METHODS FOR AFFINITY MATURATION-BASED ANTIBODY OPTIMIZATION

• US 20160069894A1
• Filed: 04/21/2015
• Published: 03/10/2016
• Est. Priority Date: 11/04/2009
• Status: Active Grant

• Current Assignee: Taurus Biosciences LLC (Ligand Pharmaceuticals, Inc.)
• Original Assignee: Fabrus, LLC. (Eloxx Pharmaceuticals, Inc.)
• Inventors: SMIDER, Vaughn, MAO, Helen Hongyuan
• Granted Patent: US 10,101,333 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/33 Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Provided herein is a rational method of affinity maturation to evolve the activity of an antibody or portion thereof based on the structure/affinity or activity relationship of an antibody. The resulting affinity matured antibodies exhibit improved or optimized binding affinity for a target antigen.

Claims

1-97. -97. (canceled)

98. A method of affinity maturation of a first antibody, or antigen-binding portion thereof, for a target antigen, comprising:

a) identifying a related antibody, or antigen-binding portion thereof, that exhibits a reduced activity for the target antigen than the corresponding form of a first antibody by screening an addressable combinatorial antibody library, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or a related variable light chain that is either;

one in which the corresponding variable heavy chain and variable light chain of the related antibody, or antigen-binding portion thereof, exhibits at least 75% amino acid sequence identity to the corresponding variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, but does not exhibit 100% sequence identity therewith;

or

one in which at least one of the V_H, D_H, and J_H germline segments of a nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof, is identical to one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, and at least one of the V_κ

and J_κ

or at least one of the V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain is identical to one of the V_κ

and J_κ

or V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof; and

b) comparing the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, to the amino acid sequence of the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

c) identifying a target region within the variable heavy chain or variable light chain of a first antibody, or antigen-binding portion thereof, wherein the target region exhibits at least one amino acid difference compared to the same region in the related antibody, or antigen-binding portion thereof;

d) producing a plurality of modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is modified in its target region by replacement of a single amino acid residue, whereby the target region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first antibody, or antigen-binding portion thereof;

e) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and

f) selecting those modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof.

99. A method according to claim 98 that further includes at least one of the following:

a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in part (d) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the target region that encodes a different amino acid as compared to the unmodified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in its target region by replacement of a single amino acid residue;

b) wherein the target region in the first antibody, or antigen-binding portion thereof, exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences compared to the corresponding region in the related antibody, or antigen-binding portion thereof;

c) wherein the related antibody, or antigen-binding portion thereof, is 1, 2, 3, 4, or 5 related antibodies, or antigen-binding portions thereof;

d) wherein the activity is selected from the group consisting of;

i. binding, optionally binding as assessed by a method selected from the group consisting of an immunoassay, optionally an immunoassay selected from the group consisting of a radioimmunoassay, an enzyme linked immunosorbent assay (ELISA), and an electrochemiluminescence assay, wherein the electrochemiluminescence assay optionally is meso-scale discovery (MSD);

whole cell panning; and

surface Plasmon resonance (SPR);

ii. signal transduction;

iii. differentiation;

iv. alteration of gene expression;

v. cellular proliferation;

vi. apoptosis;

vii. chemotaxis;

viii. cytotoxicity;

ix. cancer cell invasion;

x. endothelial cell proliferation; and

xi. tube formation;

e) wherein the first antibody, or antigen-binding portion thereof, binds to the target antigen when in a Fab form with a binding affinity that is about 10<sup>−

4</sup> M or lower, about 10<sup>−

4</sup> M to about 10<sup>−

8</sup> M, or at or about 10<sup>−

4</sup> M, 10<sup>−

5</sup> M, 10<sup>−

6</sup> M, 10<sup>−

7</sup> M, 10<sup>−

8</sup> M, or lower;

f) wherein the related antibody, or antigen-binding portion thereof, exhibits a binding affinity that is less than the binding affinity of the first antibody, or antigen-binding portion thereof, whereby the binding affinity of the related antibody, or antigen-binding portion thereof, in its Fab form is about 10<sup>−

