Antibodies and methods for making and using them

Abstract

The present invention provides methods for producing humanized antibodies and increasing the yield of antibodies and/or antigen binding fragments when produced in cell culture. In one aspect of the invention, at least one framework region amino acid residue of the variable domain is substituted by a corresponding amino acid from a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and/or HVR2 amino acid sequence of the variable domain. In another aspect, an amino acid is placed at a position proximal to a cys residue that participates in an intrachain variable domain disulfide bond that corresponds to an amino acid found at that position in a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and/or HVR2 amino acid sequence of the variable domain.

1 . A method for producing an antibody or antigen binding fragment having at least one modified framework region (FR) comprising:
 (A) (a) expressing a variable domain of the antibody or antigen binding fragment comprising at least one modified FR in a host cell, a cell extract or a cell culture, wherein the modified FR has a substitution of at least one amino acid position with a different amino acid, wherein the different amino acid is the amino acid found at the corresponding FR position of a human subgroup variable domain consensus sequence that has a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence with the most sequence identity with a corresponding HVR1 and/or HVR2 sequence of the variable domain; and   b) recovering the antibody or antigen binding fragment variable domain comprising the at least one modified FR from the host cell, cell extract or cell culture;   (B) the method of (A), wherein the antibody or antigen binding fragment comprises or consists of a humanized antibody, a chimeric antibody, a monoclonal antibody, a human antibody, a multispecific antibody, diabodies, or an antibody generated by phage display;   (C) the method of (A) or (B), wherein the antigen binding fragment comprises or consists of a Fab fragment, F(ab′) 2  fragment, scFV fragment, or sc(Fv) 2  fragment, a single arm antibody or single chain antibody;   (D) the method of any of (A) to (C), wherein the antibody or antigen binding fragment comprises or consists of an anti-VEGF antibody or an anti-IgE, IgA, IgD, IgG or IgM antibody, or an antigen binding fragment thereof;   (E) the method of any of (A) to (C), wherein the antibody or antigen binding fragment comprises or consists of an anti-IgE, IgA, IgD, IgG or IgM antibody, or an antigen binding fragment thereof;   (F) the method of any of (A) to (E), wherein the antibody or antigen binding fragment binds specifically to a transmembrane molecule, a receptor, a ligand, a growth factor, a growth hormone, a clotting factor, an anti-clotting factor, a plasminogen activator, a serum albumin, a receptor for a hormones or a growth factor, a neurotrophic factor, a nerve growth factor, a fibroblast growth factor, transforming growth factor (TGF), a CD protein, an interferon, a colony stimulating factor (CSF), an interleukin (IL), a T-cell receptor, a surface membrane protein, a viral protein, a tumor associated antigen, an integrin or an interleukin;   (G) the method of any of (A) to (F), wherein the antibody or antigen binding fragment binds specifically to: VEGF; a renin; a human growth hormone; a bovine growth hormone; a growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; a lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factor VIIIC; clotting factor IX; tissue factor (TP); von Willebrands factor; Protein C; atrial natriuretic factor; a lung surfactant; urokinase; human urine; tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha or -beta; enkephalinase; RANTES (Regulated on Activation Normally T-cell Expressed and Secreted); human macrophage inflammatory protein (MIP-1)-alpha; human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA); CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); protein A or D; a rheumatoid factor; bone-derived neurotrophic factor (BDNE); neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); NGF-beta; platelet-derived growth factor (PDGF); aFGF; bFGF; epidermal growth factor (EGE); TGP-alpha; TGF-beta, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta14, or TGF-beta5; insulin-like growth factor-I or -II (IGF-I or IGF-II); des(1-3)-IGF-I (brain IGF-I), an insulin-like growth factor binding