Antibody libraries and methods

Abstract

The present disclosure relates to methods for generating antibody libraries, antibody libraries produced using such methods, and variant antibodies. Currently, methods for improving antibody binding (affinity maturation assays) involve screening large libraries of antibody variants (often >10 nucleotides) to identify a small subset of variants with improved properties. 10 The method involves obtaining the nucleotide sequence of the framework and complementarity-determining regions of a target antibody and identifying motifs recognized by a deaminating somatic hypermutation enzyme. A small library of variants incorporating one or more of these mutations is then generated. A relatively high percentage of the variants were found to have increased affinity. The technology of the present invention has been demonstrated with trastuzumab and cathepsin S antibodies, and the variants produced are also claimed.

Description

[Technical Field]

[0001] The present invention relates to a method for generating an antibody library, an antibody library produced using such a method, and a variant antibody. [Background technology]

[0002] Antibody affinity is a measure of the strength of the interaction between an antibody and the protein to which it specifically binds, as the ratio of the association rate to the dissociation rate. There are many reasons why optimizing this ratio is desirable. Increased 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 become more sensitive. Conversely, decreasing affinity can 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 typically involve generating large libraries of variants through random mutagenesis focused within antibody complementarity-determining regions (CDRs). This process involves a very large number of variants (often >10) to identify only a small fraction of variants with improved properties. 10 It has the disadvantage of screening for ) [Overview of the Initiative]

[0004] The present invention addresses many of the problems of the prior art. As described in the examples, the inventors have shown that, surprisingly, a library of variants of a given antibody sequence can be created by restricting mutations in the nucleotide sequence encoding a given antibody sequence to sites corresponding to DNA motifs targeted by enzymes involved in somatic hypermutation, and that this library contains variants having a relatively small but relatively high proportion of increased affinity, or aggregation, or melting point, or expression level in CHO cells, or a combination thereof, compared to those prepared by many existing techniques. The inventors have illustrated the present invention using two unrelated antibodies: an anti-cathepsin S antibody, Fsn0503h (Fusion Antibodies Ltd), and an 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 the amino acid sequence of the reference antibody in one or more amino acid residues, each of which is independently encoded from a DNA segment of the DNA sequence encoding the variant, and the DNA segment of the variant differs from the corresponding DNA segment encoding the reference antibody by a point mutation in a DNA motif sensitive to deamination by somatic hypermutagenic enzymes.

[0006] A second aspect of the present invention provides a method for generating / producing a library of variant antibody molecules, wherein the variant antibody molecule is a variant of a reference antibody, and the method includes the following steps such that a library comprising multiple variants of the reference antibody is generated: a) Providing a nucleotide sequence encoding a reference antibody, b) A step of identifying one or more DNA motifs in the nucleotide sequence that are sensitive to deamination by somatic hypermutagenic enzymes; c) a step of selecting at least one variant nucleotide residue to replace a residue of one or more of the DNA motifs, wherein the substitution results in a variant nucleotide sequence encoding a variant antibody molecule having a change in the amino acid sequence encoded by the DNA motif compared to a reference antibody; and d) A step that repeats steps (b) and (c).

[0007] The reference antibody may 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 for generating a variant antibody molecule, wherein the variant antibody molecule is a variant of a reference antibody, and the method comprises the following steps: a) Providing a nucleotide sequence encoding a reference antibody, b) A step of identifying one or more DNA motifs in the nucleotide sequence that are sensitive to deamination by somatic hypermutagenic enzymes; c) A step of selecting at least one variant nucleotide residue to replace a residue of one or more of the DNA motifs, wherein the substitution results in a variant nucleotide sequence encoding a variant antibody molecule having a change in the amino acid sequence encoded by the DNA motif compared to a reference antibody.

[0009] In one embodiment of the present invention, the somatic hypermutagenic enzyme is an activation-inducible deaminase (AID).

[0010] In embodiments of the present invention, the DNA motif is DGYW or WRCH, for example, RGYW or WRCY, where D is adenine, guanine, or thymine, R is adenine or guanine, G is guanine, C is cytosine, H is adenine, cytosine, or thymine, W is adenine or thymine, and Y is cytosine or thymine.

[0011] The DNA motif may be located on any strand of DNA. If there are multiple DNA motifs, the motifs may be duplicated.

[0012] The inventors have shown that antibody libraries of the present invention or antibody libraries produced according to the method of the present invention can be further refined by limiting the members of the library to variants resulting from mutations in DNA motifs targeted by somatic hypermutagenic enzymes, which do not introduce any other motifs into the variant antibody molecule that are potentially undesirable, for example, in terms of stability or binding, compared to the reference antibody, such as stop codons.

[0013] Therefore, in certain embodiments of the present invention, the DNA sequence of each variant does not contain (or encode) a deamination site, isomerization site, N-linked glycosylation site, or oxidation site originating from the point mutation of the DNA motif.

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

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

[0016] In such an embodiment, the variant has no change in the light chain CDR1 as compared to the corresponding CDR of the reference array.

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

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

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

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

[0021] In another such embodiment, the variant has no change in the heavy chain CDR3 as compared to the corresponding CDR of the parental array.

[0022] Furthermore, as shown in the examples, in some variants of the reference antibody, many of the mutations characterizing the variant can be in the framework region of the variant antibody molecule.

[0023] Thus, in certain embodiments of the library or method of the present invention, one or more of the DNA motifs are in the DNA sequence encoding the framework region of the antibody molecule. In some such embodiments, all of the DNA motifs are in the DNA sequence encoding the framework region of the antibody molecule.

[0024] In one embodiment of the present invention, more than 20%, for example, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the nucleotide residues of each variant antibody that are different from the nucleotide residues at the corresponding positions of the reference antibody are residues of a DNA motif that is sensitive to deamination by somatic hypermutagenic enzymes.

[0025] In some embodiments of the present invention, the nucleotide sequence encoding each of the antibody molecules is identical to the nucleotide sequence of the reference antibody in all residues except for the residues of the DNA motif.

[0026] The antibody library of the present invention and / or the antibody library produced using the method of the present invention may be further refined to increase the proportion of high-affinity and / or stable variants.

[0027] The antibody library of the present invention and / or the antibody library produced using the method of the present invention may be further refined to increase the proportion of variants having different aggregation properties, i.e., variants with a lower ability to aggregate with each other. The antibody library of the present invention and / or the antibody library produced using the method of the present invention may be further refined to increase the proportion of variants having specific melting point properties. The antibody library of the present invention and / or the antibody library produced using the method of the present invention may be further refined to increase the proportion of variants exhibiting a preferred or desired expression level in CHO cells. The antibody library of the present invention and / or the antibody library produced using the method of the present invention may be further refined to increase the proportion of variants having high affinity, stability, desired aggregation properties, desired melting point properties, desired expression level properties, or a combination thereof.

[0028] Accordingly, in the present invention, the method may further include a step of determining the affinity and / or stability of the binding of the variant antibody molecule to the binding target of the reference antibody, the melting point compared to the reference antibody, the agglutination property compared to the reference antibody, the expression level compared to the reference antibody, or a combination thereof. Accordingly, in one embodiment of the method of the present invention, the method further includes a step of screening the library of variants to determine binding to an epitope to which the reference antibody binds. Appropriately, the method of the present invention may further include a step of screening the library of variants to determine the melting point, expression level, agglutination level, or stability of the variants compared to the reference antibody. An optimized library of variant antibody molecules can be generated using those variants that are determined to bind to the epitope with affinity and / or stability lower or higher than a predetermined value compared to the reference antibody, or to have melting properties, agglutination properties, or expression properties. 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 performed using in silico techniques, for example, by using molecular docking software implemented on a computer to model the binding of variants to the epitopes of reference antibodies.

