Antibody libraries and methods

Abstract

To provide a method for generating an antibody library.SOLUTION: (i) has at least two amino acid alterations in the light chain sequence when compared to the light chain amino acid sequence of the reference antibody, (ii) has at least two amino acid alterations in the heavy chain sequence when compared to the heavy chain amino acid sequence of the reference antibody, or (iii) has at least one amino acid alteration in the light chain sequence when compared to the light chain amino acid sequence of the reference antibody; And a variant of the reference antibody having at least one amino acid change in the heavy chain sequence when compared to the heavy chain amino acid sequence of the reference antibody, wherein each of the amino acid changes is in an amino acid residue independently encoded from a segment of the variant DNA sequence, and wherein the variant DNA segment differs from the corresponding segment of the reference antibody encoding DNA sequence by a point mutation in a DNA motif susceptible to deamination by a somatic hypermutation inducing enzyme.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for generating antibody libraries, antibody libraries produced using such methods, and variant antibodies. [Background technology]

[0002] Antibody affinity is a measure of the strength of the interaction between an antibody and the protein it specifically binds, expressed as the ratio of the association rate to the dissociation rate. There are many reasons why optimizing this ratio is desirable. Increasing affinity may mean that antibody therapy is more effective at a particular dose, or that less drug is needed per dose, and diagnostic tests may have improved sensitivity. Reducing affinity may also be beneficial for some drugs that require tissue penetration and therefore faster dissociation. It has also been shown that bispecific antibodies require complex tuning of the affinity of each binding site to maximize efficacy.

[0003] Existing affinity maturation platforms generally involve generating large libraries of variants through focused random mutagenesis within antibody complementarity-determining regions (CDRs). This process involves generating a very large number of variants (often >10) to identify a small subset of variants with improved properties. 10 ) has the disadvantage of screening. Summary of the Invention

[0004] The present invention addresses many of the problems of the prior art. As described in the Examples, the inventors surprisingly demonstrated that by restricting mutations to the nucleotide sequence encoding a given antibody sequence to sites corresponding to DNA motifs targeted by enzymes involved in somatic hypermutation, it is possible to generate a library of variants of a given antibody sequence, which, although relatively small compared to those prepared by many existing techniques, contains a relatively high proportion of variants with increased affinity, aggregation tendency, melting temperature, expression level in CHO cells, or combinations thereof. The inventors exemplified the invention using two unrelated antibodies: an anti-cathepsin S antibody, Fsn0503h (Fusion Antibodies Ltd), and the anti-HER2 antibody trastuzumab (Herceptin®, Roche).

[0005] Accordingly, a first aspect of the present invention provides a library of antibody molecules, each antibody molecule being a variant of a reference antibody, wherein the amino acid sequence of each antibody molecule differs from that of the reference antibody at one or more amino acid residues, each of which is independently encoded by a DNA segment of a DNA sequence encoding the variant, wherein said DNA segment of the variant differs from the corresponding segment of the DNA sequence encoding the reference antibody by a point mutation in a DNA motif that is susceptible to deamination by somatic hypermutase.

[0006] A second aspect of the present invention provides a method for generating / producing a library of variant antibody molecules, wherein said variant antibody molecules are variants of a reference antibody, said method comprising the steps of: a) providing a nucleotide sequence encoding a reference antibody; b) identifying one or more DNA motifs in said nucleotide sequence that are susceptible to deamination by somatic hypermutator enzymes; 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, said substitution resulting in a variant nucleotide sequence encoding a variant antibody molecule having an alteration in the amino acid sequence encoded by said DNA motif compared to a reference antibody; and d) Repeating steps (b) and (c).

[0007] The reference antibody can be any antibody molecule for which a variant is desired or required. In one embodiment, the reference antibody is a humanized antibody molecule.

[0008] A third aspect of the present 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 of: a) providing a nucleotide sequence encoding a reference antibody; b) identifying one or more DNA motifs in said nucleotide sequence that are susceptible to deamination by somatic hypermutator enzymes; 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, said substitution resulting in a variant nucleotide sequence encoding a variant antibody molecule having an alteration in the amino acid sequence encoded by said DNA motif compared to a reference antibody.

[0009] In one embodiment of the invention, the somatic hypermutator enzyme is activation-induced deaminase (AID).

[0010] In an embodiment of the invention, the DNA motif is DGYW or WRCH, such as 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.

[0011] The DNA motif may be on either strand of the DNA, and if there is more than one of the DNA motifs, the motifs may overlap.

[0012] The inventors have shown that antibody libraries of the invention or produced according to the methods of the invention can be refined by further limiting the members of the library to those variants that result from mutations in DNA motifs targeted by somatic hypermutatase that do not introduce stop codons or indeed any other motifs in the variant antibody molecule that may be potentially undesirable, e.g., in terms of stability or binding, compared to the reference antibody.

[0013] Thus, in certain embodiments of the invention, the DNA sequence of each variant does not contain (or encode) any deamination, isomerization, N-linked glycosylation or oxidation sites originating from said point mutations in said DNA motif.

[0014] In some embodiments, one or more of the DNA motifs are in the DNA sequence encoding the CDRs of the antibody molecule. However, as described in the Examples, the inventors have used this method with several reference antibodies to show that the generated variants may not have mutations in some CDRs compared to the corresponding CDRs of the reference antibody.

[0015] In one embodiment, the variant has no alterations in one or more of its CDRs compared to the corresponding CDRs of the reference sequence.

[0016] In one such embodiment, the variant has no alterations in the light chain CDR1 compared to the corresponding CDR of the reference sequence.

[0017] In another such embodiment, the variant has no alterations in the light chain CDR2 compared to the corresponding CDR of the reference sequence.

[0018] In another such embodiment, the variant has no alterations in the light chain CDR3 compared to the corresponding CDR of the reference sequence.

[0019] In another such embodiment, the variant has no alterations in the heavy chain CDR1 compared to the corresponding CDR of the reference sequence.

[0020] In another such embodiment, the variant has no alterations in the heavy chain CDR2 compared to the corresponding CDR of the reference sequence.

[0021] In another such embodiment, the variant has no alterations in the heavy chain CDR3 compared to the corresponding CDR of the parent sequence.

[0022] Furthermore, as shown in the Examples, in some variants of a reference antibody, many of the mutations that characterize the variant may be in the framework regions of the variant antibody molecule.

[0023] Thus, in certain embodiments of the libraries or methods of the invention, one or more of said DNA motifs are in DNA sequences encoding framework regions of said antibody molecules, hi some such embodiments, all of said DNA motifs are in DNA sequences encoding framework regions of said antibody molecules.

[0024] In one embodiment of the invention, more than 20%, e.g., more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the nucleotide residues in each variant antibody that differ from the nucleotide residues at corresponding positions in the reference antibody are residues of a DNA motif that is susceptible to deamination by somatic hypermutagenase.

[0025] In some embodiments of the invention, the nucleotide sequence encoding each of said antibody molecules does not differ from the nucleotide sequence of a reference antibody at any residue other than the residues of said DNA motif.

[0026] Antibody libraries of the invention and / or produced using the methods of the invention may be further refined to increase the proportion of high affinity and / or stability variants.

[0027] The antibody libraries of the present invention and / or antibody libraries produced using the methods of the present invention may be further refined to increase the proportion of variants with different aggregation properties, i.e., less ability to aggregate with each other. The antibody libraries of the present invention and / or antibody libraries produced using the methods of the present invention may be further refined to increase the proportion of variants with specific melting point characteristics. The antibody libraries of the present invention and / or antibody libraries produced using the methods of the present invention may be further refined to increase the proportion of variants that exhibit preferred or desired expression levels in CHO cells. The antibody libraries of the present invention and / or antibody libraries produced using the methods of the present invention may be further refined to increase the proportion of variants with high affinity, stability, desired aggregation properties, desired melting point properties, desired expression level properties, or combinations thereof.

[0028] Thus, in the present invention, the method may further comprise determining the binding affinity and / or stability of the variant antibody molecule to the binding target of the reference antibody, the melting temperature relative to the reference antibody, the aggregation tendency relative to the reference antibody, the expression level relative to the reference antibody, or a combination thereof. Thus, in one embodiment of the method of the present invention, the method further comprises screening the library of variants to determine binding to the epitope bound by the reference antibody. Suitably, the method of the present invention may further comprise screening the library of variants to determine the melting temperature, expression level, aggregation level, or stability of the variants relative to the reference antibody. Those variants determined to bind to the epitope with an affinity and / or stability less than or greater than a predetermined value, or to have melting, aggregation, or expression characteristics greater than a predetermined value, can be used to generate an optimized library of variant antibody molecules. The screening method may be by conventional in vitro techniques. Such techniques may include affinity ELISA assays, BIAcore assays, kinetic methods, or equilibrium / solution methods. Alternatively, the screening may be by in silico techniques, for example using computer-implemented molecular docking software to model the binding of the variant to the epitope of a reference antibody.

[0029] As shown in the Examples, molecular docking software can be used to rank variants by predicted affinity and stability, allowing for the selection of small libraries of variants for DNA synthesis and expression. We demonstrate that within such small libraries, a significant number of variants have increased affinity compared to the number expected to be identified in libraries generated by existing techniques such as error-prone PCR or phage display.