protein, erythropoietin; an osteoinductive factor; an immunotoxin; a bone morphogenetic protein (BMP); interferon-alpha, -beta, or -gamma; M-CSF, GM-CSF or G-CSF; IL-1 to IL-10; superoxide dismutase; decay accelerating factor; an AIDS envelope protein; a transport protein; a homing receptor; an addressin; a regulatory protein; CD3, CD4, CD8, CD11a, CD11b, CD11c, CD18, CD19, CD20, CD34, CD40, or CD46, an ICAM, VLA-4 or VCAM; or HER2, HER3 or HER4 receptor; a member of the ErbB receptor family; an EGF receptor; HER2, HER3 or HER4 receptor; a cell adhesion molecule; LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin or alphav/beta3 integrin; an alpha or beta subunit of a cell adhesion molecule; antibodies); a growth factor, VEGF; tissue factor (TF); TGF-beta; alpha interferon (alpha-IFN); IL-8; IgE; blood group antigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA4 or protein C;   (H) the method of any of (A) to (G), wherein expressing a variable domain of the antibody or antigen binding fragment comprising at least one modified FR in a host cell comprises expressing a polynucleotide encoding the variable domain comprising the at least one modified FR;   (I) the method of (H), wherein the polynucleotide further comprises a polynucleotide encoding a constant region domain connected to the polynucleotide encoding the variable domain with modified FR to form a polynucleotide encoding a full-length heavy or light chain;   (J) the method of (I), wherein the polynucleotide further comprises an expression vector;   (K) the method of any of (A) to (J), further comprising recovering a full-length heavy or light chain or both from the culture;   (L) the method of any of (A) to (K), wherein the host cell is a prokaryotic host cell, or the host cell is a mammalian cell;   (M) the method of any of (A) to (L), wherein the variable domain is a heavy chain variable domain or a light chain variable domain   (N) the method of (M), wherein the HVR1 amino acid sequence is GYTFTNYGIN (SEQ ID NO: 14), GYDFTHYGMN (SEQ ID NO: 18), or GYSITSGYSWN (SEQ ID NO: 19);   (O) the method of any of (A) to (N), wherein the framework region comprises or consists of an FR1, FR2, FR3, FR4 or a mixture thereof;   (P) the method of (O), wherein the human subgroup FR consensus sequence is a heavy chain FR1 sequence with a sequence comprising SEQ. ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;   (Q) the method of any of (A) to (P), wherein the antibody or antigen binding fragment variable domain comprising the at least one modified FR has improved yield in cell culture compared to an unmodified antibody or antigen binding fragment, or the modification to the variable domain contributes to correct folding, assembly and yield of the antibodies or antigen binding fragments, or the modification to the variable domain results in an affinity matured antibody or antigen binding fragment;   (R) the method of (Q), wherein the yield of the antibody or antigen binding fragment comprising the modified FR is improved at least 2 fold compared to the unmodified antibody or antigen binding fragment; or, the yield of the antibody or antigen binding fragment comprising the modified FR is improved at least 2 fold to 16 fold compared to the unmodified antibody or antigen binding fragment;   (S) the method of any of (A) to (R), wherein at least two amino acid positions in at least one FR that have a different amino acid are substituted with the amino acids in the corresponding positions of the selected subgroup consensus sequence;   (T) the method of (R), wherein the FR is a heavy chain FR1 and one of the amino acid positions is position 6 or position 23 or both, and the other position is position 1, 11, 13, 18 or 19 or a mixture thereof, or amino acid positions 6 and 23 are substituted; or all of the amino acid positions at positions 1, 6, 11, 13, 18, 19, and 23 of the heavy chain FR1 are substituted;   (U) the method of any of (A) to (T), wherein all of the amino acid positions in a FR that have a different amino acid are each substituted with the amino acid in the corresponding FR position in the selected subgroup consensus sequence;   (V) the method of (T), wherein the FR is FR1, FR2, or FR3; or   (W) the method of any of (A) to (V), wherein all of the amino acid positions that have a different amino acid in all FR are each substituted with the amino acid in the corresponding FR position in the selected subgroup consensus sequence.   
     