[0029] As shown in the examples, molecular docking software allows variants to be ranked by their predicted affinity and stability, enabling the selection of small libraries of variants for DNA synthesis and expression. We have demonstrated that within such small libraries, a significantly larger number of variants possess increased affinity compared to the number expected to be identified in libraries generated by existing techniques such as error-prone PCR and 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 this invention. For example, suitable software includes Schrodinger's Bioluminate software, but other software is also available.

[0031] Accordingly, in embodiments of the present invention, the antibody library contains more than 1%, for example, 5%, 10%, 20%, 30%, 40%, or 50% of variants having increased affinity to the epitope compared to the affinity of the reference antibody to the epitope bound by the reference antibody.

[0032] In one embodiment of the method of the present invention, this method is a computer implementation method.

[0033] A fourth aspect of the present invention provides a computer-readable storage medium that includes instructions for performing a method for generating a library of variant antibody molecules according to a second aspect of the present invention.

[0034] The method of the present invention, whether or not it is implemented by computer, may further include a step of synthesizing a variant antibody molecule.

[0035] In embodiments of the method of the present invention, if the method includes, for example, a computer-implemented screening method using docking modeling software, the method of the present invention may further include in vitro screening of the library of variants to determine binding to an epitope to which a reference antibody binds.

[0036] A fifth aspect of the present invention is a variant of a trastuzumab antibody, the variant having (i) at least two amino acid changes in the light chain amino acid sequence when compared to the light chain amino acid sequence of a reference antibody (where the reference antibody is trastuzumab), (ii) at least two amino acid changes in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of trastuzumab, or (iii) at least one amino acid change in the light chain amino acid sequence when compared to the light chain amino acid sequence of trastuzumab, and at least one amino acid change in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of trastuzumab; each of the amino acid changes is in an amino acid residue independently encoded from a DNA segment of the variant DNA sequence, the DNA segment of the variant being different from the segment of the corresponding DNA sequence encoding the reference antibody by a point mutation in a DNA motif sensitive to deamination by somatic hypermutagenic enzymes.

[0037] In one embodiment of a fifth aspect of the present invention, the amino acid modification is 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 Selected from the group consisting of D, hc58S, hc58N, hc58I, 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.

[0038] In the context of the present invention, mutations are identified using the above nomenclature, where lc = light chain, hc = heavy chain, the number refers to an amino acid residue in the chain, and the capital letter is a single-letter amino acid code representing the amino acid mutation at that site. Thus, for example, in the amino acid modification listed above for the fourth embodiment, lc9N refers to the asparagine at position 9 of the variant trastuzumab light chain.

[0039] In a fifth embodiment of the present invention, the amino acid modification is 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 are identical to those of the reference antibody in any residue other than the amino acid modifications 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 amino acid sequence when compared to the light chain amino acid sequence of a reference antibody (where the reference antibody is Fsn503h), (ii) at least two amino acid changes in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of Fsn503h, or (iii) at least one amino acid change in the light chain amino acid sequence when compared to the light chain amino acid sequence of Fsn503h, and at least one amino acid change in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of Fsn503h; each of the amino acid changes is in an amino acid residue independently encoded from a DNA segment of the variant DNA sequence, the DNA segment of the variant being different from the segment of the corresponding DNA sequence encoding the reference antibody by a point mutation in a DNA motif sensitive to deamination by somatic hypermutagenic enzymes.

[0042] In one embodiment of the sixth aspect of the present invention, the amino acid modification is 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, l The selected model is from the group consisting of c90F, 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 present invention, the amino acid modification is 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 present invention, the light chain and heavy chain sequences of the variant are identical to those of the reference antibody in any residue other than the amino acid modifications described above in relation to the sixth aspect.

[0045] In one embodiment of the fifth aspect of the present invention, the variant antibody molecule has a combination of amino acid mutations 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 shown for any of the antibodies listed in Table 8. In such an embodiment, the variant antibody has a combination of amino acid mutations shown for any one of the antibodies listed in Table 8 and does not have any amino acid mutations other than those shown for the antibodies listed in Table 8, compared to the trastuzumab light chain and heavy chain sequences.

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

[0047] In one embodiment of a fifth aspect of the present invention, the variant antibody has a combination of amino acid mutations shown for any of variant antibodies 19, 5, or 6 in Table 8. In such an embodiment, the variant has no amino acid mutations other than those shown for the variant antibody in Table 8, compared to the trastuzumab light chain and heavy chain sequences.

[0048] In one embodiment of a fifth aspect of the present invention, the variant antibody is a variant of the trastuzumab antibody that includes amino acid modifications of lc9K, lc43F, and hc106V compared to trastuzumab. In one embodiment, the variant antibody is variant antibody 19 listed in Table 8.

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

[0050] In another embodiment of a fifth aspect of the present invention, the variant antibody is a variant of the trastuzumab antibody that includes amino acid modifications of lc9I, lc43F, lc101D, and hc79V compared to trastuzumab. In one embodiment, the variant antibody is variant antibody 6 listed in Table 8.

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

[0052] In certain embodiments of the fifth or sixth aspect of the present invention, the light chain and heavy chain sequences of the variant include at least three, for example, at least four, at least five, or at least six amino acid changes in total compared to the amino acid sequence of the reference antibody.

[0053] In a specific embodiment of the fifth or sixth aspect of the present invention, one or more of the amino acid modifications are located in the framework region of the variant antibody. In a specific embodiment of the fifth or sixth aspect of the present invention, all of the amino acid modifications are located in the framework region of the variant antibody.

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

[0055] In a specific embodiment of the fifth or sixth aspect of the present 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 a specific embodiment of the fifth or sixth aspect of the present invention, the change in affinity of the variant antibody molecule compared to the reference antibody is greater than -10, for example greater than -15, for example greater than -20, for example greater than -25.

[0056] In certain embodiments of the fifth or sixth aspect of the present invention, the change in the stability of the variant antibody molecule compared to the reference antibody is greater than -10, for example, greater than -30, for example, greater than -50, for example, greater than -60. Appropriately, the aggregation properties, melting point properties, or expression level may differ from those of the reference antibody by at least two, three, or tenfold.

[0057] To avoid any ambiguity, a higher negative affinity value indicates greater affinity. Therefore, an antibody with an affinity value of -10 is considered to have a higher affinity value than an antibody with an affinity value of -5. Similarly, a higher negative stability value indicates greater stability. Therefore, an antibody with a stability value of -10 is considered to have a higher stability value than an antibody with a stability value of -5.