4</sup> M or lower;

about 10<sup>−

4</sup> M to about 10<sup>−

8</sup> M;

or at or about 10<sup>−

4</sup> M, 10<sup>−

5</sup> M, 10<sup>−

6</sup> M, 10<sup>−

7</sup> M, 10<sup>−

8</sup> M, or lower;

g) wherein the related antibody, or antigen-binding portion thereof, exhibits about 80% or less activity than the corresponding form of the first antibody, or antigen-binding portion thereof;

about 5% to about 80% of the activity of the corresponding form of the first antibody;

or less than or about 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less activity than the corresponding form of the first antibody;

h) wherein the related antibody, or antigen-binding portion thereof, exhibits the same or similar level of activity to the target antigen compared to a negative control;

i) wherein the target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

j) wherein the target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

k) wherein;

a target region is identified within the variable heavy chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom; and

separately and independently a target region is identified within the variable light chain of the first antibody, or antigen-binding portion thereof, and steps d)-f) are performed therefrom;

l) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is different than a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;

m) wherein a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable heavy chain is the same as a related antibody, or antigen-binding portion thereof, that contains the related corresponding variable light chain;

n) wherein the amino acid sequence of the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 80% or more sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

about 80% to about 99% of the sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

or at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen-binding portion thereof;

o) wherein the variable heavy chain or variable light chain of the first antibody, or antigen-binding portion thereof, exhibits at least about 95% sequence identity with the corresponding amino acid sequence of the related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof;

p) wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light chain that is one in which at least one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen binding portion thereof, is identical to one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof; and

/or at least one of the V_κ

and J_κ

or at least one of the V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is identical to one of the V_κ

and J_κ

or V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof;

q) wherein the target antigen is selected from the group consisting of a polypeptide, carbohydrate, lipid, nucleic acid, and a small molecule;

r) wherein the target antigen is expressed on the surface of a virus, bacteria, tumor or other cell, or is a recombinant protein or peptide;

s) wherein the target antigen is a protein that is a target for therapeutic intervention, optionally selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and

     3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor;

HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and

     4), RON (recepteur d′

origine nantais;

also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1 R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-β

R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPOR, a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-α

, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO;

t) wherein the target antigen is involved in cell proliferation and differentiation, cell migration, apoptosis or angiogenesis;

u) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement;

v) wherein only the amino acid residues that differ between the first antibody and related antibody in the target region are modified by amino acid replacement;

w) wherein only the amino acid residues that are the same between the first antibody and the related antibody in the target region are modified by amino acid replacement;

x) wherein all of the amino acids residues in the target region are modified by amino acid replacement;

y) wherein each amino acid residue that is modified in the target region is modified to all 19 other amino acid residues, or a restricted subset thereof;

or

z) further comprising determining the amino acid modifications that are altered in the modified antibody compared to the first antibody not containing the amino acid replacements;

aa) wherein the method is repeated iteratively, wherein a modified antibody, or antigen-binding portion thereof, is selected and used in step a) as the first antibody, or antigen-binding portion thereof, for subsequent affinity maturation thereof;

bb) wherein one or more amino acid replacements in the target region of one or more variable heavy chains or one or more variable light chains of selected modified antibodies, or antigen-binding portions thereof, are combined to generate a further modified antibody, or antigen-binding portion thereof, whereby the further modified antibody(ies), or antigen-binding portion(s) thereof, are screened for an activity to the target antigen to identify a further modified antibody, or antigen-binding portion thereof, that exhibits an increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, and to the selected modified antibody(ies), or antigen-binding portion(s) thereof; and

cc) wherein the antibody, or antigen-binding portion thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, is selected from the group consisting of a Fab, Fab′

, F(ab′

)2, single-chain Fv (scFv), scFab, Fv, dsFv, diabody, Fd, Fd′

, Fab fragment, Fd fragment, Fd′

fragment, scFv fragment, and scFab fragment.

100. A method according to claim 98, wherein the related antibody, or antigen-binding portion thereof, contains a related variable heavy chain or variable light that is one in which at least one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the V_H, D_H, and J_H germline segments of the nucleic acid molecule encoding the variable heavy chain of the related antibody, or antigen-binding portion thereof;

and/or at least one of the V_κ

and J_κ

or at least one of the V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain of the first antibody, or antigen-binding portion thereof, is from the same gene family as one of the V_κ

and J_κ

or V_λ

and J_λ

germline segments of the nucleic acid molecule encoding the variable light chain of the related antibody, or antigen-binding portion thereof.

101. A method according to claim 98, wherein the variable heavy chain or variable light chain of the first antibody, or antigen binding portion thereof, exhibits at least 60% or more sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof;

60% to 99% of the sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof;

or at least or about 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the corresponding related variable heavy chain or variable light chain of the related antibody, or antigen binding portion thereof.