     
         2 - 24 . (canceled) 
     
     
         25 . A method for preparing a humanized antibody or antigen binding fragment, comprising:
 (A) a) expressing a variable domain comprising at least one framework region (FR) sequence from a selected human subgroup variable domain consensus sequence, and a hypervariable region-1 (HVR1) and/or a hypervariable region-2 (HVR2) sequence of a non-human antibody in a host cell, wherein the selected human subgroup consensus sequence is the human subgroup consensus sequence that has a HVR1 and/or HVR2 sequence that has the most sequence identity to the HVR1 and/or HVR2 of the non-human antibody; and   b) recovering the antibody variable domain from the host cell;   (B) the method of (A), wherein the variable domain is a heavy chain variable domain or a light chain variable domain;   (C) the method of (B), wherein the HVR1 amino acid sequence is GYTFTNYGIN (SEQ ID NO: 14), GYDFTHYGMN (SEQ ID NO: 18), or GYSITSGYSWN (SEQ ID NO: 19);   (D) the method of any of (A) to (C), wherein the FR comprises a FR1, FR2, FR3, FR4 or a mixture thereof;   (E) the method of (D), wherein the human subgroup FR consensus sequence is a heavy chain FR1 sequence with a sequence comprising SEQ. ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;   (F) the method of any of (A) to (E), wherein the variable domain comprises all of the FR of the selected human subgroup consensus sequence;   (G) the method of any of (A) to (F), wherein the humanized antibody or antigen binding fragment has improved yield when produced in cell culture as compared to a or antigen binding fragment having the same HVR1 and/or HVR2 but without the selected FR;   (H) the method of any of (A) to (G), wherein expressing comprises: expressing an expression vector comprising a polynucleotide encoding a variable domain comprising the HVR1 and/or HVR2 of the non-human antibody, and the selected FR;   (I) the method of (H), wherein the expression vector further comprises a polynucleotide encoding a constant domain connected to the polynucleotide encoding the variable domain to form a polynucleotide encoding a full-length heavy or light chain;   (J) the method of any of (A) to (I), further comprising recovering the full-length heavy or light chain or both from the culture; or   (K) the method of any of (A) to (J), wherein the host cell is a prokaryotic host cell, or the host cell is a mammalian cell.   
     
     
         26 - 38 . (canceled) 
     
     
         39 . A method for making an antibody or antigen binding fragment having at least one modified framework region (FR) in a cell, cell extract or cell culture, comprising:
 (a) (i) modifying said at least one FR sequence of a variable domain of the antibody or antigen binding fragment such that it is at least 50% identical in sequence to the corresponding FR sequence of a selected subgroup consensus sequence to form a modified FR,   wherein the modified FR has a substitution of at least one amino acid position with a different amino acid, wherein the different amino acid is the amino acid found at the corresponding FR position of the selected human subgroup variable domain consensus sequence,   wherein the selected human subgroup consensus sequence has a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence with the most sequence identity with a corresponding HVR1 and/or HVR2 sequence of the variable domain; and   (i) recovering the variable domain with the modified FR from the cell, cell extract or cell culture;   (b) the method of (a), wherein the antibody or antigen binding fragment variable domain comprising the at least one modified FR has improved yield in the cell culture compared to an unmodified antibody or antigen binding fragment, or the modification to the FR contributes to correct folding, assembly and yield of the antibodies or antigen binding fragments, or the modification to the FR results in an affinity matured antibody or antigen binding fragment;   (c) the method of (a) or (b), wherein the HVR1 amino acid sequence is GYTFTNYGIN (SEQ ID NO: 14), GYDFTHYGMN (SEQ ID NO: 18), or GYSITSGYSWN (SEQ ID NO: 19);   (d) the method of any of (a) to (c), wherein at least two amino acid positions that have a different amino acid in at least one FR are substituted with amino acids in the corresponding position of the selected subgroup consensus sequence;   (e) the method of (d), wherein amino acid positions 6 and 23 of heavy chain FR1 are substituted, or amino acid positions 1, 6, 11, 13, 18, 19 and 23 of the heavy chain FR1 are substituted;   (f) the method of any of (a) to (e), wherein expressing comprises expressing an expression vector comprising a first polynucleotide that encodes a variable domain comprising the HVR1 and/or HVR2 amino acid sequence of the antibody or antigen binding fragment and at least one modified FR;   (g) the method of (f), wherein the expression vector further comprises a second polynucleotide encoding a constant domain, wherein the first and second polynucleotide are operably linked to a promoter; a heat stable enterotoxin sequence that can direct secretion to the periplasm; and a terminator sequence;   (h) the method of any of (a) to (g), wherein the host cell is a prokaryotic host cell, or the host cell is a mammalian cell;   (i) the method of any of (a) to (h), wherein the step of modifying comprises substituting all of the FRs of the variable domain with each of the corresponding FRs of the selected subgroup; or   (j) the method of any of (a) to (i), wherein the framework region sequence is FR1, FR2, FR3, FR4 or a mixture thereof.   
     
