阅读说明：本技术 抗体选择方法 (Antibody selection method ) 是由 H·克腾伯格 W·理查特 L·拉里维尔 T·克拉夫特 T·埃姆里希 于 2018-04-25 设计创作，主要内容包括：本文中报道了一种用于选择在食蟹猴中具有小于8mL/kg/天的全身清除率的抗体的方法,所述方法包括步骤：采用阳性线性pH梯度进行FcRn亲和色谱和采用阳性线性电导率/盐梯度上进行肝素亲和色谱,测量抗体的保留时间,并且选择抗体,所述抗体具有比SEQ ID NO:03和04的氧化型抗Her3抗体制备物的峰2和峰3保留时间之间的保留时间差异的1.78倍小的FcRn亲和色谱柱上相对保留时间,和比SEQ ID NO:01和02的抗pTau抗体的保留时间的0.87倍小的肝素亲和色谱柱上相对保留时间。(Herein is reported a method for selecting antibodies having a systemic clearance in cynomolgus monkeys of less than 8 mL/kg/day, said method comprising the steps of: FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography on a positive linear conductivity/salt gradient, the retention times of the antibodies were measured and antibodies were selected having a relative retention time on the FcRn affinity chromatography column that was less than 1.78 times the retention time difference between the retention times of peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NOs 03 and 04 and a relative retention time on the heparin affinity chromatography column that was less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NOs 01 and 02.)

1. a method for producing an antibody comprising the steps of:

a) Cultivating the mammalian cell expressing the antibody, and

c) Recovering the antibody from the cells or the culture medium,

Wherein the antibody (selected from a plurality of antibodies and/or antibody formats) has been selected to have i) a relative retention time on an FcRn affinity chromatography column that is less than 1.78 times the retention time difference between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NOs 03 and 04, and ii) a relative retention time on a heparin affinity chromatography column that is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NOs 01 and 02.

2. A method for selecting an antibody having a systemic clearance of 8 mL/kg/day in a cynomolgus monkey, comprising the steps of:

a) Measuring the retention time of the antibody on an FcRn affinity chromatography column with a positive linear pH gradient and on a heparin affinity chromatography column with a positive linear conductivity/salt gradient, and

b)

i) if the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

Antibodies were selected.

3. The method of any one of claims 1-2, wherein the antibody is selected from the group consisting of a full length antibody, a CrossMab, a 2:1 heterodimeric T cell bispecific antibody, an antibody-cytokine fusion polypeptide, an Fc region-cytokine fusion polypeptide, and an antibody-Fab fusion polypeptide.

4. The method of any one of claims 1 to 3, wherein the antibody comprises an Fc region selected from the group consisting of a human IgG1Fc region, a human IgG1Fc region having mutations L234A, L235A, and P329G, a human IgG1Fc region having a binding buckle mutation, and combinations thereof.

5. the method according to any one of claims 1 to 4, wherein an immobilized non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is used as affinity chromatography ligand in FcRn affinity chromatography with a positive linear pH gradient,

Wherein a non-covalent complex of a neonatal Fc receptor and beta-2-microglobulin is bound to a chromatographic material and the non-covalent complex is conjugated to a solid phase via a specific binding pair,

Wherein the pH gradient is from a first pH to a second pH, wherein the first pH is pH 3.5 to pH 6.4 and the second pH is pH7.4 to pH 9.5, and

wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is monobiotinylated and the solid phase is derivatized with streptavidin.

6. The method of claim 5, wherein the pH gradient is from a first pH to a second pH, whereby the first pH is pH 5.5 and the second pH is pH 8.8.

7. The method according to any one of claims 1 to 6, wherein the relative retention time on the FcRn affinity chromatography column is calculated according to the following equation:

Based on the peak definition (t) according to FIG. 1_rel，i: relative retention time of peak i; t is t_i: retention time of peak i; t is t_{Peak 2}: retention time of peak 2 of partially oxidized anti Her3 antibody according to figure 1; t is t_{Peak 3}: retention time of peak 3 of anti Her3 antibody according to figure 1).

8. the method according to any one of claims 1 to 7, wherein the relative retention time on the heparin affinity chromatography column is calculated according to the formula:

(t_rel，i: relative retention time of peak i; t is t_i: retention time of peak i; t is t_pTau: retention time of anti-pTau antibody peak).