[0030] Molecular docking software products are commercially available. Any suitable software or tool suitable for modeling antibody binding to an epitope can be used in the present invention. For example, suitable software includes Schrodinger's Bioluminate software, although other software is also available.

[0031] Thus, in an embodiment of the invention, the antibody library comprises more than 1%, e.g., more than 5%, 10%, 20%, 30%, 40%, or 50% variants that have increased affinity for the epitope bound by the reference antibody compared to the affinity of the reference antibody for said epitope.

[0032] In one embodiment of the inventive method, the method is a computer-implemented method.

[0033] A fourth aspect of the invention provides a computer readable storage medium comprising instructions for carrying out a method for generating a library of variant antibody molecules according to the second aspect of the invention.

[0034] In the methods of the present invention, whether computer-implemented or not, the method may further comprise the step of synthesizing the variant antibody molecule.

[0035] In embodiments of the methods of the invention, where the method comprises a computer-implemented screening method, for example, by docking modeling software, the method of the invention may further comprise in vitro screening of said library of variants to determine binding to the epitope bound by the reference antibody.

[0036] A fifth aspect of the invention is a variant of a trastuzumab antibody, said variant having (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, or (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; each of said amino acid changes is at an amino acid residue independently encoded by a DNA segment of the variant DNA sequence, and said DNA segment of the variant differs from the corresponding segment of the DNA sequence encoding the reference antibody by a point mutation in a DNA motif that is susceptible to deamination by somatic hypermutase.

[0037] In one embodiment of the fifth aspect of the invention, the amino acid changes are lc9N, lc9T, lc9I, lc9R, lc9K, lc25G, lc25V, lc25D, lc31N, lc31S, lc31I, lc32D, lc32G, lc32V, lc32T, lc32N, lc32S, lc32I, lc32P, lc32L, lc32F, lc33L, lc33I, lc34G, lc34V, lc34D, lc38E, lc38K, lc40A, lc40S, lc40T, lc43G, lc43V, lc43T, lc43N, lc43S, lc43I, lc43 P, lc43L, lc43F, lc46V, lc46I, lc47V, lc51S, lc51P, lc51T, lc76R, lc76N, lc76T, lc76K, lc76I, lc79K, lc79E, lc80T, lc80S, lc80A, lc85S, lc85N, lc 85I, lc89H, lc90E, lc90A, lc91N, lc91D, lc91Y, lc93S, lc93N, lc93I, lc94S, lc94N, lc94I, lc101D, lc102S, lc102N, hc2L, hc2I, hc3H, hc4M, hc4V, hc 13K, hc13E, hc14A, hc14T, hc14S, hc16A, hc16V, hc16D, hc23E, hc23G, hc23 V, hc23T, hc23K, hc23R, hc23I, hc23P, hc23L, hc23S, hc24D, hc24G, hc24V, hc24T, hc24N, hc24S, hc24I, hc24P, hc24L, hc24F, hc26A, hc26V, hc26D, hc 28K, hc35N, hc35D, hc35Y, hc48L, hc48I, hc49G, hc49S, hc56A, hc56V, hc56 hc82E, hc82K, hc85R, hc88D, hc88T, hc88S, hc88P, hc88G, hc92G, hc92V, hc92D, hc103A, hc103V, hc103D, hc106D, hc106G, hc106V, hc106T, hc106N, hc106S, hc106I, hc106P, hc106L, hc106F, hc114S, hc114N, and hc114I.

[0038] In the context of the present invention, mutations are identified using the nomenclature above, where lc = light chain, hc = heavy chain, the numbers refer to amino acid residues in the chain, and the capital letters are the single letter amino acid code representing the amino acid mutation at that position. Thus, for example, in the amino acid changes listed above for the fourth aspect, lc9N refers to the asparagine at position 9 of the variant trastuzumab light chain.

[0039] In an embodiment of the fifth aspect of the invention, the amino acid changes are selected from the group consisting of lc9T, lc9I, lc9R, lc9K, lc43F, lc47V, lc51P, lc51T, lc101D, hc2L, hc3H, hc14S, hc16V, hc24P, hc26A, hc26V, hc48I, hc58S, hc61V, hc79V, hc85R, hc88G, hc92V, hc92D, hc103A, hc103V, hc106V, hc114S, hc114N, and hc114I.

[0040] In one embodiment of the fifth aspect, the light and heavy chain sequences of the variant do not differ from the sequences of the reference antibody at any residue other than the amino acid changes described above in relation to the fifth aspect.

[0041] A sixth aspect of the present invention provides a variant of a Cathepsin S antibody, the variant having (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 the reference antibody is Fsn503h, or (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; each of the amino acid changes is at an amino acid residue independently encoded by a DNA segment of the variant DNA sequence, and the DNA segment of the variant differs from the corresponding segment of the DNA sequence encoding the reference antibody by a point mutation in a DNA motif that is susceptible to deamination by somatic hypermutase.

[0042] In one embodiment of the sixth aspect of the invention, the amino acid changes are lc12A, lc12S, lc12T, lc19V, lc28R, lc32T, lc32I, lc45A, lc45S, lc45T, lc50H, lc51V, lc51F, lc51I, lc56L, lc56F, lc56I, lc58K, lc66S, lc69A, lc69V, lc81T, lc81I, lc81N, lc85P, lc85S, lc85T, lc90L, lc91A, lc91B, lc91C, lc91D, lc91E, lc91F, lc91F, lc91H, lc91I, lc91F, lc91I, lc91L, lc91F, lc91I, lc91P, lc91S, lc91T, lc91H, lc91I, lc91F, lc91H, lc91I ... hc90F, lc96I, lc96S, lc96I, lc96N, lc108N, hc3H, hc4V, hc4M, hc10A, hc10V, hc14A, hc14S, hc24G, hc24V, hc30T, hc30I, hc31R, hc31T, hc37L, hc37F, hc40P, hc40S, hc52S, hc52I, hc53S, hc53I, hc84T, hc84I, hc92G, and hc92V.

[0043] In one embodiment of the sixth aspect of the invention, the amino acid changes are selected from the group consisting of lc12A, lc12S, lc12T, lc19V, lc45S, lc45T, lc50H, lc51V, lc56I, lc81I, lc96I, lc96S, lc96I, lc96N, lc108N, hc10A, hc10V, hc14S, hc30I, hc31R, hc37L, hc37F, hc40P, hc40S, hc52I, and hc92G.

[0044] In one embodiment of the sixth aspect of the invention, the light and heavy chain sequences of the variant do not differ from the sequences of the reference antibody at any residue other than the amino acid changes described above in relation to the sixth aspect.

[0045] In one embodiment of the fifth aspect of the invention, the variant antibody molecule has a 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, the variant antibody molecule has a combination of amino acid mutations as shown for any of the antibodies listed in Table 8. In one such embodiment, the variant antibody has a combination of amino acid mutations as shown for any one of the antibodies listed in Table 8, and has no amino acid mutations other than those shown for the antibodies listed in Table 8 compared to the trastuzumab light and heavy chain sequences.

[0046] In one embodiment of the fifth aspect of the invention, the variant antibody has the mutation lc43F.

[0047] In one embodiment of the fifth aspect of the invention, the variant antibody has a combination of amino acid mutations as shown for any of variant antibodies 19, 5 or 6 in Table 8. In one such embodiment, the variant does not have any amino acid mutations compared to the trastuzumab light and heavy chain sequences other than those shown for said variant antibody in Table 8.

[0048] In one embodiment of the fifth aspect of the invention, the variant antibody is a variant of the trastuzumab antibody comprising the following amino acid changes compared to trastuzumab: lc9K, lc43F, and hc106V. In one embodiment, the variant antibody is variant antibody 19 listed in Table 8.

[0049] In another embodiment of the fifth aspect of the invention, the variant antibody is a variant of the trastuzumab antibody comprising the following amino acid changes compared to trastuzumab: lc9R, lc43F, and hc114S. In one embodiment, the variant antibody is variant antibody 5 listed in Table 8.

[0050] In another embodiment of the fifth aspect of the invention, the variant antibody is a variant of the trastuzumab antibody which comprises the following amino acid changes compared to trastuzumab: lc9I, lc43F, lc101D and hc79V. In one embodiment, the variant antibody is variant antibody 6 listed in Table 8.

[0051] In one embodiment of the sixth aspect of the invention, the variant antibody molecule has a combination of amino acid mutations as shown for any of the antibodies listed in Table 1.

[0052] In particular embodiments of the fifth or sixth aspect of the invention, the light and heavy chain sequences of the variant comprise a total of at least three, such as at least four, at least five, or at least six amino acid changes compared to the amino acid sequences of the reference antibody.

[0053] In certain embodiments of the fifth or sixth aspect of the invention, one or more of the amino acid changes are in a framework region of the variant antibody. In certain embodiments of the fifth or sixth aspect of the invention, all of the amino acid changes are in a framework region of the variant antibody.

[0054] In particular embodiments of the fifth or sixth aspect of the invention, one or more of said amino acid alterations are in a CDR of said variant antibody.