     
         40 - 49 . (canceled) 
     
     
         50 . A method for producing an antibody or antigen binding fragment having at least one modified framework region (FR) in a cell, cell extract or cell culture comprising:
 (A) a) expressing a modified variable domain of the antibody or antigen binding fragment in a host cell, cell extract or cell culture, wherein the modified variable domain has a substitution of at least one amino acid position proximal to a cys residue that participates in an intrachain variable domain disulfide bond with a different amino acid, wherein the different amino acid is the amino acid found at corresponding position of a human subgroup variable domain consensus sequence that has a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence with the most sequence identity with a corresponding HVR1 and/or HVR2 amino acid sequence of the variable domain; and   b) recovering the antibody or antigen binding fragment comprising the modified variable domain from the host cell, cell extract or cell culture;   (B) the method of (A), wherein the antibody or antigen binding fragment variable domain comprising the at least one modified FR has improved yield in the cell culture compared to an unmodified antibody or antigen binding fragment, or the modification to the FR contributes to correct folding, assembly and yield of the antibodies or antigen binding fragments, or the modification to the FR results in an affinity matured antibody or antigen binding fragment;   (C) the method of (A) or (B), wherein the variable domain is a heavy chain variable domain or a light chain variable domain;   (D) the method of (C), wherein said at least one position is selected from the group consisting of the amino acid position 4 of the light chain, the amino acid position 6 of the light chain, the amino acid position 33 of the light chain, the amino acid position 35 of the light chain, the amino acid position 71 of the light chain and mixtures thereof;   (E) the method of (C), wherein said at least one position is selected from the group consisting of the amino acid position 4 of the heavy chain, the amino acid position 6 of the heavy chain, the amino acid position 34 of the heavy chain, the amino acid position 36 of the heavy chain, the amino acid position 78 of the heavy chain, the amino acid position 104 of the heavy chain and mixtures thereof;   (F) the method of (C), wherein said at least one position selected from the group consisting of amino acid position 4 of the light chain, amino acid position 6 of the light chain, amino acid position 33 of the light chain, amino acid position 35 of the light chain, amino acid position 71 of the light chain, and at least one position is selected from the group consisting of amino acid position 4 of the heavy chain, amino acid position 6 of the heavy chain, amino acid position 34 of the heavy chain, amino acid position 36 of the heavy chain, amino acid position 78 of the heavy chain, and amino acid position 104 of the heavy chain;   (G) the method of any of (A) to (F), wherein the at least one amino acid position is an amino acid position adjacent to the cys residue that forms an intra chain variable domain disulfide bond;   (H) the method of (G), wherein the at least one amino acid position is selected from group consisting of amino acid position 21, amino acid position 22, amino acid position 24, amino acid position 25, amino acid position 86, amino acid position 87, amino acid position 89 and amino acid position 90 in a light chain variable domain;   (I) the method of (G), wherein the at least one amino acid position is selected from the group consisting of amino acid position 20, amino acid position 21, amino acid position 23, amino acid position 24, amino acid position 90, amino acid position 91, amino acid position 93 and amino acid position 94 in a heavy chain variable domain;   (J) the method of any of (A) to (I), wherein the variable domain is from an anti-VEGF antibody or anti-IgE, anti-IgM, anti-IgD, anti-IgG or anti-IgA antibody;   (K) the method of any of (A) to (J), wherein the variable domain is from a humanized antibody or antigen binding fragment;   (L) the method of any of (A) to (K), wherein the step of expressing comprises expressing an expression vector comprising a first polynucleotide that encodes the modified variable domain sequence with an amino acid substitution in at least one of the amino acids proximal to a cys residue, wherein at least one amino acid is substituted with the amino acid at the corresponding position in the selected subgroup consensus sequence;   (M) the method of (L), wherein the expression vector comprises a second polynucleotide encoding antibody constant region domains, wherein the first and second polynucleotide are operably linked to a promoter; a heat stable enterotoxin sequence that can directs secretion to the periplasm; and a terminator sequence;   (N) the method of any of (A) to (M), further comprising recovering a full-length heavy or light chain or both;   (O) the method of (N), wherein the heavy chain variable domain has a substitution in amino acid position 4, 6, 34, 78, or mixtures thereof, or the light chain variable domain has a substitution in amino acid position 4, 71, or mixtures thereof;   (P) the method of any of (A) to (O), wherein the host cell is a prokaryotic host cell, or the host cell is a eukaryotic host cell; or   (Q) the method of any of (A) to (P), wherein the antibody or antigen binding fragment with modified variable domain has increased yield of at least 2 fold when produced in cell culture as compared to the antibody or antigen binding fragment, or the yield of the antibody or antigen binding fragment with the modified variable domain is increased at least 2 to 16 fold as compared to the antibody or antigen binding fragment.   
     