[0058] Affinity and stability can be evaluated by any suitable method. In the examples, the inventors used a residue scanning affinity maturation tool as part of Schrodinger's biopharmaceutical tool Maestro. The values ​​were relative to the parent antibody, and the minimum improvement in affinity was -2 kcal / mol for both affinity and stability. [Brief explanation of the drawing]

[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). Double-enclosed amino acids were not accepted in the library because their use at that position is rare. Double-enclosed asterisks (*) represent stop codons that were similarly not accepted. Single-enclosed amino acid regions are 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). Double-enclosed amino acids were not accepted in the library because their use at that position is rare. Double-enclosed asterisks (*) represent stop codons that were similarly not accepted. Single-enclosed amino acid regions are CDRs. [Figure 2] Figure 2 is a schematic model showing the predicted protein-protein interaction 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 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-2] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-3] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-4]Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-5] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-6] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-7] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-8] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-9] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-10] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-11] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 3-12] Figure 3 shows Table 1, which lists the predicted affinity and stability of amino acid mutations in a combinatorial manner for 1 to 6 mutations. The group of mutations highlighted in blue represents variants where both stability and affinity are improved, with a change greater than -2. [Figure 4] Figure 4 shows an ELISA comparison of each expressed variant with the parental Fsn0503h antibody. [Figure 5] Figure 5 shows the affinity (KD) values ​​for each variant measured by BLI using an Octet instrument (Pall), alongside those for the parent Fsn0503h antibody. [Figure 6] Figure 6 shows the affinity on-off rate map for variants of Fsn0503h. The parent antibody is shown as a white dot. The 4.45 nM line represents affinity equivalent to the parent antibody, and any variant with increased affinity (<4.45 nM) lies in the area 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). Amino acid residues at each position in the wild-type antibody light chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. An asterisk (*) with a double line represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 7-2]Figure 7 shows the amino acid sequence of the trastuzumab antibody light chain (SEQ ID NO: 3). Amino acid residues at each position in the wild-type antibody light chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. An asterisk (*) with a double line represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 7-3] Figure 7 shows the amino acid sequence of the trastuzumab antibody light chain (SEQ ID NO: 3). Amino acid residues at each position in the wild-type antibody light chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. An asterisk (*) with a double line represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 8-1] Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4). Amino acid residues at each position in the wild-type antibody chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. A double-lined asterisk (*) represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 8-2] Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4). Amino acid residues at each position in the wild-type antibody chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. A double-lined asterisk (*) represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 8-3]Figure 8 shows the amino acid sequence of the trastuzumab antibody heavy chain (SEQ ID NO: 4). Amino acid residues at each position in the wild-type antibody chain are shown in bold, and potential mutations are indicated in the square to the right of each residue, where applicable. Amino acids shown with a double line are not accepted in the library because their use at that position is rare. A double-lined asterisk (*) represents a stop codon that was similarly not accepted. The complementarity-determining region (CDR) and framework region (FR) are shown. [Figure 9] Figure 9 schematically illustrates how somatic hypermutation can be used to induce changes in the antibody DNA sequence that result in amino acid changes in the antibody protein structure, leading to increased affinity. [Figure 10] Figure 10 shows the fully elucidated crystal structure of trastuzumab complexed 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 represents the light chain and H represents the heavy chain. Δ affinity is the predicted change in free energy (ΔΔG) in kcal / mol compared to the affinity of trastuzumab. Δ stability is the predicted change in free energy (ΔΔG) compared to the stability of trastuzumab. Negative Δ affinity and negative Δ stability values ​​are thought 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 represents the light chain and H represents the heavy chain. Δ affinity is the predicted change in free energy (ΔΔG) in kcal / mol compared to the affinity of trastuzumab. Δ stability is the predicted change in free energy (ΔΔG) compared to the stability of trastuzumab. Negative Δ affinity and negative Δ stability values ​​are thought 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents 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 represents the light chain and H represents the heavy chain. [Figure 12-1] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-2]Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-3] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-4] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-5] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-6] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-7] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-8] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 12-9] Figure 12 shows Table 8, which lists 100 trastuzumab variants from Tables 1-7 that have both improved affinity and improved stability, ranked according to improved affinity. [Figure 13A]Figure 13 schematically shows 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 2 and 4 have four mutations each, and finally rank 5 contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while scFv is colored green and CDR is colored red. Mutations are highlighted in yellow. [Figure 13B] Figure 13 schematically shows 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 2 and 4 have four mutations each, and finally rank 5 contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while scFv is colored green and CDR is colored red. Mutations are highlighted in yellow. [Figure 13C] Figure 13 schematically shows 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 2 and 4 have four mutations each, and finally rank 5 contains three amino acid mutations. Domain IV of HER2, to which trastuzumab binds, is colored cyan, while scFv is colored green and CDR is colored red. Mutations are highlighted in yellow. [Figure 14] Figure 14 shows sensorgrams of 20 samples displaying the report points used for ranking. The assay was performed at 25°C. [Figure 15]Figure 15 shows a graph ranking the off-rates of trastuzumab variants that bind to HER2, indicating stability_early and stability_late to identify stable conjugates (the best conjugates are circled 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. The wild type is an antibody with the trastuzumab sequence that was transiently expressed concurrently with all trastuzumab variants / mutants. Mutants are trastuzumab variants. Mutants 5, 6, and 19 are the trastuzumab variants listed as numbers 5, 6, and 19 in Table 8. The assay was performed at 25°C. [Figure 16] Figure 16 shows a graph of the off-rate ranking of HER2-binding trastuzumab variants, indicating stability_early and stability_late to identify stable conjugates from a small subset of the top conjugates shown in Figure 15 (these conjugates are closest to the 100% retention line). In total, 14 trastuzumab variants were analyzed and ranked for binding stability. Trastuzumab is a commercially available antibody. Wild-type is the substance transiently expressed concurrently with all trastuzumab variants / mutants. Mutants are trastuzumab variants. Mutants 5, 6, and 19 are the 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 on the surface of non-covalently immobilized trastuzumab (a), wild-type (b), mutant 5 (c), and (d) mutant 19 antibodies for various HER2 concentrations (2-fold serial dilutions; highest concentration shown on the curve) in running buffer at 37°C. The apparent dissociation rate constant (kd) and association rate (ka) constants were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The global fitting results are summarized in Table 9. [Figure 17-2]Figure 17 shows reference-corrected BLI binding curves (black) monitored on the surface of non-covalently immobilized trastuzumab (a), wild-type (b), mutant 5 (c), and (d) mutant 19 antibodies for various HER2 concentrations (2-fold serial dilutions; highest concentration shown on the curve) in running buffer at 37°C. The apparent dissociation rate constant (kd) and association rate (ka) constants were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The global fitting results are summarized in Table 9. [Figure 18] Figure 18 schematically illustrates the correlation between predicted and measured affinity of trastuzumab variants. [Figure 19-1] Figure 19 shows the blank-corrected BLI binding curves (black) monitored on the surface of non-covalently immobilized HER2 at 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 constant (kd) and association rate constant (ka) were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The global fitting results are summarized in Table 10. [Figure 19-2] Figure 19 shows the blank-corrected BLI binding curves (black) monitored on the surface of non-covalently immobilized HER2 at 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 constant (kd) and association rate constant (ka) were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The global fitting results are summarized in Table 10. [Figure 20-1]Figure 20 shows the dual-reference corrected BLI binding curves (black) monitored on a non-covalently immobilized HER2 surface coated with a range of concentrations of wild-type (a), mutant 5 (b), and (c) mutant 19 antibodies presented in a range of concentrations in running buffer at 37°C. The apparent dissociation rate constant (kd) and association rate (ka) constants were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The results of the global fitting are summarized in Tables 11, 12, and 13. [Figure 20-2] Figure 20 shows the dual-reference corrected BLI binding curves (black) monitored on a non-covalently immobilized HER2 surface coated with a range of concentrations of wild-type (a), mutant 5 (b), and (c) mutant 19 antibodies presented in a range of concentrations in running buffer at 37°C. The apparent dissociation rate constant (kd) and association rate (ka) constants were determined by globally fitting a simple 1:1 interaction model A+B=AB to the sensorgram (red) using the instrument's software. The results of the global fitting are summarized in Tables 11, 12, and 13. [Figure 21] Figure 21 shows the expression yield of the Fsn0503 variant by Octet immunoassay, expressed as mg of purified IgG obtained per 1 ml of supernatant of transfected CHO. The wild type is shown in orange. [Figure 22] Figure 22 shows the melting point measurements (TM1 and TM2) of the Fsn0503 variant in Celsius. The wild type is ranked 1st. [Figure 23] Figure 23 shows the measured percentage of monodisperse (non-aggregated) molecules of the Fsn0503 variant by size exclusion chromatography. The wild type is shown in orange. [Modes for carrying out the invention]

[0060] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art of the present invention.