[0055] In certain embodiments of the fifth or sixth aspect of the invention, the change in affinity of the variant antibody molecule compared to the reference antibody is greater than -2 and the change in stability of the variant antibody molecule compared to the reference antibody is greater than -2. In certain embodiments of the fifth or sixth aspect of the invention, the change in affinity of the variant antibody molecule compared to the reference antibody is greater than -10, such as greater than -15, such as greater than -20, such as greater than -25.

[0056] In particular embodiments of the fifth or sixth aspect of the invention, the change in stability of the variant antibody molecule compared to the reference antibody is greater than -10, such as greater than -30, such as greater than -50, such as greater than -60. Suitably, the aggregation properties, melting temperature properties or expression levels may differ from the reference antibody by at least 2-fold, 3-fold, 10-fold.

[0057] For the avoidance of doubt, the more negative the affinity value, the greater the affinity. Thus, an antibody with an affinity value of -10 would be considered to have a greater affinity value than an antibody with an affinity value of -5. Similarly, the more negative the stability value, the greater the stability. Thus, an antibody with a stability value of -10 would be considered to have a greater stability value than an antibody with a stability value of -5.

[0058] Affinity and stability can be assessed by any suitable method. In the examples, we used the residue-scanning affinity maturation tool as part of Schrodinger's Maestro biologics tool. Values ​​are relative to the parent antibody, and the minimum affinity improvement was -2 kcal / mol for both affinity and stability. [Brief explanation of the drawings]

[0059] [Figure 1A]Figure 1 shows the amino acid sequences of the light and heavy chains of the Fsn0503h antibody, indicating potential mutations on each chain (SEQ ID NOS: 1 and 2). Doubly enclosed amino acids were not allowed in the library due to their rare use at that position. A doubly enclosed asterisk (*) represents a stop codon that was also not allowed. The simply enclosed amino acid regions are the CDRs. [Figure 1B] Figure 1 shows the amino acid sequences of the light and heavy chains of the Fsn0503h antibody, indicating potential mutations on each chain (SEQ ID NOS: 1 and 2). Doubly enclosed amino acids were not allowed in the library due to their rare use at that position. A doubly enclosed asterisk (*) represents a stop codon that was also not allowed. The simply enclosed amino acid regions are the CDRs. [Figure 2] FIG. 2 is a schematic model showing the predicted protein-protein interactions between Fsn0503h and cathepsin S. [Figure 3-1] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-2] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-3] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-4]Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-5] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-6] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-7] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-8] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-9] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-10] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-11] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 3-12] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for mutations 1–6. The group of mutations highlighted in blue represents variants with changes greater than −2, improving both stability and affinity. [Figure 4] Figure 4 shows ELISA comparison of each expressed variant with the parental Fsn0503h antibody. [Figure 5] Figure 5 shows the affinity (KD) measurements of each variant alongside that of the parent Fsn0503h antibody, measured by BLI using an Octet instrument (Pall). [Figure 6] Figure 6 shows affinity on-off rate maps for Fsn0503h variants. The parent antibody is shown as an open dot. The 4.45 nM line represents affinity equivalent to the parent antibody, and any variants with increased affinity (<4.45 nM) fall within the region to the left of the 4.45 nM line. [Figure 7-1] Figure 7 shows the amino acid sequence of the trastuzumab antibody light chain (SEQ ID NO: 3), with the amino acid residue at each position of the wild-type antibody light chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library because they are rarely used at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 7-2]Figure 7 shows the amino acid sequence of the trastuzumab antibody light chain (SEQ ID NO: 3), with the amino acid residue at each position of the wild-type antibody light chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library because they are rarely used at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 7-3] Figure 7 shows the amino acid sequence of the trastuzumab antibody light chain (SEQ ID NO: 3), with the amino acid residue at each position of the wild-type antibody light chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library because they are rarely used at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 8-1] Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4), with the amino acid residue at each position of the wild-type antibody chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library due to their rare use at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 8-2] Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4), with the amino acid residue at each position of the wild-type antibody chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library due to their rare use at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 8-3]Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4), with the amino acid residue at each position of the wild-type antibody chain shown in bold and potential mutations shown in boxes to the right of each residue, where applicable. Amino acids shown with a double underline are not allowed in the library due to their rare use at that position. A double underline asterisk (*) represents a stop codon that was also not allowed. The complementarity-determining regions (CDRs) and framework regions (FRs) are shown. [Figure 9] FIG. 9 shows a schematic of how somatic hypermutation can be used to generate changes in the DNA sequence of an antibody that result in amino acid changes in the antibody's protein structure, leading to increased affinity. [Figure 10] Figure 10 shows the fully solved 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-1] Figure 11a shows Table 2, which lists trastuzumab variants with one mutation compared to the reference trastuzumab antibody. In the second column, L indicates light chain and H indicates heavy chain. Δaffinity is the predicted free energy change (ΔΔG) in kcal / mol relative to the affinity of trastuzumab. Δstability is the predicted free energy change (ΔΔG) relative to the stability of trastuzumab. Negative Δaffinity and negative Δstability values ​​are considered to represent improved affinity and improved stability compared to trastuzumab. [Figure 11A-2] Figure 11a shows Table 2, which lists trastuzumab variants with one mutation compared to the reference trastuzumab antibody. In the second column, L indicates light chain and H indicates heavy chain. Δaffinity is the predicted free energy change (ΔΔG) in kcal / mol relative to the affinity of trastuzumab. Δstability is the predicted free energy change (ΔΔG) relative to the stability of trastuzumab. Negative Δaffinity and negative Δstability values ​​are considered to represent improved affinity and improved stability compared to trastuzumab. [Figure 11B-1] Figure 11b shows Table 3, which lists trastuzumab variants that have two mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11B-2] Figure 11b shows Table 3, which lists trastuzumab variants that have two mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11B-3] Figure 11b shows Table 3, which lists trastuzumab variants that have two mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11B-4] Figure 11b shows Table 3, which lists trastuzumab variants that have two mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11B-5] Figure 11b shows Table 3, which lists trastuzumab variants that have two mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-1] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-2] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-3] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-4] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-5] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-6] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-7] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-8] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11C-9] Figure 11c shows Table 4, which lists trastuzumab variants with three mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-1] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-2] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-3] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-4] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-5]Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-6] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-7] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-8] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-9] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-10] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-11] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-12] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11D-13] Figure 11d shows Table 5, which lists trastuzumab variants with four mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-1]Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-2] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-3] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-4] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-5] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-6] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-7] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-8] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-9] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-10]Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-11] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-12] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-13] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-14] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-15] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11E-16] Figure 11e shows Table 6, which lists trastuzumab variants with five mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-1] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-2] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-3]Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-4] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-5] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-6] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-7] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-8] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-9] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-10] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-11] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-12]Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-13] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-14] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-15] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-16] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-17] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-18] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 11F-19] Figure 11f shows Table 7, which lists trastuzumab variants with six mutations compared to the reference trastuzumab antibody. In the second column, L indicates the light chain and H indicates the heavy chain. [Figure 12-1] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-2]FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-3] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-4] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-5] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-6] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-7] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-8] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-9] FIG. 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1 to 7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 13A]Figure 13 shows a schematic of the top five trastuzumab variants, scored based on affinity using the predicted free energy change (ΔΔG) in kcal / mol. The first and third ranked variants have a total of five mutations, ranks two and four have four mutations, and finally rank five contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while the scFv is colored green and the CDRs are colored red. Mutations are highlighted in yellow. [Figure 13B] Figure 13 shows a schematic of the top five trastuzumab variants, scored based on affinity using the predicted free energy change (ΔΔG) in kcal / mol. The first and third ranked variants have a total of five mutations, ranks two and four have four mutations, and finally rank five contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while the scFv is colored green and the CDRs are colored red. Mutations are highlighted in yellow. [Figure 13C] Figure 13 shows a schematic of the top five trastuzumab variants, scored based on affinity using the predicted free energy change (ΔΔG) in kcal / mol. The first and third ranked variants have a total of five mutations, ranks two and four have four mutations, and finally rank five contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while the scFv is colored green and the CDRs are colored red. Mutations are highlighted in yellow. [Figure 14] Figure 14 shows the sensorgrams of the 20 samples displaying the reporting points used for ranking. Assays were performed at 25°C. [Figure 15]Figure 15 shows a graph depicting the off-rate ranking of trastuzumab variants binding to HER2, showing stability_early and stability_late to identify stable binders (best binders are boxed in blue). A total of 89 samples were analyzed and ranked for binding stability, but only 29 are shown for clarity. Trastuzumab is a commercially available antibody material. Wild-type is an antibody with the sequence of trastuzumab that was transiently expressed simultaneously with all trastuzumab variants / mutants. Mutants are trastuzumab variants. Mutants 5, 6, and 19 are trastuzumab variants listed as numbers 5, 6, and 19 in Table 8. Assays were performed at 25°C. [Figure 16] Figure 16 shows a graph depicting the off-rate ranking of trastuzumab variants binding to HER2, with stability_early and stability_late shown to identify stable binders from the small subset of top binders shown in Figure 15 (these binders are closest to the 100% remaining line). In total, 14 trastuzumab variants were analyzed and ranked for binding stability. Trastuzumab is a commercially available antibody. Wild-type is material transiently expressed alongside all trastuzumab variants / mutants. Mutants are trastuzumab variants. Mutants 5, 6, and 19 are trastuzumab variants listed as numbers 5, 6, and 19 in Table 8. Assays were performed at 37°C. [Figure 17-1] Figure 17 shows reference-corrected BLI binding curves (black) monitored at non-covalently immobilized trastuzumab (a), wild-type (b), mutant 5 (c), and mutant 19 (d) antibody surfaces for various HER2 concentrations (2-fold serial dilutions; the highest concentration is indicated on the curve) in running buffer at 37 °C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by globally fitting (red) a simple 1:1 interaction model, A + B = AB, to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Table 9. [Figure 17-2]Figure 17 shows reference-corrected BLI binding curves (black) monitored at non-covalently immobilized trastuzumab (a), wild-type (b), mutant 5 (c), and mutant 19 (d) antibody surfaces for various HER2 concentrations (2-fold serial dilutions; the highest concentration is indicated on the curve) in running buffer at 37 °C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by globally fitting (red) a simple 1:1 interaction model, A + B = AB, to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Table 9. [Figure 18] FIG. 18 shows a schematic of the correlation between predicted and measured affinities of trastuzumab variants. [Figure 19-1] Figure 19 shows blank-corrected BLI binding curves (black) monitored at a non-covalently immobilized HER2 surface for a range of concentrations of wild-type (a), mutant 5 (b), and (c) mutant 19 antibodies in running buffer at 37°C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by global fitting (red) a simple 1:1 interaction model A + B = AB to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Table 10. [Figure 19-2] Figure 19 shows blank-corrected BLI binding curves (black) monitored at a non-covalently immobilized HER2 surface for a range of concentrations of wild-type (a), mutant 5 (b), and (c) mutant 19 antibodies in running buffer at 37°C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by global fitting (red) a simple 1:1 interaction model A + B = AB to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Table 10. [Figure 20-1]Figure 20 shows double-reference-corrected BLI binding curves (black) monitored at non-covalently immobilized HER2 surfaces coated at a range of concentrations presented with wild-type (a), mutant 5 (b), and mutant 19 (c) antibodies in running buffer at 37°C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by globally fitting (red) a simple 1:1 interaction model, A + B = AB, to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Tables 11, 12, and 13. [Figure 20-2] Figure 20 shows double-reference-corrected BLI binding curves (black) monitored at non-covalently immobilized HER2 surfaces coated at a range of concentrations presented with wild-type (a), mutant 5 (b), and mutant 19 (c) antibodies in running buffer at 37°C. The apparent dissociation rate constants (kd) and association rate constants (ka) were determined by globally fitting (red) a simple 1:1 interaction model, A + B = AB, to the sensorgrams using the software provided with the instrument. The global fitting results are summarized in Tables 11, 12, and 13. [Figure 21] Figure 21 shows the expression yield of Fsn0503 variants by Octet immunoassay expressed as mg of purified IgG obtained per ml of supernatant of transfected CHO, with the wild type shown in orange. [Figure 22] Figure 22 shows the measured melting points (TM1 and TM2) of Fsn0503 variants in degrees Celsius, with the wild type at position 1. [Figure 23] Figure 23 shows the proportion of monodisperse (non-aggregated) molecules measured by size exclusion chromatography for Fsn0503 variants, with the wild type shown in orange. DETAILED DESCRIPTION OF THE INVENTION