102. A method according to claim 98, wherein the target region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4.

103. A method according to claim 98, wherein:

a) the first antibody, or antigen-binding portion thereof, is identified by screening a combinatorial antibody library or combinatorial antigen-binding antibody fragment library;

b) the combinatorial library is produced by a method comprising;

i) combining a V_H, a D_H, and a J_H human germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VH chain or a portion thereof;

ii) combining a V_κ

and a J_κ

human germline segment or portion thereof, or a V_λ

and a J_λ

germline segment or portion thereof in frame to generate a sequence of a nucleic acid molecule encoding a VL chain or a portion thereof, wherein;

in steps i) and ii), each of the portions of the V_H, D_H, J_H, V_κ

, J_κ

, V_λ

or J_λ

are sufficient to produce an antibody or antigen-binding portion thereof containing a VH or VL or portion thereof that forms a sufficient antigen binding site;

iii) repeating steps i) and ii) a plurality of times to generate sequences of a plurality of different nucleic acid molecules;

iv) synthesizing the nucleic acid molecules to produce two libraries, wherein;

the first library comprises nucleic acid molecules encoding a VH chain or a portion thereof; and

the second library comprises nucleic acid molecules encoding a VL chain or a portion thereof;

v) introducing a nucleic acid molecule from the first library and from the second library into a cell and repeating this a plurality of times to produce a library of cells, wherein each cell contains nucleic acid molecules encoding a different combination of VH and VL from at least some of the other cells in the library of cells; and

vi) growing the cells to express the antibodies, or antigen-binding portions thereof, thereby producing a plurality of antibodies, or antigen-binding portion thereof, wherein the different antibodies, or antigen-binding portions thereof, in the library each comprise a different combination of a VH and a VL chain or a sufficient portion thereof to form an antigen binding site; and

c) screening of the library is effected by;

i) contacting an antibody, or antigen-binding portion thereof, in the library with a target protein;

ii) assessing binding of the antibody, or antigen-binding portion thereof, with the target protein and/or whether the antibody, or antigen-binding portion thereof, modulates a functional activity of the target protein; and

iii) identifying an antibody, or antigen-binding portion thereof, that exhibits an activity for the target protein, wherein the identified antibody, or antigen-binding portion thereof, is a first antibody.

104. A method according to claim 103 that further includes at least one of the following:

a) the related antibody also is identified by screening a combinatorial antibody library by steps a)-c), whereby the related antibody exhibits reduced activity for the target antigen compared to the first antibody;

b) the library is an addressable library, whereby;

in step iv), the synthesized nucleic acid sequences are individually addressed, thereby generating a first addressed nucleic acid library and a second addressed nucleic acid library;

in step v), the cells are addressed, wherein each locus comprises a cell that contains nucleic acid molecules encoding a different combination of a VH and a VL from every other cell in the addressed library of cells; and

in step vi) the plurality of antibodies or portions thereof are addressed, wherein;

the antibodies or portions thereof at each locus in the library are the same antibody and are different from those at each and every other locus;

and the identity of the antibody or portion thereof is known by its address, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein each individual locus of the array corresponds to a different antibody member;

c) wherein the antibodies are in an addressable library, wherein optionally the antibodies in the addressable library are arranged in a spatial array, optionally a multiwell plate, wherein optionally each individual locus of the array corresponds to a different antibody member are attached to a solid support selected from the group consisting of a filter, chip, slide, bead or cellulose, and the different antibody members are immobilized to the surface thereof;

d) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient hast strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair;

or

e) wherein the plurality of nucleic acid molecules are generated by a method selected from the group consisting of NNK, NNS, NNN, NNY, or NNR mutagenesis.

105. A method according to claim 98, further comprising before step d),

g) performing scanning mutagenesis of the first antibody, or antigen-binding portion thereof, comprising producing a plurality of modified antibodies, or antigen-binding portions thereof, comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain, or portion thereof, is one that is modified by replacement of a single amino acid residue with a scanned amino acid residue in the target region, whereby each of the plurality of antibodies, or antigen-binding portion thereof, contains replacement of an amino acid in the target region compared to the first antibody, or antigen-binding portion thereof, wherein the scanned amino acid optionally is selected from the group consisting of alanine, threonine, proline, glycine, and a non-natural amino acid;

h) screening each of the plurality of modified antibodies, or antigen-binding portions thereof, for an activity to the target antigen; and

i) selecting a second antibody, or antigen-binding portion thereof, from among the modified antibodies, or antigen-binding portions thereof, that exhibits retained or increased activity for the target antigen compared to the first antibody, or antigen-binding portion thereof, not containing the amino acid replacement, whereby the second antibody, or antigen-binding portion thereof, is used in place of the first antibody, or antigen-binding portion thereof, in step b).