[0061] Throughout the specification, unless otherwise required by context, the terms “comprise” or “include,” or variations such as “comprises” or “comprising,” “includes” or “including,” are understood to mean including the integer or group of integers described, but not excluding any other integer or group of integers.

[0062] As used herein, terms such as “a,” “an,” and “the” include singular and plural referents unless otherwise clearly required by the context. Therefore, for example, a reference to “activator” or “pharmacological activator” includes a single activator and combinations of two or more different activators, while a reference to “carrier” includes a single carrier as well as mixtures of two or more carriers.

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

[0064] As used herein, the term “essentially consisting of” means that the present invention necessarily includes the enumerated items, but may also include unenumerated items that do not substantially affect the fundamental and novel characteristics of the present 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 possess specific three-dimensional structural properties, along with specific charge properties that contribute to the three-dimensional structure of the epitope.

[0066] The antibody molecule of the present invention or the antibody molecule for use in the present invention may be bound to a non-adjacent epitope. A "non-adjacent epitope" is an epitope consisting of a series of amino acid residues that are not linear in sequence, such that the residues are discontinuously spaced or grouped along the length of the polypeptide sequence.

[0067] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to describe a set of at least two amino acids covalently linked by a peptide bond or a modified peptide bond such as an isoster. No limit is placed on the maximum number of amino acids that may constitute a peptide or protein. Furthermore, the term polypeptide extends to peptide fragments, analogs, and derivatives, where 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 in this specification to describe polypeptide components follows the conventional practice of presenting the amino group (N) to the left of each amino acid residue and the carboxyl group to the right.

[0069] Antibodies and antibody molecules An "antibody" is an immunoglobulin, whether naturally occurring or partially or entirely synthetically produced. The term also encompasses any polypeptide, protein, or peptide that has an antibody-binding domain or a binding domain homologous to an antibody-binding domain. These may originate from natural sources or be partially or entirely synthetically produced. Examples of antibodies include immunoglobulin isotypes and their isotype subclasses, and fragments containing antigen-binding domains 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 as encompassing any binding member or substance having a binding domain with the desired specificity. The antibody molecule of the present invention or the antibody molecule for use in the antibody molecule may be a monoclonal antibody, or a fragment, derivative, functional equivalent, or homolog. This term includes any polypeptide containing an immunoglobulin-binding domain, whether natural or entirely or partially synthesized. Therefore, it includes chimeric molecules containing an immunoglobulin-binding domain or equivalent fused to another polypeptide.

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

[0072] Fragments of an antibody can perform antigen-binding functions. Examples of such binding fragments include Fab fragments containing or consisting of 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 containing two linked Fab fragments; single-stranded Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to bind and form an antigen-binding site; and bispecific antibodies, which may be multivalent or multispecific fragments constructed by gene fusion.

[0073] In certain embodiments, humanized antibodies may be used. These humanized antibodies may be modified antibodies having a hypervariable region of a non-human antibody and a constant region of a human antibody. Therefore, the binding member may include the human constant region. Variable regions other than the hypervariable region may also originate from the variable region of a human antibody and / or from a non-human antibody. In other cases, the entire variable region may originate from a non-human antibody, and the antibody is said to be chimeric.

[0074] It is possible to obtain monoclonal antibodies or other antibodies and use recombinant DNA techniques to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the constant region of a different immunoglobulin, or the immunoglobulin variable region of an antibody against the constant region and framework region, or the complementarity-determining region (CDR). Hybridomas or other antibody-producing cells may undergo genetic mutations or other alterations, which may or may not alter the binding specificity of the antibodies produced.

[0075] The antibody may be selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, camel antibodies, shark antibodies, and in vitro antibodies. In certain embodiments, antigen-binding fragments 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 Fab fragments, scFv fragments, Fv fragments, and dAb fragments. In certain embodiments, the antibody comprises two complete heavy chains and two complete light chains, or their antigen-binding fragments. In certain embodiments, the antibody is of isotype IgG, IgA, IgE, or IgM, or their antigen-binding fragments. In certain embodiments where the antibody is of isotype IgG, the antibody may be of subtype IgG1, IgG2, or IgG3, or their antigen-binding fragments. In certain embodiments, the antibody is of subtype IgG4, or its antigen-binding fragment.

[0076] Antibody production Antibodies can be provided by many techniques. For example, combinatorial screening techniques, such as phage display-based biopanning assays, can be used to identify amino acid sequences with specificity for binding to antigens. Such phage display biopanning techniques involve the use of phage display libraries, which are utilized to identify suitable epitope-binding ligands in a procedure that mimics immunoselection by presenting antibody-binding fragments on the surface of filamentous fungi. Phages with specific binding activity are selected. The selected phages can then be used for chimeras, CDR grafting, humanization, or the production of 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 whole or partially 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 well known to those skilled in the art. Furthermore, they may be prepared in solution, by liquid-phase methods, or by any combination of solid-phase, liquid-phase, and solution chemistry.

[0078] Another convenient method for producing antibodies or antibody fragments suitable for use in the present invention is to express the encoding nucleic acid by using nucleic acid in an expression system.

[0079] Antibodies may be produced by mutagenesis of antibody genes to produce an artificial repertoire of antibodies. This technique enables the preparation of antibody libraries. An artificial repertoire of immunoglobulins, such as an artificial scFv repertoire, may be used as an immunoglobulin source to identify binding molecules that are specific to a particular epitope.

[0080] Methods for generating repertoire are well-characterized in the relevant field.

[0081] Any suitable means for generating antibody libraries may be used in conjunction with the present invention. Selection protocols for isolating desired members of large libraries, such as phage display techniques, are known in the art. Such systems, in which diverse peptide sequences are presented on the surface of fibrous bacteriophages, have proven useful for creating libraries of antibody fragments (and encoding nucleotide sequences) for in vitro selection and amplification of specific antibody fragments that bind to target antigens. Nucleotide sequences encoding the VH and VL regions are ligated to gene fragments encoding a leader signal that is induced in the periplasmic space of Escherichia coli (E. coli), and the resulting antibody fragments are presented on the surface of the bacteriophage, typically as fusions to a bacteriophage coat protein (e.g., pIII or pVIII). Alternatively, the antibody fragments are presented outside the lambda phage capsid (the phage body). The advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing phages containing the selected library members in bacterial cells. Furthermore, because the nucleotide sequences encoding polypeptide library members are contained within the phage or phagemid vector, sequencing, expression, and subsequent genetic manipulation are relatively straightforward.