[0060] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0061] Throughout the specification, unless the context requires otherwise, the words "comprise" or "include" or variations such as "comprises" or "comprising", "includes" or "including" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0062] As used herein, terms such as "a," "an," and "the" include singular and plural referents unless the context clearly requires otherwise. Thus, for example, reference to an "active agent" or "pharmacologically active agent" includes a single active agent and combinations of two or more different active agents, while reference to a "carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

[0063] The term "DNA segment" refers to a portion of a DNA sequence. In the context of the present invention, a DNA segment may be a portion of DNA residues that are part of a longer DNA sequence encoding an antibody molecule. The DNA segment may consist of DNA residues that form a DNA motif that is a sequence-specific binding site for somatic hypermutase.

[0064] As used herein, the term "consisting essentially of" means that the invention necessarily includes the recited items, and may include unrecited items that do not materially affect the basic and novel characteristics of the invention.

[0065] As defined herein, an "epitope" refers to a group of amino acid residues that can be recognized and bound by an antibody molecule. Epitopes generally consist of chemically active surface groups and have specific three-dimensional structural characteristics, as well as specific charge characteristics that contribute to the three-dimensional structure of the epitope.

[0066] An antibody molecule of or for use in the present invention may bind to a non-contiguous epitope. A "non-contiguous epitope" is an epitope made up of a series of amino acid residues that is not linear in sequence, such that the residues are non-contiguously spaced or grouped along the length of the polypeptide sequence.

[0067] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to describe a series of at least two amino acids covalently linked by a peptide bond or a modified peptide bond, such as an isostere. No limit is imposed on the maximum number of amino acids that may constitute a peptide or protein. Furthermore, the term polypeptide extends to fragments, analogs, and derivatives of peptides, wherein the fragment, analog, or derivative retains the same biological functional activity as the peptide from which the fragment, derivative, or analog is derived.

[0068] The nomenclature used herein to describe polypeptide components follows the conventional practice of presenting the amino group (N) to the left and the carboxyl group to the right of each amino acid residue.

[0069] Antibodies and antibody molecules An "antibody" is an immunoglobulin, whether natural or partially or wholly synthetically produced. The term also includes any polypeptide, protein, or peptide having a binding domain which is, or is homologous to, an antibody binding domain. These may be derived from natural sources or partially or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses, and fragments which contain an antigen-binding domain, such as Fab, scFv, Fv, dAb, or Fd, and bispecific antibodies.

[0070] Antibodies can be modified in many ways, and the terms "antibody" and "antibody molecule" should be interpreted to encompass any binding member or substance having a binding domain with the required specificity. Antibody molecules of the invention or antibody molecules for use in the antibody molecules may be monoclonal antibodies, or fragments, derivatives, functional equivalents, or homologs. The term includes any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are included.

[0071] The constant region of the antibody can be of any suitable immunoglobulin subtype. In certain embodiments, when human immunoglobulin molecules are used, the antibody subtype can be of the IgA, IgM, IgD, and IgE classes. Such antibodies can further belong to any subclass, for example, IgG1, IgG2a, IgG2b, IgG3, and IgG4.

[0072] Fragments of whole antibodies can perform the function of antigen binding. Examples of such binding fragments are Fab fragments comprising or consisting of the VL, VH, CL, and CH1 antibody domains; Fv fragments consisting of the VL and VH domains of a single antibody; F(ab')2 fragments; bivalent fragments comprising two linked Fab fragments; single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to bind to form an antigen-binding site; and bispecific antibodies, which can be multivalent or multispecific fragments constructed by gene fusion.

[0073] In certain embodiments, humanized antibodies may be used. Humanized antibodies may be engineered antibodies having hypervariable regions from a non-human antibody and constant regions from a human antibody. Thus, the binding member may contain 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 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 chimerized.

[0074] It is possible to take a monoclonal or other antibody and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity-determining regions (CDRs), of the antibody to the constant region, or constant regions and framework regions, of a different immunoglobulin. Hybridomas or other cells that produce antibodies may undergo genetic mutation or other changes that may or may not alter the binding specificity of the antibody produced.

[0075] The antibody may be selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a camelid antibody, a shark antibody, and an in vitro antibody. In certain embodiments, an antigen-binding fragment may be used. The antigen-binding fragment may be derived from any of the aforementioned antibodies. In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, an scFv fragment, an Fv fragment, and a dAb fragment. In certain embodiments, the antibody comprises two complete heavy chains and two complete light chains, or an antigen-binding fragment thereof. In certain embodiments, 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 IgG1, IgG2, or IgG3, or an antigen-binding fragment thereof. In certain embodiments, the antibody is of the subtype IgG4, or an antigen-binding fragment thereof.

[0076] antibody production Antibodies can be produced by a variety of techniques. For example, combinatorial screening techniques such as phage display-based biopanning assays can be used to identify amino acid sequences with antigen-binding specificity. Phage display biopanning techniques involve the use of phage display libraries, which display antibody-binding fragments on the surface of filamentous fungi to identify suitable epitope-binding ligands in a procedure mimicking immune selection. Phages with specific binding activity are selected. The selected phage can then be used to produce chimeric, CDR-grafted, humanized, or human antibodies. Antibodies can be tested for their ability to bind to antigens using methods known in the art.

[0077] Antibodies or antigen fragments for use in the present invention may also be produced in whole or in part by chemical synthesis. Antibodies can be readily prepared according to well-established standard liquid-phase or, preferably, solid-phase peptide synthesis methods, general descriptions of which are widely available and familiar to those skilled in the art. Furthermore, they may be prepared in solution, by solution-phase methods, or by any combination of solid-phase, liquid-phase, and solution chemistries.

[0078] Another convenient way to produce antibodies or antibody fragments suitable for use in the present invention is to express the nucleic acids encoding them by using nucleic acids in an expression system.

[0079] Antibodies may be generated by mutagenesis of antibody genes to produce an artificial repertoire of antibodies. This technique allows for the preparation of antibody libraries. To identify binding molecules with specificity for a particular epitope, an artificial repertoire of immunoglobulins, such as an artificial scFv repertoire, may be used as an immunoglobulin source.