9. A method for selecting an antibody binding to at least one antigen with systemic clearance suitable for use as a therapeutic agent, said method comprising the steps of:

a) providing antibodies in different patterns selected from

i) Full length antibodies, CrossMab, 2:1 heterodimeric T cell bispecific antibodies and any of the foregoing fused to one, two or three additional Fab, scFv, scFab, CrossFab molecules, either directly or via a peptide linker, and/or

ii) a human IgG1Fc region, a human IgG1Fc region with mutations L234A, L235A, and P329G, a human IgG1Fc region with a knot-in mutation, and combinations thereof.

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using each different antibody pattern of a), and

c)

i) If the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

antibody patterns were selected.

10. A method for selecting an antibody binding to at least one antigen with systemic clearance suitable for use as a therapeutic agent, said method comprising the steps of:

a) Providing at least two antibodies that bind to at least one antigen, said antibodies

i) have different CDR sequences, or

ii) have the same CDR sequences and different variable domain sequences, or

iii) have the same CDR sequences in different patterns,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient with each different antibody of a), and

c) Selecting an antibody having

i) The relative retention time on the FcRn affinity chromatography column relative to the retention time of the first reference antibody on the FcRn affinity chromatography column is less than a first threshold, and

ii) the ratio of the retention time on the heparin affinity chromatography column to the retention time of the second reference antibody on the heparin affinity chromatography column is less than a second threshold.

11. The method according to any one of claims 9 to 10, further comprising the step of:

d) If none of the provided antibodies or antibody patterns meet the criteria of step c), providing at least one other antibody pattern or antibody and repeating steps b) and c).

Background

Human immunoglobulin (IgG) of class G contains two antigen binding (Fab) regions conferring specificity to a target antigen and one constant region (Fc region) responsible for interaction with an Fc receptor (see, e.g., Edelman, g.m., scand.j.immunol.34(1991) 1-22; Reff, m.e., and Heard, c., crit.rev.oncol.hematol.40(2001) 25-35). Human IgG subclasses IgG1, IgG2, and IgG4 have a mean serum half-life of 21 days, which is longer than the mean serum half-life of any other known serum protein (see, e.g., Waldmann, t.a. and Strober, w., prog.allergy 13(1969) 1-110). This long half-life is primarily mediated by the interaction between the Fc region and the neonatal Fc receptor (FcRn) (see, e.g., Ghetie, v. and Ward, e.s., annu. rev. immunol.18(2000) 739-. This is one of the reasons why IgG or Fc-containing fusion proteins are used as a class of broad therapeutic agents.

The neonatal Fc receptor FcRn is a membrane-bound receptor involved in IgG and albumin homeostasis, translocation of maternal IgG across the placenta, and phagocytosis of antigen-IgG immune complexes (see, e.g., Brambell, F.W. et al, Nature 203(1964) 1352-351354; Ropeenian, D.C. et al, J.Immunol.170(2003) 3528-3533). Human FcRn is a major organization by glycosylation class ICompatible Complex-like protein (alpha-FcRn) and beta₂Microglobulin (beta)₂m) heterodimers of subunits (see, e.g., Kuo, t.t. et al, j.clin.immunol.30(2010) 777-. FcRn and C of Fc region_H2-C_HSite binding within region 3 (see, e.g., Ropeenian, D.C. and Akilesh, S., Nat. Rev. Immunol.7(2007) 715. 725; Martin, W.L. et al, mol.cell 7(2001) 867. 877; Goebl, N.A. et al, mol.biol.cell 19(2008) 5490. 5505; Kim, J.K. et al, Eur.J.Immunol.24(1994) 542. 548.) and two n molecules can bind to the Fc region simultaneously (see, e.g., Sanchez, L.M. et al, Biochemistry 38 (38) (9471) 941999; Huber, A.H. et al, J.mol.Biol.230(1993) 1077. 1083.). The affinity between FcRn and the Fc region is pH dependent, exhibiting nanomolar affinity at in vivo pH 5-6 and relatively weak binding at physiological pH7.4 (see, e.g., Goebl, N.A. et al, mol.biol.Cell 19(2008) 5490-. The basic mechanism that confers IgG a long half-life can be explained by three basic steps. First, IgG is subject to nonspecific engulfment by a variety of cell types (see, e.g., akinesh, s. et al, j. immunol.179(2007) 4580-. Secondly, IgG encounters and binds FcRn in acidic endosomes at pH 5-6, thus protecting IgG from lysosomal degradation (see, e.g., Ropeenian, d.c. and Akilesh, s., nat. rev. immunol.7(2007) 715-66725; Rodewald, r., j.cell biol.71(1976) 666-669). Finally, IgG is released in extracellular clearance at physiological pH7.4 (see, e.g., Ghetie, v. and Ward, e.s., annu.rev.immunol.18(2000) 739-. This strictly pH-dependent binding and release mechanism is important for IgG recycling and any deviation in binding characteristics at different pH values may strongly influence the circulating half-life of IgG (see, e.g., vaccarao, c. et al, nat. biotechnol.23(2005) 1283-1288).