     
         51 - 68 . (canceled) 
     
     
         69 . The method of  claim 50  further comprising:
 (i) a) identifying at least one amino acid position in a second variable domain of the antibody or antigen binding fragment that is proximal to a cys residue that forms an intrachain variable domain disulfide bond in the second variable domain;   b) selecting a variable domain subgroup consensus sequence having the most sequence identity with a HVR1 and/or HVR2 amino acid sequence of the second variable domain; and   c) determining whether the amino acid in the amino acid position identified in the second variable domain is different than the amino acid in the selected subgroup consensus sequence; and   d) placing at said at least one position in the second variable domain the amino acid found at the corresponding position in the selected subgroup consensus sequence to form a modified variable domain; or   (ii) the method of (i), wherein the variable domain is a heavy chain variable domain and the second variable domain is a light chain variable domain.   
     
     
         70 . (canceled) 
     
     
         71 . A method for preparing a humanized antibody or antigen binding fragment, comprising:
 (A) (i) a) expressing a variable domain in a host cell, cell extract or cell culture, wherein the variable domain is formed by substituting at least one amino acid position proximal to a cys residue that participates in an intrachain variable domain disulfide bond with a different amino acid, wherein the different amino acid is the amino acid found at corresponding position of a human subgroup variable domain consensus sequence that has a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence with the most sequence identity with a corresponding HVR1 and/or HVR2 amino acid sequence of the variable domain; and   b) recovering the antibody variable domain from the host cell, cell extract or cell culture; or   (ii) the method of (i), wherein the variable domain is a heavy chain variable domain, or the variable domain is a light chain variable domain; or   (B) (a) identifying at least one amino acid position in a heavy chain variable domain that is proximal to a cys residue that participates in an intrachain disulfide bond in the heavy chain variable domain;   (b) selecting a first human antibody heavy chain variable domain subgroup consensus sequence having the most identity with a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence of the heavy chain variable domain; and   (c) placing at said at least one position in the heavy chain variable domain an amino acid found at the corresponding position in the selected first subgroup;   (d) identifying at one amino acid position in a light chain variable domain that is proximal to a cys residue that participates in an intrachain disulfide bond in the light variable domain;   (e) selecting a second human antibody light chain variable domain subgroup consensus sequence having the most sequence identity with a HVR1 and/or HVR2 amino acid sequence of the light chain variable domain; and   (f) placing at said at least one position in the light chain variable domain an amino acid found at the corresponding position in the second selected subgroup.   
     
     
         72 - 78 . (canceled) 
     
     
         79 . An isolated, synthetic or recombinant antibody comprising:
 (a) a heavy chain variable domain comprising a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) and at least one framework region (FR), wherein the FR has an amino acid residue of the corresponding FR of a human subgroup consensus sequence with the most sequence identity to the HVR1 and/or HVR2 amino acid sequence in at least two amino acid positions selected from the group consisting of 1, 6, 11, 13, 18, 19, 23, and mixtures thereof;   (b) the antibody of (a), wherein each of the amino acid positions has an amino acid of the corresponding position in FR of the human subgroup consensus sequence with the most sequence identity to the HVR1; or   (c) the antibody of (a) or (b), wherein amino acid positions 6 and 23 each has an amino acid found at the corresponding position in the FR of a human subgroup consensus sequence with the most sequence identity to the HVR1.   
     