[0080] Methods for generating repertoires are well characterized in the art.

[0081] Any suitable means for generating antibody libraries can be used in conjunction with the present invention. Selection protocols for isolating desired members of large libraries, as exemplified by phage display technology, are known in the art. Such systems, in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, have proven useful for generating libraries of antibody fragments (and the nucleotide sequences encoding them) for in vitro selection and amplification of specific antibody fragments that bind to target antigens. Nucleotide sequences encoding the VH and VL regions are linked to a gene fragment encoding a leader signal that targets the periplasmic space of Escherichia coli (E. coli), and the resulting antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pIII or pVIII). Alternatively, antibody fragments are displayed on the exterior of the lambda phage capsid (phage body). An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by propagating phage containing the selected library members in bacterial cells. Furthermore, because the nucleotide sequences encoding the polypeptide library members are contained on phage or phagemid vectors, sequencing, expression and subsequent genetic manipulation are relatively straightforward.

[0082] Methods for constructing bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art.

[0083] Methods for producing a polypeptide may include culturing host cells transformed with a recombinant expression vector encoding the polypeptide under conditions that promote expression of the polypeptide, and then recovering the expressed polypeptide from the culture. Those skilled in the art will recognize that procedures for purifying the expressed polypeptide will vary depending on factors such as the type of host cell used and whether the polypeptide is intracellular, membrane-bound, or soluble and secreted from the host cell.

[0084] Any suitable expression system may be used. The vector may contain DNA encoding the polypeptide or fragment of the present invention operably linked to appropriate transcriptional or translational regulatory nucleotide sequences, such as those derived from mammalian, avian, microbial, viral, bacterial, or insect genes. A nucleotide sequence is operably linked when it is functionally related to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if it 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 for identifying transformants are generally incorporated into the expression vector.

[0085] Additionally, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into an expression vector. The DNA sequence of a signal peptide (secretory leader) can be fused in-frame to a nucleic acid sequence of the invention such that the DNA is first transcribed and the mRNA is translated into a fusion protein containing the signal peptide. A signal peptide that is functional in the intended host cell promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, allowing for secretion of the polypeptide from the cell.

[0086] Suitable host cells for expression of the polypeptide include higher eukaryotic cells and yeast, although prokaryotic systems are also suitable.

[0087] Mammalian cells, particularly CHO cells, are particularly preferred for use as host cells. CHO cells are widely used for protein production due to their ease of culture and transfection. ExpiCHO-S cells are a suspension cell line that can be grown to very high densities, enabling high protein expression. Plasmid DNA of interest can be transfected into ExpiCHO cells by complexing with ExpiFectamine (a cationic lipid-based transfection reagent) to condense the DNA. This condensed DNA enters the ExpiCHO cells by endocytosis and is expressed in the nucleus. The expressed protein is present in the cell culture supernatant and can be harvested after a suitable period of time.

[0088] nucleic acid Nucleic acids for use in accordance with the present invention can comprise DNA or RNA and can be wholly or partially synthetic. In a preferred embodiment, a nucleic acid for use in the present invention encodes an antibody or antibody fragment of the present invention as defined above. One skilled in the art will be able to determine substitutions, deletions, and / or additions to a nucleic acid that will still provide an antibody molecule of the present invention or an antibody molecule for use in the present invention.

[0089] Nucleic acid sequences encoding antibodies or antibody fragments for use in the present invention can be readily prepared by one of skill in the art. These techniques include (i) using the polymerase chain reaction (PCR) to amplify samples of such nucleic acids, for example, from genomic sources; (ii) chemical synthesis; or (iii) preparation of cDNA sequences. DNA encoding antibody fragments can be produced and used by any suitable method known to those of skill in the art, including obtaining the encoding DNA, identifying appropriate restriction enzyme recognition sites on either side of the portion to be expressed, and excising the 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 DNA with appropriate PCR primers. Sequence modifications can be made, for example, using site-directed mutagenesis, to direct expression of modified peptides or to take into account codon preferences in the host cell used to express the nucleic acid.

[0090] The nucleic acids may be included as constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. The constructs may be contained within recombinant host cells comprising one or more constructs as described above. Expression may conveniently be achieved by culturing recombinant host cells comprising the nucleic acids under appropriate conditions. Following production by expression, the antibody or antibody fragment may be isolated and / or purified using any suitable technique and then used as appropriate.

[0091] Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, insect, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, and NS0 mouse myeloma cells. A common, preferred bacterial host is Escherichia 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 producing binding members. General techniques for producing antibodies are well known to those skilled in the art.

[0092] In certain embodiments of the invention, recombinant nucleic acids are provided comprising an insert encoding an antibody heavy and / or light chain variable domain. By definition, such nucleic acids include the coding single-stranded nucleic acid, the double-stranded nucleic acid consisting of the coding nucleic acid and its complementary nucleic acid, or these complementary (single-stranded) nucleic acids themselves.

[0093] Furthermore, the nucleic acid encoding the antibody heavy and / or light chain variable domain may be an enzymatically or chemically synthesized nucleic acid having an authentic sequence encoding a naturally occurring heavy and / or light chain variable domain, or a variant thereof. Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: [Example]

[0094] Materials and Methods Library Design Antibody libraries were generated for (i) a humanized anti-cathepsin S antibody, Fsn0503h (Fusion Antibodies, Belfast) (Kwok et al., Molecular Cancer 2011, 10:147) and (ii) trastuzumab (Roche) by searching for specific DNA sequence motifs, RGYW in the 3' to 5' strand and WRCY in the 5' to 3' strand, recognized by the AID enzyme, which is responsible for generating mutations in antibodies in humans, thereby mimicking natural somatic hypermutation in humans.

[0095] The DNA sequences had point mutations introduced into the guanine at position 2 of the DNA sequence motif RGYW or the cytosine at position 3 of the DNA sequence motif WRCY, changing the nucleotide to any other nucleotide. The human AID enzyme introduces spontaneous mutations at these positions, which can result in a possible change in the amino acid at a single position. To generate all naturally possible amino acids throughout the antibody sequence and form an initial library, all possible mutations were identified throughout the antibody DNA sequence in both the 3' to 5' and 5' to 3' strands.

[0096] The size of each library was then controlled by removing any recognized sequence burden, such as deamidation sites, isomerization sites, n-linked glycosylation sites, and oxidation sites, from the newly generated amino acid sequences.

[0097] Molecular docking For each library, homology modeling was performed using the antibody prediction tool in Maestro 11.7 (Schrodinger) to generate antibody models based on the amino acid sequences of the heavy and light chain variable regions. Antigens were imported from the PDB. For Fsn0503h, we imported the crystal structure of human cathepsin S (CatS) with a C25S mutation and the bound drug (PDB code: 3MPE). For the trastuzumab variant library, we used the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB code: 1N8Z).

[0098] For both the antibody and antigen models, the Protein Preparation Wizard (Bioluminate, Schrodinger) was used to assign bond orders (using the Chemical Component Dictionary (CCD) database), add hydrogens, create zero-order bonds to metals, create disulfide bonds, and convert selenomethionine to methionine. Prime was used to fill missing side chains and loops, and Epik was used to generate het states. ProtAssign (Bioluminate, Schrodinger) was used to further refine the structures and define hydroxyl, asparagine, glutamine, and histidine states. Waters with fewer than three non-water bonds were removed. Finally, the structures were minimized using the OPLS3e force field (Bioluminate, Schrodinger).

[0099] Cathepsin S was docked onto the surface of an 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. In vitro information about the epitope of Cathepsin S was used to select suitable docked configurations based on rank, shape complementarity (using a protein interaction analysis tool), and insight into surface interactions.

[0100] Similarly, for trastuzumab variants, the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB code: 1N8Z) was docked onto 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. Based on the rank and our insights into shape complementarity and surface interactions (using protein interaction analysis tools), suitable docked configurations were selected.

[0101] Combinatorial mutation analysis Residue scanning was performed on the docked configuration of the antibody-antigen complex, and informed mutations were made to the antibody, avoiding highly conserved residues, to increase the affinity of the antibody for the antigen and enhance stability.

[0102] Residue scanning was performed by first generating a model in which a single amino acid was mutated from the original structure, and then repeating iterations of up to six simultaneous mutations (with the same mutation) from the wild-type Ab. The residue mutation tool calculated the stability and affinity of the mutants relative to the original wild-type antibody-antigen complex.

[0103] Variants were then classified by affinity and stability differences relative to the wild type: scores below a threshold of -2 for stability and affinity differences were selected, and the variants were ranked based on the combination of the two scores (prioritizing affinity differences).

[0104] The best variants of the Fsn503h and trastuzumab antibodies were synthesized and analyzed in vitro.