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

[0083] A method for producing polypeptides may include culturing host cells transformed with a recombinant expression vector encoding the polypeptide under conditions that promote polypeptide expression, and then recovering the expressed polypeptide from the culture. Those skilled in the art will recognize that the procedure for purifying the expressed polypeptide varies depending on factors such as the type of host cell used and whether the polypeptide is intracellular, membrane-bound, or soluble, 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 ligated to a suitable transcriptional or translational regulatory nucleotide sequence, such as those derived from mammalian, avian, microorganism, virus, bacterium, or insect genes. The nucleotide sequence is operably ligated if the regulatory sequence is functionally related to the DNA sequence. Thus, if the promoter nucleotide sequence controls the transcription of the DNA sequence, the promoter nucleotide sequence is operably ligated to the DNA sequence. A replication origin that provides the ability to replicate in a desired host cell, and a select gene for identifying transformants, are generally incorporated into the expression vector.

[0085] Furthermore, sequences encoding a suitable signal peptide (native or heterologous) can be incorporated into the expression vector. The DNA sequence of the signal peptide (secretion leader) can be fused in-frame to the nucleic acid sequence of the present invention so that the DNA is first transcribed, and then the mRNA is translated into a fusion protein containing the signal peptide. The signal peptide, functioning in the target host cell, promotes the extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide during translation, but still allows for the secretion of the polypeptide from the cell.

[0086] Suitable host cells for polypeptide expression include higher eukaryotic cells and yeast. Prokaryotes are also suitable.

[0087] Mammalian cells, particularly CHO cells, are especially 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. The target plasmid DNA can be transfected into ExpiCHO cells by condensing it by forming a complex with ExpiFectamine (a cationic lipid-based transfection reagent). This condensed DNA enters the ExpiCHO cells via endocytosis and is expressed in the nucleus. The expressed protein is present in the cell culture supernatant and is harvested after an appropriate number of days.

[0088] nucleic acid Nucleic acids for use in accordance with the present invention may include DNA or RNA and may be synthesized whole or partially. In a preferred embodiment, the nucleic acid for use in the present invention encodes the antibody or antibody fragment of the present invention as defined above. Those skilled in the art will be able to determine substitutions, deletions, and / or additions to nucleic acids that still provide antibody molecules of the present invention or antibody molecules 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 those skilled in the art. These techniques include (i) the use of polymerase chain reaction (PCR) to amplify a sample of such nucleic acid from, for example, a genomic source, (ii) chemical synthesis, or (iii) the preparation of a cDNA sequence. DNA encoding an antibody fragment can be generated and used by any suitable method known to those skilled in the art, including obtaining encoding DNA, identifying a suitable restriction enzyme recognition site on either side of the portion to be expressed, and excising the portion from the DNA. The portion may then be operably ligated to a suitable promoter in a standard commercial expression system. Another recombination approach is to amplify the relevant portion of the DNA with a suitable PCR primer. Sequence modification can be performed, for example, using site-directed mutagenesis, to lead to the expression of a modified peptide or to take into account codon preference in host cells used to express the nucleic acid.

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

[0091] Systems for the cloning and expression of polypeptides in various different host cells are well known. Suitable host cell systems include bacterial, mammalian, yeast, insect, and baculovirus systems. Mammalian cell lines available in the art for heterologous polypeptide expression include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, and NS0 mouse myeloma cells. A generally preferred bacterial host is Escherichia coli. The expression of antibodies and antibody fragments in prokaryotic cells such as Escherichia 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 the production of binding members. General techniques for antibody production are well known to those skilled in the art.

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

[0093] Furthermore, the nucleic acids encoding the heavy chain variable domain and / or light chain variable domain of an antibody may be enzymatically or chemically synthesized nucleic acids that have authentic sequences encoding naturally occurring heavy chain variable domains and / or light chain variable domains, or variants thereof. Embodiments of the present invention are described herein by reference only to the accompanying drawings: [Examples]

[0094] Materials and methods Library design By searching for specific DNA sequence motifs RGYW in the 3' to 5' strand and WRCY in the 5' to 3' strand, which are recognized by the AID enzyme that plays a role in inducing mutations in antibodies in the human body, antibody libraries were generated for (i) the humanized anti-cathepsin S antibody, Fsn0503h (Fusion Antibodies, Belfast) (Kwok et al., Molecular Cancer 2011, 10:147) and (ii) trastuzumab (Roche).

[0095] The DNA sequences have point mutations introduced at position 2 of the DNA sequence motif RGYW (guanine) or position 3 of the DNA sequence motif WRCY (cytosine), where the nucleotide is mutated to any other nucleotide. The human AID enzyme introduces spontaneous mutations at these positions. This can cause possible changes to amino acids at a single position. To generate all naturally possible amino acids across the entire antibody sequence and form an initial library, all these possible mutations were identified across the entire antibody DNA sequence in both directions: 3' to 5' and 5' to 3'.

[0096] Next, the size of each library was controlled by removing the burden of any recognized sequence, 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 variable chain regions. Antigens were imported from the PDB. In the case of Fsn0503h, the inventors imported the crystal structure of human cathepsin S (CatS) with the C25S mutation accompanied by the conjugated drug (PDB code: 3MPE). For the trastuzumab variant library, the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB code: 1N8Z) was used.

[0098] For both 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, convert selenomethionine to methionine, fill in missing side chains and loops using Prime, and generate the het state using Epik. ProtAssign (Bioluminate, Schrodinger) was used to further refine the structure and define the hydroxyl, asparagine, glutamine, and histidine states. Water molecules with fewer than three non-water bonds were removed. Finally, the structure was minimized using the OPLS3e force field (Bioluminate, Schrodinger).

[0099] Cathepsin S was docked to the surface of an antibody model using the protein-protein docking tool Prime. Only the CDR region of the antibody was considered for molecular docking; non-CDR regions were masked. Using in vitro information on the Cat S epitope, appropriate docked configurations were selected based on rank, shape complementarity (using a protein interaction analysis tool), and insights into surface interactions.

[0100] Similarly, for trastuzumab variants, the extracellular domain of human epidermal growth factor receptor 2 (HER2) (PDB code: 1N8Z) was docked to the surface of antibody models using the protein-protein docking tool Prime. Only the CDR region of the antibody was considered for molecular docking; non-CDR regions were masked. Based on rank, appropriate docked configurations were selected from our insights into shape complementarity and surface interactions (using protein interaction analysis tools).

[0101] Combinatorial Variation Analysis Residue scanning was performed on the docked configuration of the antibody-antigen complex. To enhance the affinity of the antibody to the antigen and improve its stability, information-based mutations were introduced into the antibody, avoiding highly conserved residues.

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

[0103] Next, variants were classified based on differences in affinity and stability compared to the wild type. Scores below a threshold of -2 were selected for both stability and affinity differences, and the variants were ranked based on the combination of these two scores (prioritizing affinity differences).