     
         80 - 99 . (canceled) 
     
     
         100 . A method for improving the yield of antibody or antigen binding fragments in a cell, cell extract or cell culture, comprising:
 (i) (a) expressing a polynucleotide molecule encoding a modified variable domain of a parent antibody or antigen binding fragment in a host cell, wherein the modified variable domain has a substitution of at least one amino acid proximal to a cys reside that participates in an intrachain variable domain disulfide bond with a different amino acid,   wherein the different amino acid is the amino acid found at corresponding position of a human subgroup variable domain consensus sequence that has a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence with the most sequence identity with a corresponding HVR1 and/or HVR2 amino acid sequence of the parent variable domain,   wherein the antibody or antigen binding fragment comprising the modified variable domain has improved yield in cell culture compared to the parent antibody or antigen binding fragment; and   (b) recovering the antibody or antigen binding fragment comprising the modified variable domain from the cell, cell extract or host cell;   (ii) the method of (i), wherein the antibody or antigen binding fragment comprising the modified variable domain has improved yield in cell culture compared to the parent antibody or antigen binding fragment, or the modification to the variable domain contributes to correct folding, assembly and yield of the antibodies or antigen binding fragments, or the modification to the variable domain results in an affinity matured antibody or antigen binding fragment;   (iii) the method of (i) or (ii), wherein the polynucleotide molecule comprises an expression vector that comprises a polynucleotide molecule encoding the modified variable domain and at least one constant region domain operably linked to a promoter, a heat stable enterotoxin sequence that can direct secretion to the periplasm, and a terminator sequence; or   (iv) the method of any of (i) to (iii), wherein the host cell is a prokaryotic host cell or a eukaryotic host cell.   
     
     
         101 - 103 . (canceled) 
     
     
         104 . A method for making an antibody or an antigen binding fragment, comprising:
 (A) a) comparing a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence of a variable domain of a parent antibody or antigen binding fragment to a corresponding HVR1 and/or HVR2 amino acid sequence of each human subgroup variable domain consensus sequences and selecting a human subgroup variable domain consensus sequence that has the most sequence identity with the HVR1 and/or HVR2 sequence of the variable domain;   b) identifying at least one amino acid position in at least one framework region (FR) in the variable domain comprising consisting of FR1, FR2, FR3, FR4 or mixtures thereof, wherein the amino acid position has a different amino acid than the amino acid at a corresponding position of the selected human subgroup variable domain consensus sequence; and   c) substituting the at least one amino acid position identified in step (b) with the amino acid in the corresponding position of the selected human subgroup variable domain consensus sequence to form a variable domain with a modified FR;   (B) the method of (A), wherein the antibody or antigen binding fragment with the modified FR has improved yield in cell culture compared to the parent antibody or antigen binding fragment;   (C) the method of (A) or (B), wherein the parent antibody comprises or consists of a humanized antibody, a chimeric antibody, a monoclonal antibody, a human antibody, a multispecific antibody, diabodies, or an antibody generated by phage display;   (D) the method of any of (A) to (C), wherein the parent antigen binding fragment comprises or consists of a Fab fragment, F(ab′) 2  fragment, scFV fragment, sc(Fv) 2  fragment, single arm antibody or a single chain antibody;   (E) the method of any of (A) to (D), wherein the parent antibody is an anti-VEGF antibody or an anti-IgE, anti-IgD, anti-IgM, anti-IgA or anti-IgG antibody, or the parent antibody is a humanized antibody;   (F) the method of any of (A) to (E), wherein step (c) comprises modifying a polynucleotide encoding the parent variable domain to form a polynucleotide encoding a variable domain with a modified FR, wherein the modified FR has at least one amino acid position substituted with the amino acid in the corresponding position of the selected human subgroup variable domain consensus sequence;   (G) the method of any of (A) to (F), wherein the polynucleotide further comprises a polynucleotide encoding a constant region domain connected to the polynucleotide encoding the variable domain with modified FR parent to form a polynucleotide encoding a full-length heavy or light chain;   (H) the method of any of (A) to (G), wherein the polynucleotide further comprises an expression vector;   (I) the method of any of (A) to (H), further comprising culturing a host cell comprising the expression vector; and recovering a full-length heavy or light chain or both from the culture;   (J) the method of any of (A) to (I), wherein the host cell is a prokaryotic host cell, or the host cell is a mammalian cell;   (K) the method of any of (A) to (J), wherein the variable domain is a heavy chain variable domain or a light chain variable domain;   (L) the method of any of (A) to (I), wherein the HVR1 amino acid sequence is GYTFTNYGIN (SEQ ID NO: 14), GYDFTHYGMN (SEQ ID NO:18), or GYSITSGYSWN (SEQ ID NO:19);   (M) the method of any of (A) to (L), wherein the framework region comprises or consists of FR1, FR2, FR3, FR4, or a mixture thereof;   (N) the method of (M), wherein the human subgroup FR consensus sequence is a heavy chain FR1 sequence with a sequence comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3;   (O) the method of any of (A) to (N), wherein the yield of the antibody or antigen binding fragment with the modified FR is improved at least 2 fold compared to the parent antibody or antigen binding fragment;   (P) the method of any of (A) to (O), wherein the yield of the antibody or antigen binding fragment with the modified FR is improved at least 2 fold to 16 fold compared to the parent antibody or antigen binding fragment;   (Q) the method of any of (A) to (P), wherein at least two identified amino acid positions in at least one FR are substituted with amino acids in the corresponding position of the selected subgroup consensus sequence;   (R) the method of (Q), wherein the FR is a heavy chain FR1 and one of the identified amino acid positions is position 6 or position 23 or both, and the other position is position 1, 11, 13, 18, 19 or a and mixture thereof, or amino acid positions 6 and 23 are substituted, or all of the amino acid positions at position, 1, 6, 11, 13, 18, 19, and 23 of the heavy chain FR1 are substituted;   (S) the method of any of (A) to (P), wherein all of the identified amino acid positions in a FR are each substituted with the amino acid in the corresponding position in the selected subgroup consensus sequence;   (T) the method of (S), wherein the FR is FR1, FR2, or FR3; or   (U) the method of any of (A) to (P), wherein all of the identified amino acid positions in all FR are each substituted with the amino acid in the corresponding position in the selected subgroup consensus sequence.   
     