[0105] antibody synthesis Transient transfection: Culture 4–6 × 10 suspension-adapted ExpiCHO cells in ExpiCHO expression medium in a 500 ml aerated Erlenmeyer flask at 130 rpm, 37 °C, and 8% CO 6 The cells were routinely cultured at 1000 cells / ml. For each Fsn0503h variant, 1 μg / ml of DNA was diluted in 4% (v / v) OptiPRO SFM in a centrifuge tube. In a separate 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 being cultured in a 125 ml vented Erlenmeyer flask at a final density of 6 x 10 cells / ml. 6The transfected cells were added to 25 ml of ExpiCHO cells at 1000 cells / ml. Each transfected culture was grown overnight at 37°C, 8% CO2, and 130 rpm. 20 hours after transfection, the cells were supplemented with 0.6% (v / v) ExpiCHO enhancer and 24% (v / v) ExpiCHO feed. The cultures were then transferred to an incubator at 32°C, 5% CO2, and 130 rpm. The cultures were harvested by centrifugation at 4000 rpm for 40 minutes at 18°C.

[0106] purification: Purification of Fsn0503h wild-type and variant antibodies was performed in two steps using a Tricorn 5 / 50 column (GE) packed with 1 ml of MabSelect™ PrismA (GE), followed by 10 ml (2 x 5 ml) Hitrap Desalting (Desalt) columns (GE). MabSelect™ PrismA affinity media was chosen for its high mAb binding and specificity properties, as well as its alkaline resistance for efficient clean-in-place (CIP). Unless otherwise noted, all steps were performed at room temperature using a flow rate of 4 ml / min. After loading (performed using an AKTA sample pump), the Protein A column was washed with 10 column volumes (CV) of PBS (reverse flow mode), followed by a one-step elution with 100 mM glycine, pH 3.0 (reverse flow mode). Protein A elution was collected in a 2 ml loop (AKTA with a 10 mm flow cell) when the absorbance at 280 nm exceeded 120 mAU and immediately injected onto a pre-equilibrated Desalt column. The Desalt peak elution was collected in a 96-well 2 ml block at 2-8 °C when the absorbance of the eluate exceeded 100 mAU. Additionally, to avoid cross-contamination, the automated process included clean-up of both the affinity column and the desalting column. Clean-up was performed between each sample for all contact paths using 0.2 M NaOH (reverse flow mode was used to wash the column).

[0107] The level of expression was determined as the total yield of purified material per ml of culture medium.

[0108] Trastuzumab variants were synthesized using similar techniques.

[0109] Affinity Ranking Enzyme-linked immunosorbent assay: MaxiSorp 96-well plates were coated with 66 0503 variants at 1 μg / ml in PBS for 24 hours at 4°C. To obtain EC50 results, variants were serially diluted in PBS from 1000 ng / ml to 1 ng / ml and coated in duplicate. A standard curve was prepared using the parent 0503 antibody coated at 1 μg / ml in PBS for 24 hours at 4°C. After 24 hours, MaxiSorp plates were washed three times with PBS-T. 200 μl of SuperBlock was added to each well, removed, and replaced three times. 100 μl of 200 ng / ml Cat S antigen was added to each well and shaken at 150 rpm at RT for 1 hour 30 minutes. The plates were washed three times with PBS-T and allowed to dry. 100 μl of 5 μg / ml anti-his-HRP was added to each well and shaken at 150 rpm at RT for 1 hour 30 minutes. The plate was washed three times with PBS-T and once with PBS, then dried. 100 μl of TMB was added to each well and incubated at 37°C for 10 minutes. 50 μl of 1 M HCl was then added, and the absorbance of the plate was measured at 450 nm.

[0110] Affinity ranking using the Octet RED96 system (Fsn503h variants): The affinity ranking assay was performed by first capturing IgG using an anti-human Octet biosensor (ForteBio part number 18-5060), followed by a 2-minute baseline step in HBS-EBT buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / ml BSA, and 0.05% Tween-20, pH 7.4). The mAb-captured biosensor was then submerged in a well containing 200 ng / ml recombinant cathepsin S antigen for 10 minutes (association step), followed by a 10-minute dissociation step in running buffer. To allow for double-referencing, the IgG-captured sensor was immersed in a well containing buffer alone, and a blank sensor was also immersed in a well containing antigen. This reference provided a means of compensating for both spontaneous dissociation of the captured IgG and nonspecific binding of the antigen to the sensor surface. All steps were performed in HBS-EBT buffer at 25°C with a constant flow rate of 1000 rpm. A new sensor was used for each sample. Dissociation rate constants (koff) were calculated using ForteBio Data Analysis software. All consumables used were as recommended by ForteBio.

[0111] Antibody quantification using Biolayer Interferometry (Octet RED96 System): To measure IgG content, 200 μL volumes (ranging from 0.06 to 512 μg / ml) of antibody standards and IgG-containing cell supernatants (diluted within the calibration curve range) were prepared in duplicate using 1x HBS-EBT buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / ml BSA, and 0.05% Tween-20, pH 7.4) and placed in wells of a 96-well black microtiter plate (Greiner Bio-One part number 655209). All samples and standards were measured in duplicate using a Protein A biosensor (Fortebio PN 18-5010). The plate was placed in an Octet and equilibrated to 25°C in a thermostatic chamber. A run was initiated by placing the sensor in the well, and the change in layer thickness (nanometers, nm) over time was measured, all under computer control. Data were acquired for 180–600 seconds at flow rates of 400–1000 rpm (orbital flow) for each set of eight samples at a time (one plate column measured simultaneously). Data were processed automatically using Octet User Software version 3.1. Measurement time and flow rate were varied depending on the required sensitivity.

[0112] Affinity ranking (Trastuzumab variants) using Biolayer Interferometry (Octet RED96 system): The affinity ranking assay was first performed using an anti-human Octet biosensor (ForteBio part number 18-5060). *IgG capture was followed by a 2-minute baseline step in HBS-P+ buffer (10 mM HEPES, 150 mM NaCl, 1 mg / ml BSA, and 0.05% Tween-20, pH 7.4). The mAb-captured biosensor was then submerged in a well containing 5 nM recombinant HER2 (Acro Biosystems; P / N.H5225) antigen for 15 minutes (association step), followed by a 20-minute dissociation step in running buffer. To allow for double-referencing, the IgG-captured sensor was immersed in a well containing buffer alone, and a blank sensor was also immersed in a well containing antigen. This reference provided a means of compensating for both spontaneous dissociation of the captured IgG and nonspecific binding of the antigen to the sensor surface. The steps were performed at a constant flow rate of 1000 rpm in HBS-EBT buffer at either 25°C or 37°C. A new sensor was used for each sample. The dissociation rate constant (koff) was calculated using ForteBio Data Analysis software. All consumables used were recommended by ForteBio.

[0113] * To allow for similar loading levels, quantified IgG-containing cell supernatants (as described above) were diluted to the same concentration.

[0114] K using biolayer interferometry (Octet RED96 system) D measurement: Kinetic assays were performed by first capturing IgG using an anti-human Fc Octet biosensor, followed by two baseline steps of 2 min each in HBS-P+ buffer running buffer. The mAb-captured biosensors were then submerged in wells containing various concentrations of HER2 for 15 min, followed by 20 min of dissociation in running buffer. To allow for double-referencing, the IgG-captured sensors were submerged in wells containing buffer alone, and blank sensors were also submerged in wells containing a range of antigen concentrations. This reference provided a means of compensating for both spontaneous dissociation of the captured IgG and nonspecific binding of antigen to the sensor surface. All steps were performed at a constant flow rate of 1000 rpm in kinetic buffer at 37°C.

[0115] Melting point determination Antibodies typically exhibit two measurable melting temperatures, termed TM1 and TM2, in analyses resulting from the thermal denaturation of different portions of the assembled molecule. These values ​​were determined by thermal shift assay. Five microliters of a solution containing Sypro Orange (diluted 1 / 200 in PBS, pH 7.4; Molecular Probes) and 45 μl of 0.3 mg / ml antibody were added to a low-profile PCR tube (Bio-Rad; TLS0851). The tube was sealed with an optically ultra-clear sealing cap (Bio-Rad; TCS0803) and heated in 1°C increments from 20°C to 90°C in an i-Cycler iQ5 Real-Time PCR Detection System (Bio-Rad). Fluorescence changes in the plate wells were simultaneously monitored with a charge-coupled device (CCD) camera. The excitation and emission wavelengths were 485 nm and 575 nm, respectively. The midpoint of the protein unfolding transition temperature, T, was determined. m was calculated using Bio-Rad iQ5 software.

[0116] Determination of monodispersity The level of monodispersity of each variant, defined as the individual free molecules of immunoglobulin within the antibody preparation, was also shown to differ from the wild-type molecule. This measurement is commonly used as an indicator of the aggregation tendency of antibody molecules. Aggregation is the tendency of protein molecules to associate into multimeric complexes, reducing the solubility and activity of an antibody preparation over time, and is an important attribute that contributes to the stability of antibody molecules in solution.

[0117] This was determined in antibody solutions purified using size-exclusion chromatography. All samples were diluted to a final concentration of 0.1 mg / ml using phosphate-buffered saline. Highly purified samples of antibody were individually loaded onto a Superdex 200 Increase 10 / 300GL gel filtration column. 50 μl of sample was injected, and the column flow rate was maintained at 0.75 μl / min. Separation and equilibration steps were performed in phosphate-buffered saline at 22°C. Protein peaks were monitored using absorbance at 280 and 214 nm, and spectra were analyzed using the Unicorn emulation software package (GE healthcare). Results were expressed as the V of each peak. r (ml) and relative peak area (%).