106. A method according to claim 105 that further includes at least one of the following:

a) wherein the plurality of modified antibodies, or antigen-binding portions thereof, in step g) are produced by producing a plurality of nucleic acid molecules that encode modified forms of a variable heavy chain or a variable light chain of the first antibody, or antigen-binding portion thereof, containing the target region, wherein the nucleic acid molecules contain one codon that encodes a scanned amino acid in the target region compared to the corresponding codon of the unmodified variable heavy or variable light chain that does not encode the scanned amino acid, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified by replacement of a single amino acid residue to the same scanned amino acid residue in the target region;

b) wherein a second antibody is, or antigen-binding portion thereof, selected that exhibits an activity that is at least 75% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;

is at least 75% to 200% of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;

or is at least or about 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 200% or more of the activity of the corresponding form of the first antibody, or antigen-binding portion thereof;

c) further comprising after step i) determining the amino acid residue position that is modified in the second antibody, or antigen-binding portion thereof, to contain a neutral amino acid compared to the first antibody not containing the amino acid replacement;

d) wherein a subset of the amino acid residues in the target region are modified by amino acid replacement to a scanned amino acid;

e) wherein only the amino acid residues that differ between the first antibody, or antigen-binding portion thereof, and related antibody, or antigen-binding portion thereof, in the target region are modified by amino acid replacement to a scanned amino acid;

f) wherein all of the amino acids in the target region are modified by amino acid replacement to a scanned amino acid;

g) wherein the selected modified antibody, or antigen-binding portion thereof, exhibits about 2-fold, 5-fold, 10-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 10000-fold, or more improved activity for the target antigen compared to the first antibody, or antigen-binding portion thereof;

or

h) wherein the modified antibody, or antigen-binding portion thereof, exhibits a binding affinity that is greater than the binding affinity of the first antibody, or antigen-binding portion thereof, and is about 1×

10<sup>−

9</sup>M or less;

1×

10<sup>−

9</sup>M to 1×

10<sup>−

11</sup> M;

or is or is about 1×

10<sup>−

9</sup> M, 2×

10<sup>−

9</sup> M, 3×

10<sup>−

9</sup> M, 4×

10<sup>−

10</sup> M, 5×

10<sup>−

9</sup> M, 6×

10<sup>−

9</sup> M, 7×

10<sup>−

9</sup> M, 8×

10<sup>−

9</sup> M, 9×

10<sup>−

9</sup> M, 1×

10<sup>−

10</sup> M, 2×

10<sup>−

10</sup> M, 3×

10<sup>−

10</sup> M, 4×

10<sup>−

10</sup> M, 5×

10<sup>−

10</sup> M, 6×

10<sup>−

10</sup> M, 7×

10<sup>−

10</sup> M, 8×

10<sup>−

10</sup> M, 9×

10<sup>−

10</sup> M, or less,

107. A method according to claim 98, comprising:

performing steps a)-f) on the variable heavy chain of the first antibody, or antigen-binding portion thereof, and selecting first modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;

performing steps a)-f) independently and separately on the variable light chain of the first antibody and selecting second modified antibodies, or antigen-binding portions thereof, each containing an amino acid replacement in the target region;

combining the variable heavy chain of a first modified antibody, or antigen-binding portion thereof, with the variable light chain of a second modified antibody, or antigen-binding portion thereof, to generate a plurality of different third modified antibodies, or antigen-binding portions thereof, each comprising an amino acid replacement in the target region of the variable heavy chain and variable light chain; and

screening each of the plurality of third modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

selecting those third modified antibodies, or antigen-binding portions thereof, that exhibit an increased activity for the target antigen compared to the first and second modified antibodies.

108. A method according to claim 98, further comprising after selecting a first modified antibody, or antigen-binding portion thereof, in step f):

j) selecting another different region within the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, for further mutagenesis, wherein optionally the further different region is selected from the group consisting of a CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4;

k) producing a plurality of nucleic acid molecules that encode modified forms of the variable heavy chain or variable light chain of the first modified antibody, or antigen-binding portion thereof, wherein the nucleic acid molecules contain one codon encoding an amino acid in the selected region that encodes a different amino acid from the first modified variable heavy or variable light chain, whereby each nucleic acid molecule of the plurality encodes a variable heavy chain or variable light chain that is modified in the selected region by replacement of a single amino acid residue;

l) producing a plurality of further modified antibodies, or antigen-binding portions thereof, each comprising a variable heavy chain and a variable light chain, or a portion thereof, wherein at least one of the variable heavy chain or variable light chain is one produced in step k), whereby the selected region in each of the plurality of antibodies, or antigen-binding portions thereof, contains replacement of an amino acid to a different amino acid compared to the first modified antibody, or antigen-binding portion thereof;

m) screening each of the plurality of further modified antibodies, or antigen-binding portions thereof, for binding to the target antigen; and

n) selecting those further modified antibodies, or antigen-binding portions thereof, that exhibit increased activity for the target antigen compared to the first modified antibody, or antigen-binding portion thereof.