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

[0105] antibody synthesis Transient transfection: In a 500 ml ventilated Erlenmeyer flask of ExpiCHO expression medium, 4–6 × 10⁶ suspension-compatible ExpiCHO cells were expressed at 130 rpm, 37°C, and 8% CO₂. 6 Cells were routinely cultured at a concentration of cells / ml. For each variant of Fsn0503h, 1 μg / ml of DNA was diluted with 4% (v / v) OptiPRO SFM in a centrifuge tube. In a separate tube, 0.32% (v / v) ExpiFectamine was diluted with 3.7% OptiPRO SFM. The ExpiFectamine / OptiPRO mix was then added to the DNA / OptiPRO mix and incubated at room temperature for 3 minutes. Finally, the mixture was transferred to a 125 ml ventilated Erlenmeyer flask to a final density of 6 × 10⁶. 6The transfected cells were added to 25 ml of ExpiCHO cells at a cell / ml concentration. Each transfected culture was incubated overnight at 37°C, 8% CO2, and 130 rpm. Twenty 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 collected by centrifugation at 4000 rpm for 40 minutes at 18°C.

[0106] purification: Fsn0503h WT and variant antibodies were purified in two steps using a Tricorn 5 / 50 column (GE) packed with 1 ml of MabSelect® PrismA (GE), followed by a 10 ml (2 × 5 ml) Hitrap Desalting (Desalt) column (GE). The MabSelect® PrismA affinity medium was selected for its high mAb binding and specificity properties, as well as its alkali resistance for efficient clean-in-place (CIP) washing. Unless otherwise specified, all steps were performed at room temperature using a flow rate of 4 ml / min. After loading (using an AKTA sample pump), the Protein A column was washed with 10 column volumes (CV) of PBS (backflow mode), followed by one-step elution with 100 mM glycine, pH 3.0 (backflow mode). When the absorbance at 280 nm exceeded 120 mAU, the protein A eluate was collected in a 2 ml loop (AKTA with a 10 mm flow cell) and immediately injected into a pre-equilibrated desalt column. Desalt peak elution was collected in a 2 ml block of 96 wells at 2–8°C when the absorbance of the eluate exceeded 100 mAU. To avoid cross-contamination, the automated process included CIP (clean-in-place) of both affinity and desalting columns. CIP was performed between each sample for all contact pathways using 0.2 M NaOH (backflow mode was used for column washing).

[0107] Expression levels were determined as the total yield of purified material per 1 ml of culture medium.

[0108] Trastuzumab variants were synthesized using a similar technique.

[0109] Affinity Ranking Enzyme-linked immunosorbent assay: MaxiSorp 96-well plates were coated with 66 different O503 variants at 1 g / ml in PBS for 24 hours at 4°C. To obtain EC50 results, the variants were serially diluted in PBS from 1000 ng / ml to 1 ng / ml, and two copies of each were coated. A standard curve was prepared using parental O503 antibody coated at 1 μg / ml in PBS for 24 hours at 4°C. After 24 hours, the MaxiSorp plates were washed three times with PBS-T. 200 μl of SuperBlock was added to each well, removed, and the plates were replaced three times. 100 μl of 200 ng / ml Cat S antigen was added to each well, and the plates were shaken at RT, 150 rpm for 1 hour and 30 minutes. The plates were washed three times with PBS-T and dried. 100 μl of 5 μg / ml anti-his-HRP was added to each well, and the plates were shaken at 150 rpm, RT for 1 hour and 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. After that, 50 μl of 1 M HCl was added, and the absorbance of the plate was measured at 450 nm.

[0110] Affinity ranking using the Octet RED96 system (Fsn503h variant): The affinity ranking assay was performed by first capturing IgG using an anti-human Octet biosensor (ForteBio part number 18-5060), followed by a baseline step of 2 minutes 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). Next, the mAb capture biosensor was immersed in a well containing 200 ng / ml recombinant cathepsin S antigen for 10 minutes (association step), followed by a dissociation step of 10 minutes in running buffer. To allow for dual-reference correction, the IgG capture sensor was immersed in a well containing only buffer, and a blank sensor was also immersed in a well containing antigen. This reference provided a means of compensating for both the natural dissociation of captured IgG and the nonspecific binding of the antigen to the sensor surface. All steps were performed in HBS-EBT buffer at 25°C at a constant flow rate of 1000 rpm. A new sensor was used for each sample. The dissociation rate constant (koff) was calculated using ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.

[0111] Antibody quantification using biolayer interferometry (Octet RED96 system): To measure IgG content, two 200 μL volumes (ranging from 0.06 to 512 μg / ml) of antibody standards and IgG-containing cell supernatant (diluted within the calibration curve's measurement range) were prepared using 1× 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 the wells of a 96-well black microtiter plate (Greiner Bio-One part number 655209). All samples and standards were measured twice using a Protein A biosensor (Fortebio PN 18-5010). The plate was placed in an Octet and equilibrated at 25°C in a thermostat-controlled chamber. The run was initiated by placing the sensor in a well, and the change in layer thickness (nanometers, nm) over time was measured under computer control. Data was acquired for each set of eight samples (measuring one plate column simultaneously) at flow velocities (orbital flow) of 400–1000 rpm for 180–600 seconds. Data was automatically processed using Octet User Software version 3.1. Measurement time and flow velocity were varied according to the required sensitivity.

[0112] Affinity ranking using biolayer interferometry (Octet RED96 system) (trastuzumab variants): The affinity ranking assay first uses an anti-human Octet biosensor (ForteBio part number 18-5060) *IgG was captured and then subjected to 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). Next, the mAb capture biosensor was immersed 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 dual-reference correction, the IgG capture sensor was immersed in a well containing only buffer, and a blank sensor was also immersed in a well containing antigen. This reference provided a means of compensating for both the natural dissociation of captured IgG and the nonspecific binding of the antigen to the sensor surface. The steps were performed in HBS-EBT buffer at either 25°C or 37°C at a constant flow rate of 1000 rpm. A new sensor was used for each sample. The dissociation rate constant (koff) was calculated using ForteBio Data Analysis software. All consumables used were those recommended by ForteBio.

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

[0114] K using biolayer interferometry (Octet RED96 system) D measurement: The kinetic assay was performed by first capturing IgG using an anti-human Fc Octet biosensor, followed by two baseline steps of 2 minutes each in a running buffer of HBS-P+ buffer. Next, the mAb capture biosensor was immersed in wells containing various concentrations of HER2 for 15 minutes, followed by dissociation in the running buffer for 20 minutes. To allow for dual-reference correction, the IgG capture sensor was immersed in a well containing only buffer, and a blank sensor was also immersed in wells containing a range of antigen concentrations. This reference provided a means of compensating for both the natural dissociation of captured IgG and the nonspecific binding of the antigen to the sensor surface. All steps were performed in kinetic buffer at 37°C at a constant flow rate of 1000 rpm.

[0115] Determination of the melting point Antibodies typically exhibit two measurable melting points, called TM1 and TM2, in analysis as a result of thermal denaturation of different parts of the assembled molecule. These values ​​were determined by thermal shift assay. 5 μl of a solution containing Sypro Orange ([PBS, pH 7.4, diluted 1 / 200); Molecular Probes) and 45 μl of 0.3 mg / ml antibody were added to low-profile PCR tubes (Bio-Rad; TLS0851). The tubes were sealed with optical ultra-clear sealing caps (Bio-Rad; TCS0803) and heated in 1°C increments from 20°C to 90°C using an i-Cycler iQ5 real-time PCR detection system (Bio-Rad). Fluorescence changes in the plate wells were simultaneously monitored with a charge-coupled (CCD) camera. The excitation and emission wavelengths were 485 nm and 575 nm, respectively. The midpoint T of the protein unfolding transition temperature was determined. m This was calculated using Bio-Rad iQ5 software.