     
         105 - 127 . (canceled) 
     
     
         128 . A recombinant, synthetic or isolated polypeptide comprising or consisting of
 (A) a variable domain of an antibody or antigen binding fragment produced by the method of  claim 1 ; or   (B) (a) a first variable domain with at least one amino acid position that is proximal to a cys residue that forms an intrachain disulfide bond in the first variable domain   wherein the at least one position having an amino acid is found at a corresponding position of a human antibody subgroup consensus sequence selected for the most identity with a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence of the first variable domain; and   (b) a second variable domain with at least one amino acid position that is proximal to a cys residue that forms an intrachain disulfide bond in the second variable domain;   wherein the at least one position having an amino acid at a corresponding position of a human antibody subgroup consensus sequence is selected for the most identity with a HVR1 and/or HVR2 amino acid sequence of the second variable domain;   wherein the first variable domain is a heavy chain and the second variable domain is a light chain; or   (C) a first variable domain comprising:   (A) a) at least one amino acid position that is proximal to a cys residue that forms an intrachain disulfide bond in the first variable domain;   b) said at least one position having an amino acid found at a corresponding position of a human antibody subgroup consensus sequence selected for the most sequence identity with a hypervariable region-1 (HVR1) and/or hypervariable region-2 (HVR2) amino acid sequence of the first variable domain; or   (B) the polypeptide of (A), wherein the first variable domain is a heavy chain and the positions are amino acid positions 4, 6, 34, 78 or mixtures thereof, or the first variable domain is a light chain and the amino acid positions are 4, 71, or both.   
     
     
         129 . A recombinant, synthetic or isolated nucleic acid comprising: (i) a polynucleotide encoding the polypeptide of claim  75 ; (ii) a polynucleotide encoding the antibody of  claim 79 ; (iii) a polynucleotide encoding the polypeptide of  claim 128 ; (iv) a polynucleotide encoding the polypeptide of  claim 129 ; or (v) any combination of (i) to (iv).