[0118] Example 1 - Cathepsin S antibody variants Library Design The DNA sequences of the light and heavy chain variable domains of the Fsn0503h antibody were analyzed for motifs susceptible to mutation during somatic hypermutation, and the potential amino acids corresponding to these mutational outcomes were plotted on top of the parental sequences, as shown in Figure 1. Undesirable amino acids or stop codons generated as a result of DNA mutations were identified.

[0119] It was a surprising finding that there were more mutations in the framework regions than in the CDRs, and in particular, CDR-H3 had no functional mutations.

[0120] Molecular docking The parent Fsn0503h antibody variable domain (Kwok et al., Molecular Cancer 2011, 10:147) was docked with the Cathepsin S protein using Schrodinger molecular docking software as described in Methods. The results of the docking procedure are shown in Figure 2.

[0121] Combinatorial mutagenesis Mutations of amino acid residues were introduced within the docked structure, and the relative differences in both affinity and stability were predicted. The number of mutations introduced was increased until no further benefit in stability or affinity was predicted. The results of combinatorial mutagenesis are shown in Figure 3. We identified 66 variants with predicted improvements in both affinity and stability. DNA encoding these variants was then synthesized, and the antibodies were expressed and purified as described in the methods.

[0122] ELISA Each of the purified variants was analyzed by ELISA for binding to recombinant cathepsin S protein. The results (Figure 4) show that 12 variants had higher relative ODs than the parent Fsn0503h, potentially indicating a higher affinity for the cathepsin S target.

[0123] Affinity Ranking To determine the exact affinity of each variant compared to the parent Fsn0503h antibody, interactions with cathepsin S were measured by BLI using an Octet instrument as described in the methods.

[0124] The results show that approximately 50% of the variants have improved affinity as measured against the average reading of the Fsn0503h antibody (Figure 6). The association (Kon) and dissociation (Kdis) rates are shown in Figure 5.

[0125] Effects on expression The 66 variants expressed in CHO as described above also showed a range of expression levels compared to the wild type. IgG levels were determined by quantitative human IgG immunoassay on a BLI Octet instrument after purification and represent the total amount relative to the amount of purified supernatant.

[0126] Melting point change The 66 variants also exhibited a range of variability in stability characteristics associated with antibody molecules, including changes in the melting temperature profile across the two melting points typically observed in immunoglobulin molecules. The results of the melting point measurements are shown in Figure 22. It is noteworthy that some variants, such as Mut 6 and Mut 49, appear to lack a clear two-phase melting pattern, with only a single melting temperature being observable.

[0127] Change in monodispersity This property was analyzed by analytical size exclusion chromatography of 66 variants and shows a range of values ​​among the variants analyzed, as shown in FIG. Example 2 - Trastuzumab variants

[0128] Library Design A library of trastuzumab antibody variants was generated based on somatic hypermutation that occurs naturally in humans. Using the method described above, we replicated somatic hypermutation to generate naturally occurring mutations in the trastuzumab DNA sequence, which were then translated into their respective amino acid mutations, as shown in Figure 9. These mutations were compiled into a library of potential mutations at specific sites in both the complementarity-determining regions (CDRs) and framework regions (Figures 7 and 8), with the potential amino acid results of this method detailed next to the parent sequence. Any undesirable amino acids or stop codons generated as a result of DNA mutations were identified.

[0129] The fully solved crystal structure of trastuzumab in complex with the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB code: 1N8Z) is shown in Figure 10. The library of trastuzumab mutations shown in Figures 7 and 8 was used for mutant scanning. Mutants were sequentially scanned by first analyzing single amino acid mutations, followed by analysis of the heavy and light chains from two mutations to a total of six mutations, and then the variants were ranked based on affinity and stability. As with the Fsn503h library, Schrodinger molecular docking software was used as described in the Methods.

[0130] Mutations of amino acid residues were introduced into the docked structure, and the relative differences in both affinity and stability were predicted. The number of mutations introduced was increased until no further benefit in stability or affinity was predicted. The results of combinatorial mutagenesis are detailed in the tables in Figures 11 and 12. A total of 558 potential variants of trastuzumab were found to have both improved affinity and improved stability. The affinities of the top-ranked five variants are shown in Figure 13. Among these variants, a total of five mutations across the heavy and light chains were found to be the most common. The predicted free energy change (ΔΔG) was used to score the variants relative to the affinity of the parent antibody.

[0131] Off Rate Ranking A panel of 89 trastuzumab variants was screened for binding to the HER2 antigen using biolayer interferometry (see Materials and Methods for details). Figure 14 shows sensorgrams for 20 samples, with report points displayed as color bars. Figure 15 shows a scatter plot of the report points for stability_early plotted against stability_late. The best binders (14 in total) with high binding stability and slow dissociation are highlighted with blue circles. To provide more biologically relevant data, we repeated the off-rate ranking experiment for these variants at 37 °C (Figure 16). Variant 19 appeared to have improved stability compared to WT trastuzumab and was used for kinetic analysis.

[0132] KD determination All samples were diluted in freshly prepared running buffer. Either trastuzumab or a variant was immobilized on a series of biosensor surfaces using the described capture method (see Materials and Methods). HER2 was passed over the surface to generate a binding response. Binding data for HER2 interactions were collected by the biosensors at 37°C. Results were globally fitted and k a , k d , and K D A dilution series of HER2 antigen (5 nM to 0.078 nM) was used in the association step to obtain the best possible value of K. The response data for antigen binding to surface-immobilized IgG were fitted to a 1:1 binding model to generate data traces (red - see Figure 17). Experiments were performed twice in duplicate, and the average kinetic parameters are summarized in Table 9. For example, the data show that variant 19 exhibits an apparent approximately 2-fold increase in affinity compared to the wild-type control and commercial trastuzumab. The affinity increase is primarily due to a slower Kd, 6.04E-05, compared to 1.20E-04 for the wild-type antibody. Figure 18 shows a schematic comparison of the actual and predicted affinities of trastuzumab variants selected by in silico prediction of improved affinity and stability. The actual Octet affinity ranking correlates fairly well with the top 20 predictions.

[0133] [Table 1]

[0134] R 2 The value indicates how well the fit correlates with the experimental data, with a fit greater than 0.95 being considered good; X 2 is the sum of the squares of the deviations and should generally be less than 3; X 2 is a measure of the error between the experimental data and the fitted curve. 2 The smaller the value, the better the fit.

[0135] Determination of KD of monomers To further validate the affinities of the top variants and understand the contribution of avidity to affinity measurements within the top two variants (MUT 5 and MUT 19), these molecules were prepared by enzymatic cleavage into monomeric fragment antigen-binding (Fab) fragments, consisting of the heavy and light chain variable domains and the CH1 constant domain, with a single antigen-binding domain per molecule.

[0136] Initial measurements were performed after coating the probe with 2.5 μg / ml of biotinylated Her2 recombinant Fc fusion protein. A range of concentrations of each of the three monomeric antibodies was evaluated for binding to the Her2 surface. No double referencing was applied in this case. The sensorgram data are shown in Figure 19, and the resulting kinetic calculations (1:1 model) are listed in Table 10.

[0137] [Table 2]

[0138] Sensors were then prepared by coating with 0.625, 1.25, or 2.5 μg / ml of biotinylated Her2 recombinant Fc fusion protein. Purified Fab fragments were then tested for binding to these probes. Sensorgram traces of these measurements are shown in FIG. 20 and are detailed in Tables 11, 12, and 13. Unlike previous experiments, this data was double-referenced to rule out any drift due to nonspecific interactions at the probe surface.

[0139] [Table 3]

[0140] [Table 4]

[0141] [Table 5]

[0142] From these monomer analyses, it is difficult to assign a definitive value to the increased affinity of MUT 5 and MUT 19 compared to the WT trastuzumab molecule, as affinity values ​​appear to change as a result of coating concentration. Without wishing to be bound by theory, this is thought to be due to issues with operating at the sensitivity limit of the ForteBio Octet Biosensor instrument.

[0143] conclusion Using the methods of the present invention, the inventors have successfully demonstrated that very large physical libraries of antibody variants can be generated to improve the affinity of the antibody for its target without the need for a subsequent selection / screening process. Furthermore, expression generates a pool of variants that display a range of interesting attributes, including affinity, expression level, and physicochemical properties, all of which are of interest for the potential developability of the antibody molecule.

[0144] While the present invention has been particularly shown and described with reference to specific examples, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention.

Claims

1. (i) has at least two amino acid changes in the light chain sequence when compared to the light chain amino acid sequence of a reference antibody; (ii) has at least two amino acid changes in the heavy chain sequence when compared to the heavy chain amino acid sequence of a reference antibody; or (iii) a variant of a reference antibody, which has at least one amino acid alteration in its light chain sequence compared to the light chain amino acid sequence of the reference antibody and at least one amino acid alteration in its heavy chain sequence compared to the heavy chain amino acid sequence of the reference antibody; each of said amino acid changes is at an amino acid residue independently encoded by a DNA segment of the variant DNA sequence; A variant of a reference antibody, wherein said DNA segment of the variant differs from the corresponding segment of the DNA sequence encoding the reference antibody by a point mutation in a DNA motif that is susceptible to deamination by somatic hypermutase.