With the majority of human therapeutic antibody candidates showing pharmacokinetic properties suitable for clinical use, Hoetzel, i. et al (mAbs 4(2012) 753-. This document describes an assay based on ELISA to detect binding to Baculovirus (BV) particles to assess non-specific binding of therapeutic proteins.

Analytical FcRn affinity chromatography for the functional characterization of monoclonal antibodies is disclosed in WO 2013/120929. Putnam, W.S. et al disclose a pharmacokinetic, pharmacodynamic and immunogenicity comparability evaluation strategy for monoclonal antibodies (Trends Biotechnol.28(2010) 509-516).

Sampei, z. et al (PLoS One 8(2013) e57479) disclose the identification and multidimensional optimization of asymmetric bispecific IgG antibodies that mimic the function of factor VIII cofactor activity.

WO 2015/140126 discloses a method for predicting the in vivo half-life of an antibody based on the retention time determined on an FcRn affinity chromatography column.

The current literature discloses the use of FcRn chromatography (see, e.g., Schoch, a. et al, proc.natl.acad.sci.usa 112 (2015)) 5997-. Alternatively, heparin binding is disclosed, e.g., in an ELISA format (see, e.g., Datta-Mannan, a., et al, MAbs 7(2015) 1084-.

brief description of the invention

with the method of the invention, an increased number, i.e. more, antibodies with pharmacokinetic properties suitable for therapeutic use can be identified; in particular, antibodies having pharmacokinetic properties suitable for therapeutic applications can be selected more accurately. This was done by evaluating antibodies from the various antibodies provided based on the results obtained by the methods as reported herein.

it has been found that the selection of antibodies can be improved by using a combination of FcRn affinity chromatography and heparin affinity chromatography relative to/based on the clearance of antibodies in cynomolgus monkey Single Dose Pharmacokinetic (SDPK) studies compared to isolated (1-dimensional) FcRn or isolated heparin chromatography, respectively. The improvement resides in, among other things, reducing the number of rejected antibodies with acceptable PK profiles.

It has been found that the combination of FcRn affinity chromatography and heparin affinity chromatography allows defining an FcRn affinity chromatography column and a heparin affinity chromatography column retention time threshold and thus a two-dimensional retention time region, wherein antibodies with slow clearance (i.e. long systemic circulation half-life) can be found. Thus, this combination allows for improved selection of antibodies with long systemic circulation half-life, improved accuracy of pharmacokinetic predictions, and reduced number of rejected antibodies despite having long systemic circulation half-life, among other things.

It has been found that when the retention times on the FcRn affinity chromatography column and on the heparin affinity chromatography column are normalized based on the retention times of the respective on-column reference antibodies, a relative retention time region of the antibodies is defined which predominates to comprise a slow clearance. This region has a relative retention time (in oxidized form (H) of less than 1.78 on an FcRn affinity chromatography column₂O₂Treated) anti-Her 3 antibody preparation as reference antibody) and a relative retention time on a heparin affinity chromatography column of less than 0.87 (with anti-pTau antibody as reference antibody).

Accordingly, the invention includes a method of selecting an antibody having systemic clearance in cynomolgus monkeys suitable for use as a therapeutic agent (in humans), the method comprising the steps of:

a) Optionally providing an antibody-containing (ex vivo; manual) of the samples are taken,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient, and

c)

i) if/when the relative retention time of the antibody on the FcRn affinity chromatography column is less than the first threshold relative to the retention time of the first reference antibody on the FcRn affinity chromatography column, and,

ii) if/when the ratio of the retention time of the antibody on the heparin affinity chromatography column to the retention time of the second reference antibody on the heparin affinity chromatography column is less than the second threshold,

Antibodies were selected.

