
• Fab Fragment Antigen Binding
• Table 12 Kinetic parameters (fit to 1:1 interaction model) for Trastuzumab variants as monomeric Fab and HER2 interaction at a HER2 coating concentration of 1.25 pg/ml (double-referenced)
• the affinity value appears to vary as a result of coating concentration so it is difficult to assign a definitive value for the increase in affinity for MUT 5 and MUT 19 compared to the WT Trastuzemab molecule from these monomeric analysis. Without wishing to be bound by theory, this is thought to be due to the problem of working at the limits of sensitivity of the ForteBio Octet Biosensor instrument.
• the inventors have successfully demonstrated that the affinity of an antibody for its target can be improved without the need to generate a very large physical library of antibody variants and subsequent selection/screening process.
• a pool of variants are generated which, upon expression, display a range of attributes of interest including affinity, expression level and physiochemical characteristics, which are all of interest to the potential developability of an antibody molecule.

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