2. 2. The variant antibody of claim 1, wherein the somatic hypermutator is activation-induced deaminase (AID) and the DNA motif is DGYW or WRCH, e.g., 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.

3. 3. The variant antibody of claim 1 or claim 2, wherein the reference antibody is trastuzumab.

4. The amino acid changes are lc9N, lc9T, lc9I, lc9R, lc9K, lc25G, lc25V, lc25D, lc31N, lc31S, lc31I, lc32D, lc32G, lc32V, lc32T, lc32N, lc32S, lc32I, lc32P, lc32L, lc32F, lc33L, lc33I, lc34G, lc34V, lc34D, lc38E, lc38K, lc40A, lc40S, lc40T, lc43G, lc43V, lc43T, lc43N, lc43S, lc43I, lc43P, lc43L, lc43F, lc4 6V, lc46I, lc47V, lc51S, lc51P, lc51T, lc76R, lc76N, lc76T, lc76K, lc76I , lc79K, lc79E, lc80T, lc80S, lc80A, lc85S, lc85N, lc85I, lc89H, lc90E, l c90A, lc91N, lc91D, lc91Y, lc93S, lc93N, lc93I, lc94S, lc94N, lc94I, lc1 01D, lc102S, lc102N, hc2L, hc2I, hc3H, hc4M, hc4V, hc13K, hc13E, hc14A, h c14T, hc14S, hc16A, hc16V, hc16D, hc23E, hc23G, hc23V, hc23T, hc23K, hc 23R, hc23I, hc23P, hc23L, hc23S, hc24D, hc24G, hc24V, hc24T, hc24N, hc24 S, hc24I, hc24P, hc24L, hc24F, hc26A, hc26V, hc26D, hc28K, hc35N, hc35D, hc35Y, hc48L, hc48I, hc49G, hc49S, hc56A, hc56V, hc56D, hc58S, hc58N, hc 4. The variant antibody of claim 3, selected from the group consisting of 58I, hc61G, hc61V, hc61D, hc79G, hc79V, hc79D, hc82E, hc82K, hc85R, hc88D, hc88T, hc88S, hc88P, hc88G, hc92G, hc92V, hc92D, hc103A, hc103V, hc103D, hc106D, hc106G, hc106V, hc106T, hc106N, hc106S, hc106I, hc106P, hc106L, hc106F, hc114S, hc114N, and hc114I.

5. 5. The variant antibody of claim 4, wherein the amino acid changes are selected from the group consisting of lc9T, lc9I, lc9R, lc9K, lc43F, lc47V, lc51P, lc51T, lc101D, hc2L, hc3H, hc14S, hc16V, hc24P, hc26A, hc26V, hc48I, hc58S, hc61V, hc79V, hc85R, hc88G, hc92V, hc92D, hc103A, hc103V, hc106V, hc114S, hc114N, and hc114I.

6. A variant antibody according to any one of claims 3 to 5, wherein the light and heavy chain sequences of the variant do not differ from the sequences of the reference antibody at any residue other than the amino acid changes described in claim 4.

7. 7. The variant antibody of any one of claims 3 to 6, wherein the variant antibody molecule has a combination of amino acid mutations as shown for variant number 19 in Table 8 or any of the other antibodies listed in Table 8.

8. 7. The variant antibody of any one of claims 3 to 6, wherein the variant antibody molecule has a combination of amino acid mutations as shown for any of the antibodies listed in Tables 2 to 7.

9. 9. The variant antibody of any one of claims 3 to 8, which is a variant of the trastuzumab antibody comprising the amino acid changes lc9K, lc43F, and hcl06V compared to trastuzumab.

10. 3. The variant antibody of claim 1 or claim 2, wherein the variant is a variant of a cathepsin S antibody and the reference antibody is Fsn503h.

11. The amino acid changes are lc12A, lc12S, lc12T, lc19V, lc28R, lc32T, lc32I, lc45A, lc45S, lc45T, lc50H, lc51V, lc51F, lc51I, lc56L, lc56F, lc56I, lc58K, lc66S, lc69A, lc69V, lc81T, lc81I, lc81N, lc85P, lc85S, lc85T, lc90L, lc90F, lc96I, lc96S, lc90H, lc9 ...91I, lc96L, lc96F, lc96I, lc96K, lc90H, lc91V, lc51F, lc51I, lc56L, lc56F, lc56I, lc58K, lc66S, lc69A, lc69V, lc81T, lc81I, lc81N, lc85P, lc85S, lc85T, lc90L, lc90F, lc96I, lc96S, lc90H, lc91V, lc51F, lc 11. The variant antibody of claim 10, selected from the group consisting of c96I, lc96N, lc108N, hc3H, hc4V, hc4M, hc10A, hc10V, hc14A, hc14S, hc24G, hc24V, hc30T, hc30I, hc31R, hc31T, hc37L, hc37F, hc40P, hc40S, hc52S, hc52I, hc53S, hc53I, hc84T, hc84I, hc92G, and hc92V.

12. 12. The variant antibody of claim 11, wherein the amino acid changes are selected from the group consisting of lc12A, lc12S, lc12T, lc19V, lc45S, lc45T, lc50H, lc51V, lc56I, lc81I, lc96I, lc96S, lc96I, lc96N, lc108N, hc10A, hc10V, hc14S, hc30I, hc31R, hc37L, hc37F, hc40P, hc40S, hc52I, and hc92G.

13. A variant antibody according to any one of claims 10 to 12, wherein the light and heavy chain sequences of the variant do not differ from the sequences of the reference antibody at any residue other than the amino acid changes described in claim 11.

14. 14. The variant antibody of any one of claims 10 to 13, wherein the variant antibody molecule has a combination of amino acid mutations as shown for any of the antibodies listed in Table 1.

15. 15. The variant antibody of any one of claims 1 to 14, wherein the light and heavy chain sequences of the variant comprise a total of at least three, such as at least four, at least five, or at least six amino acid changes compared to the amino acid sequences of the reference antibody.

16. The variant antibody of any one of claims 1 to 15, wherein one or more of said amino acid alterations are in a framework region of said variant antibody.

17. The variant antibody of any one of claims 1 to 16, wherein one or more of said amino acid alterations are in a CDR of said variant antibody.

18. 17. The variant antibody of claim 16, wherein all of the amino acid changes are in framework regions of the variant antibody.

19. 19. The variant antibody of any one of claims 1 to 18, wherein the change in affinity of said variant antibody molecule compared to a reference antibody is greater than -2 and the change in stability of said variant antibody molecule compared to a reference antibody is greater than -2.

20. A library of antibody molecules, each antibody molecule being 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, each of which is independently encoded by a DNA segment of a variant DNA sequence, wherein the variant DNA segment differs from the corresponding segment of the DNA sequence encoding the reference antibody by a point mutation in a DNA motif that is susceptible to deamination by somatic hypermutase.

21. 21. The library of claim 20, wherein the somatic hypermutator is activation-induced deaminase (AID).

22. 22. The library of claim 21, wherein the DNA motif is DGYW or WRCH, e.g., 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.

23. 23. The library of any one of claims 20 to 22, wherein the nucleotide sequence encoding each of the antibody molecules does not differ from the nucleotide sequence of a reference antibody at any residue other than the residues of the DNA motif.

24. The library of any one of claims 20 to 23, wherein one or more of the DNA motifs are in a DNA sequence encoding a framework region of the antibody molecule.

25. The library of any one of claims 20 to 24, wherein one or more of the DNA motifs are in a DNA sequence encoding a CDR of the antibody molecule.

26. 25. The library of claim 24, wherein all of the DNA motifs are in DNA sequences encoding framework regions of the antibody molecules.

27. 27. The library of any one of claims 20 to 26, wherein the DNA sequence of each variant does not contain (or encode) a deamination site, an isomerization site, an N-linked glycosylation site, or an oxidation site originating from said point mutation in said DNA motif.

28. A library of nucleotide sequences, each member of the library encoding an antibody molecule of the library of antibody molecules according to any one of claims 20 to 27.

29. 1. A method for generating / producing a library of variant antibody molecules, wherein said variant antibody molecules are variants of a reference antibody, said method comprising: a) providing a nucleotide sequence encoding a reference antibody; b) identifying one or more DNA motifs in said nucleotide sequence that are susceptible to deamination by somatic hypermutator enzymes; 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, said substitution resulting in a variant nucleotide sequence encoding a variant antibody molecule having an alteration in the amino acid sequence encoded by said DNA motif compared to a reference antibody; and d) repeating steps (b) and (c). A method comprising:

30. 30. The method of claim 29, further comprising screening said library of variants to determine binding to the epitope bound by the reference antibody.

31. 31. The method of claim 30, wherein those variants determined to bind to the epitope with an affinity and / or stability greater than a predetermined value relative to the reference antibody are used to generate an optimized library of variant antibody molecules.

32. The method of any one of claims 29 to 31, which is a computer-implemented method.

33. The method of any one of claims 29 to 32, further comprising the step of synthesizing the variant antibody molecule.

34. The method of any one of claims 29 to 33, wherein the library is a library of antibody molecules according to any one of claims 21 to 27.

35. The library of any one of claims 20 to 27 or the method of any one of claims 29 to 34, wherein the reference antibody is trastuzumab or the anti-cathepsin S antibody Fsn0503h.

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