[0116] Determination of monovariance 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 that of 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, which reduces the solubility and activity of the 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 antibody samples were individually loaded onto Superdex 200 increased 10 / 300 GL gel filtration columns. 50 μl of sample was injected, and the column flow rate was maintained at 0.75 μl / min. The 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 the spectra were analyzed using the Unicorn emulation software package (GE Healthcare). The results were obtained for each peak. r It is reported as (ml) and relative peak area (%).

[0118] Example 1: Cathepsin S antibody variant Library design As shown in Figure 1, the DNA sequences of the light and heavy chain variable domains of the Fsn0503h antibody were analyzed to identify motifs susceptible to mutation during somatic hypermutation, and the potential amino acids corresponding to the results of these mutations were plotted on the parent sequence. Undesirable amino acids or stop codons generated as a result of DNA mutations were identified.

[0119] The presence of more mutations within the framework region than in CDR, and the fact that CDR-H3 lacks functional mutations in particular, was a surprising discovery.

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

[0121] Combinatorial mutagenesis We introduced amino acid residue mutations into the docked structures and predicted relative differences in both affinity and stability. We increased the number of mutations introduced until no further benefits in stability or affinity were predicted. The results of combinatorial mutagenesis are shown in Figure 3. We identified 66 variants that had predicted improvements in both affinity and stability. Next, we synthesized the DNA encoding these variants, expressed antibodies, and purified them 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 have a higher relative OD than the parent Fsn0503h, potentially indicating a higher affinity for the cathepsin S target.

[0123] Affinity Ranking To determine the precise affinity comparison of each variant with the parental Fsn0503h antibody, the interaction with cathepsin S was measured by BLI using an Octet instrument as described in the methods.

[0124] The results show that approximately 50% of the variants exhibited improved affinity when measured relative to 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 As described above, the 66 variants expressed in CHO also showed expression levels within a certain range 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 purified supernatant.

[0126] Change in melting point The 66 variants also showed a range of variation in stability properties related to the antibody molecule. This includes changes in the melting temperature profile at the two melting points commonly 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 have lost their distinct biphasic melting pattern, with only a single melting temperature being observable.

[0127] Changes in monodispersity This characteristic was analyzed by analytical size exclusion chromatography of 66 variants, and as shown in Figure 23, it exhibits a range of values ​​among the analyzed variants. Example 2 - Trastuzumab Variant

[0128] Library design Based on naturally occurring somatic hypermutations in humans, a library of trastuzumab antibody variants was generated. As shown in Figure 9, the somatic hypermutations were reproduced using the method described above, generating naturally occurring mutations in the trastuzumab DNA sequence, which were then translated into their respective amino acid mutations. These mutations were compiled into a library of potential mutations at specific sites in both the complementarity-determining region (CDR) and framework region (Figures 7 and 8), with the potential amino acid results of this method shown in detail alongside the parent sequence. Any undesirable amino acids or stop codons resulting from the DNA mutations were identified.

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

[0130] Amino acid residue mutations were introduced into the docked structure, and relative differences in both affinity and stability were predicted. The number of mutations introduced was increased until no further benefits in stability or affinity were predicted. The results of combinatorial mutagenesis are shown in detail 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 five ranked variants are shown in Figure 13. Among these variants, a total of five mutations spanning the heavy and light chains were found to be the most common. Variants were scored against the affinity of the parent antibody using the predicted free energy change (ΔΔG).

[0131] Off-rate ranking A panel of 89 trastuzumab variants was screened for binding to the HER2 antigen using the biolayer interferometry method (see Materials and Methods for details). Sensorgrams of 20 samples with report points shown as a color bar are shown in Figure 14. Figure 15 shows a scatter plot of the initial report points plotted against the late stability. The top conjugates with high binding stability and slow dissociation (a total of 14) are highlighted with blue circles. To provide more biologically relevant data, off-rate ranking experiments were repeated for these mutants at 37 °C (Figure 16). Mutant 19 appears to have improved stability compared to WT trastuzumab and was used for kinetic analysis.

[0132] Determination of KD All samples were diluted with freshly prepared running buffer. Either trastuzumab or a variant (mutant) was immobilized on the surface of a series of biosensors using the described capture method (see Materials and Methods). To generate a binding response, HER2 was passed over the surface. Binding data for the HER2 interaction were collected by the biosensor at 37 °C. The results were globally fitted to obtain the best values of k a 、k d 、and K D A dilution series of the HER2 antigen (5 nM to 0.078 nM) was used in the association step. Response data regarding the binding of the antigen to the IgG immobilized on the surface were fitted to a 1:1 binding model to generate a data trace (see red - Figure 17). The experiment was repeated twice, and the average kinetic parameters are summarized in Table 9. For example, the data show that Mutant 19 appears to exhibit an approximately 2-fold increase in apparent affinity compared to the wild-type control and commercially available trastuzumab. The increased affinity is mainly due to a slower Kd, 6.04E-05, compared to 1.20E-04 for the wild-type antibody. Figure 18 schematically shows a 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; a value above 0.95 indicates a good fit; X 2 X is the sum of the squares of the deviations, and should generally be less than 3; X 2 X is a measure of the error between experimental data and the approximation curve. 2 A smaller value indicates better fit.

[0135] Determining the KD of monomers To further validate the affinity of the top variants and understand the contribution of binding strength to the affinity measurements within the top two variants (MUT 5 and MUT 19), these molecules were enzymatically cleaved to prepare monomeric fragment antigen-binding (Fab) fragments consisting of variable heavy and light chain domains and a CH1 constant domain, with each molecule having a single antigen-binding domain.

[0136] The initial measurements were performed after coating the probe with 2.5 μg / ml biotinylated Her2 recombinant Fc fusion protein. Binding to the Her2 surface was evaluated for each of three monomeric antibodies at a range of concentrations. In this case, dual reference was not applied. Sensorgram data are listed in Figure 19, and kinetic calculations (1:1 model) of the results are listed in Table 10.

[0137] [Table 2]

[0138] Next, sensors were prepared by coating with biotinylated Her2 recombinant Fc fusion protein at 0.625, 1.25, or 2.5 μg / ml. Purified Fab fragments were then tested for binding to these probes. Sensor gram traces of these measurements are shown in Figure 20, with details in Tables 11, 12, and 13. For this data, unlike previous experiments, a dual reference was used to exclude any drift due to nonspecific interactions at the probe surface.

[0139] [Table 3]

[0140] [Table 4]

[0141] [Table 5]

[0142] Because affinity values ​​appear to change as a result of coating concentration, it is difficult to assign a clear value to the increased affinity of MUT 5 and MUT 19 compared to the WT trastuzumab molecule from these monomer analyses. While we do not wish to be bound by theory, this is likely due to issues with operation at the sensitivity limits of the ForteBio Octet Biosensor instrument.

[0143] conclusion Using the method of the present invention, the inventors have successfully demonstrated that it is possible to generate a very large physical library of antibody variants and improve the affinity of the antibody to its target without the need for subsequent selection / screening processes. Furthermore, expression generates a pool of variants that exhibit various attributes of interest, including affinity, expression level, and physicochemical properties, all of which are of interest to the potential development potential of the antibody molecule.