In one embodiment, the first reference antibody is an oxidized antibody preparation. In one embodiment, the oxidized antibody preparation is a preparation comprising a reference antibody in a non-oxidized form, in a monooxidized form (only one of the two methionines at position 252 is oxidized), and in a doubly oxidized form (both methionine at position 252 are oxidized) with respect to the methionine residue at position 252 in the heavy chain CH2 domain (numbering according to Kabat). In one embodiment, the relative retention time is calculated based on the following equation

Wherein t is_rel，iRelative retention time of the antibody; t is t_iRetention time of antibody. In one embodiment, the first reference antibody is an anti-Her 3 antibody having a heavy chain with the amino acid sequence of SEQ ID NO. 03 and a light chain with the amino acid sequence of SEQ ID NO. 04. In one embodiment, the first threshold is 2. In one embodiment, the first threshold is 1.8. In one embodiment, the first threshold is 1.78.

In one embodiment, the second reference antibody is an anti-pTau antibody having a heavy chain having the amino acid sequence of SEQ ID NO. 01 and a light chain having the amino acid sequence of SEQ ID NO. 02. In one embodiment, the second threshold is 1. In one embodiment, the second threshold is 0.8. In one embodiment, the second threshold is 0.78.

in one embodiment, step c) is

i) when/if the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) when/if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

antibodies were selected.

In one embodiment, the systemic clearance rate in cynomolgus monkeys suitable for therapeutic use (i.e., the antibody may be used as a therapeutic agent) is 8 mL/kg/day or less. In one embodiment, the systemic clearance is less than 8 mL/kg/day. In one embodiment, the systemic clearance is less than 6 mL/kg/day.

The invention also comprises a method of selecting an antibody (specifically) binding to at least one antigen (in cynomolgus monkeys) having systemic clearance suitable for use (in humans) as a therapeutic agent, comprising the steps of:

a) Providing antibodies in different patterns selected from

i) Full length antibodies, CrossMab, 2:1 heterodimeric T cell bispecific antibodies and any of the foregoing fused to one, two or three additional Fab, scFv, scFab, CrossFab molecules, either directly or via a peptide linker, and/or

ii) a human IgG1Fc region, a human IgG1Fc region with mutations L234A, L235A and P329G,

human IgG1Fc region having a knot-in mutation and combinations thereof,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using each different antibody pattern of a), and

c)

i) If the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

the selection of the antibody is carried out in the presence of a suitable antibody,

And thus selecting antibodies

The invention also comprises a method of selecting an antibody (specifically) binding to at least one antigen (in cynomolgus monkeys) having systemic clearance suitable for use (in humans) as a therapeutic agent, comprising the steps of:

a) Providing at least two antibodies that bind to at least one antigen, said antibodies

i) Have different CDR sequences, or

ii) have the same CDR sequences and different variable domain sequences, or

iii) have the same CDR sequences in different antibody formats,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient with each different antibody of a), and

c) Selecting an antibody having

i) The relative retention time on the FcRn affinity chromatography column relative to the retention time of the first reference antibody on the FcRn affinity chromatography column is less than a first threshold, and

ii) the ratio of the retention time on the heparin affinity chromatography column to the retention time of the second reference antibody on the heparin affinity chromatography column is less than a second threshold.

In one embodiment, the first reference antibody is an oxidized antibody preparation. In one embodiment, the oxidized antibody preparation is a preparation comprising an antibody in a non-oxidized form, in a mono-oxidized form (only one of the two methionines at position 252 is oxidized), and in a di-oxidized form (both methionine at position 252 are oxidized) with respect to the methionine residue at position 252 in the heavy chain CH2 domain (numbering according to Kabat). In one embodiment, the relative retention time is calculated based on the following equation

wherein t is_rel，iRelative retention time of the antibody; t is t_iRetention time of antibody. In one embodiment, the first reference antibody is an anti-Her 3 antibody having a heavy chain with the amino acid sequence of SEQ ID NO. 03 and a light chain with the amino acid sequence of SEQ ID NO. 04. In one embodiment, the first threshold is 2. In one embodiment, the first threshold is 1.8. In one embodiment, the first threshold is 1.78.

In one embodiment, the second reference antibody is an anti-pTau antibody having a heavy chain having the amino acid sequence of SEQ ID NO. 01 and a light chain having the amino acid sequence of SEQ ID NO. 02. In one embodiment, the second threshold is 1. In one embodiment, the second threshold is 0.8. In one embodiment, the second threshold is 0.78.

In one embodiment, step c) is

i) When/if the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) when/if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

Antibodies were selected.

In one embodiment, the method further comprises the steps of:

d) If none of the provided antibodies or antibody patterns meet the criteria of step c), providing at least one other antibody pattern or antibody and repeating steps b) and c).