[0144] While the present invention is specifically shown and described with reference to certain examples, it will be understood by those skilled in the art that various modifications in form and detail can be made within the scope of the invention without departing from it.

Claims

1. (i) When compared with the light chain amino acid sequence of the reference antibody, the light chain sequence has at least two amino acid changes, (ii) When compared to the heavy chain amino acid sequence of the reference antibody, it has at least two amino acid changes in the heavy chain sequence, or (iii) A variant antibody of a reference antibody having at least one amino acid change in the light chain amino acid sequence when compared to the light chain amino acid sequence of the reference antibody, and having at least one amino acid change in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of the reference antibody; Each of the aforementioned amino acid modifications is located at an amino acid residue encoded from a DNA segment of the variant DNA sequence, and each of the aforementioned amino acid residues is independently encoded. The DNA segment of the variant antibody differs from the corresponding DNA sequence segment encoding the reference antibody due to a point mutation in a DNA motif that is sensitive to deamination by activation-induced deaminase (AID). The DNA motif is DGYW or WRCH, where D is adenine, guanine, or thymine, R is adenine or guanine, G is guanine, C is cytosine, H is adenine, cytosine, or thymine, W is adenine or thymine, and Y is cytosine or thymine. The aforementioned reference antibody is trastuzumab, One or more of the aforementioned amino acid changes are mutations in the framework region. The affinity value of the variant antibody is at least 2 kcal / mol less than the affinity value of the reference antibody. The light chain of trastuzumab has the amino acid sequence of SEQ ID NO: 3, and the heavy chain of trastuzumab has the amino acid sequence of SEQ ID NO:

4. The aforementioned variant antibody has a combination of amino acid mutations for trastuzumab, as shown for any one of the mutant antibodies numbered 1 to 100 below. The light chain and heavy chain sequences of the variant antibody are the residues described for the variant antibody. Table 1 Other than that, a variant antibody of the reference antibody in which neither of the residues in SEQ ID NO: 3 nor SEQ ID NO: 4 differs from the sequences of trastuzumab.

2. The variant antibody according to claim 1, having a combination of amino acid mutations compared to trastuzumab, as shown in any one of the variants numbered 1 to 20 in the table below. Table 2

3. The variant antibody according to claim 1 or 2, wherein the variant antibody comprises mutations in at least two framework regions.

4. A variant antibody according to any one of claims 1 to 3, which is a variant antibody of a trastuzumab antibody comprising amino acid modifications of lc9K, lc43F, and hc106V compared to trastuzumab.

5. A variant antibody according to any one of claims 1 to 4, which has no mutation in the light chain CDR1 compared to the light chain CDR1 of trastuzumab.

6. A variant antibody of the reference antibody, (i) When compared with the light chain amino acid sequence of the reference antibody, the light chain sequence has at least two amino acid changes, (ii) When compared to the heavy chain amino acid sequence of the reference antibody, it has at least two amino acid changes in the heavy chain sequence, or (iii) A variant antibody of a reference antibody having at least one amino acid change in the light chain amino acid sequence when compared to the light chain amino acid sequence of the reference antibody, and having at least one amino acid change in the heavy chain amino acid sequence when compared to the heavy chain amino acid sequence of the reference antibody; Each of the aforementioned amino acid modifications is located at an amino acid residue encoded from a DNA segment of the variant DNA sequence, and each of the aforementioned amino acid residues is independently encoded. The DNA segment of the variant antibody differs from the corresponding DNA sequence segment encoding the reference antibody due to a point mutation in a DNA motif that is sensitive to deamination by activation-induced deaminase (AID). The DNA motif is DGYW or WRCH, where D is adenine, guanine, or thymine, R is adenine or guanine, G is guanine, C is cytosine, H is adenine, cytosine, or thymine, W is adenine or thymine, and Y is cytosine or thymine. The variant antibody is a variant antibody of cathepsin S antibody, and the reference antibody is Fsn503h. The light chain of Fsn503h has the amino acid sequence of SEQ ID NO: 1, and the heavy chain of Fsn503h has the amino acid sequence of SEQ ID NO:

2. One or more of the aforementioned amino acid changes are mutations in the framework region. The affinity value of the variant antibody is at least 2 kcal / mol less than the affinity value of the reference antibody. The variant antibody molecule has a combination of amino acid mutations for Fsn503h, as shown for any one of the variants listed below. The light chain and heavy chain sequences of the variant antibody are the residues described for the variant antibody. Table 3 Otherwise, neither the residue in SEQ ID NO: 1 nor SEQ ID NO: 2 differs from the sequence of the reference antibody. A variant antibody of the reference antibody.

7. The variant antibody according to claim 6, wherein the variant antibody comprises mutations in at least two framework regions.

8. The variant antibody according to any one of claims 1 to 7, wherein the light chain and heavy chain sequences of the variant antibody include at least three amino acid changes in total compared to the amino acid sequence of the reference antibody.

9. The variant antibody according to any one of claims 1 to 8, wherein one or more of the aforementioned amino acid modifications are located in the CDR of the variant antibody.

10. The variant antibody according to any one of claims 1 to 9, wherein the affinity value of the variant antibody is at least 10 kcal / mol less than the affinity value of the reference antibody, and the stability value of the variant antibody is at least 2 kcal / mol less than the stability value of the reference antibody.

11. A library of antibody molecules, wherein each antibody molecule is a variant of a reference antibody, the reference antibody is trastuzumab, the amino acid sequence of each antibody molecule differs from the amino acid sequence of the reference antibody by one or more amino acid residues, each of the amino acid residues is encoded from a DNA segment of the variant DNA sequence, each of the amino acid residues is encoded independently, and the DNA segment of the variant differs from the corresponding DNA sequence segment encoding the reference antibody due to a point mutation in a DNA motif sensitive to deamination by activation-induced deaminase (AID). The nucleotide sequence encoding each of the antibody molecules is identical to the nucleotide sequence of the reference antibody in all residues except for the residues of the DNA motif. One or more of the aforementioned DNA motifs are located in the DNA sequence that encodes the framework region of the antibody molecule. A library comprising at least one variant antibody according to any one of claims 1 to 5.

12. A library of antibody molecules, wherein each antibody molecule is a variant of a reference antibody, the reference antibody being cathepsin S antibody Fsn503h, the amino acid sequence of each antibody molecule differs from the amino acid sequence of the reference antibody by one or more amino acid residues, each of the amino acid residues is encoded from a DNA segment of the variant DNA sequence, each of the amino acid residues is encoded independently, and the DNA segment of the variant differs from the corresponding DNA sequence segment encoding the reference antibody due to a point mutation in a DNA motif sensitive to deamination by activation-induced deaminase (AID). The nucleotide sequence encoding each of the antibody molecules is identical to the nucleotide sequence of the reference antibody in all residues except for the residues of the DNA motif. One or more of the aforementioned DNA motifs are located in the DNA sequence that encodes the framework region of the antibody molecule. A library comprising at least one variant antibody as described in claim 6.

13. The library according to claim 11 or 12, comprising more than 30% of variants having increased affinity to the epitope bound by the reference antibody, compared to the affinity of the reference antibody to the epitope.

14. A library of nucleotide sequences, wherein each member of the library encodes an antibody molecule of the antibody molecule library according to any one of claims 11 to 13.

Images