The invention includes a method for producing an antibody, the method comprising the steps of:

a) culturing a mammalian cell expressing an antibody comprising one or more nucleic acids encoding the antibody, and

c) Recovering the antibody from the cells or the culture medium,

Wherein the antibodies (from the various antibodies and/or antibody formats) have been selected to have i) a relative retention time on an FcRn affinity chromatography column that is less than 1.78 times the retention time difference between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NOs 03 and 04, and ii) a relative retention time on a heparin affinity chromatography column that is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NOs 01 and 02.

the invention includes a method for selecting an antibody having a systemic clearance in cynomolgus monkeys of less than 8 mL/kg/day, comprising the steps of:

a) Optionally providing a sample comprising the antibody,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient, and

c)

i) If the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

Antibodies were selected.

The invention also includes a method for producing an antibody, the method comprising the steps of:

a) Providing a cell comprising one or more nucleic acids encoding an antibody selected with a method according to the invention, and

b) Culturing the cell in a culture medium and recovering the antibody from the cell or the culture medium, and thereby producing the antibody.

In one embodiment of the overall method, the relative retention time on the FcRn affinity chromatography column is calculated according to the following equation:

Based on the peak definition (t) according to FIG. 1_rel，i: relative retention time of peak i; t is t_i: retention time of peak i; t is t_{Peak 2}: retention time of peak 2 of partially oxidized anti Her3 antibody according to figure 1; t is t_{Peak 3}: retention time of peak 3 of anti Her3 antibody according to figure 1).

In one embodiment of the overall method, the relative retention time on the heparin affinity chromatography column is calculated according to the formula:

(t_rel，i: relative retention time of peak i; t is t_i: retention time of peak i; t is t_pTau: anti-pTau antibodyRetention time of bulk peak).

In one embodiment of the overall method, the antibody or antibody format is selected from the group consisting of a full length antibody comprising two antibody light chains and two antibody heavy chains, a CrossMab, a 2:1 heterodimeric T cell bispecific antibody, an antibody-cytokine fusion polypeptide, an Fc region-cytokine fusion polypeptide, and an antibody-Fab fusion polypeptide.

in one embodiment of the overall method, the antibody comprises an Fc region selected from the group consisting of a human IgG1Fc region, a human IgG1Fc region having mutations L234A, L235A, and P329G, a human IgG1Fc region having a binding-clip mutation, and combinations thereof.

In one embodiment of FcRn affinity chromatography with a positive linear pH gradient, an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is used as affinity chromatography ligand,

Wherein a non-covalent complex of a neonatal Fc receptor and beta-2-microglobulin is bound to a chromatographic material and the non-covalent complex is conjugated to a solid phase via a specific binding pair,

Wherein the pH gradient is from a first pH to a second pH, wherein the first pH is pH 3.5 to pH 6.4 and the second pH is pH7.4 to pH 9.5, and

Wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is monobiotinylated and the solid phase is derivatized with streptavidin.

In one embodiment, the pH gradient is from a first pH value to a second pH value, whereby the first pH value is pH 5.5 and the second pH value is pH 8.8.

In one embodiment, the beta-2-microglobulin is from the same species as FcRn.

In one embodiment, the FcRn is selected from the group consisting of human FcRn, cyno FcRn, mouse FcRn, rat FcRn, sheep FcRn, canine FcRn, pig FcRn, mini-pig FcRn, and rabbit FcRn.

in one embodiment, the beta-2-microglobulin is from the same species as FcRn.

In one embodiment, the beta-2-microglobulin is from a different species than FcRn.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is a bispecific antibody.

In one embodiment, the antibody is a chimeric antibody.

typically, the soluble extracellular domain of FcRn (SEQ ID NO:31 for human FcRn) with a C-terminal His-Avi Tag (SEQ ID NO:32) is co-expressed with β 2-microglobulin (SEQ ID NO:33 for human β -2-microglobulin) in mammalian cells. The non-covalent FcRn-microglobulin complex was biotinylated and loaded on streptavidin-derivatized agarose gel.

In principle, any buffer substance may be used in the method as reported herein.

in one embodiment, the reference antibody for FcRn affinity chromatography is an anti-HER 3 antibody having SEQ ID NO 03 (heavy chain) and SEQ ID NO 04 (light chain).

In one embodiment, the reference antibody for heparin affinity chromatography is an anti-pTau antibody having SEQ ID NO:01 (heavy chain) and SEQ ID NO:02 (light chain).

In one embodiment, the antibody is a monospecific antibody or antibody fragment of a fusion polypeptide, or a bispecific antibody or antibody fragment of a fusion polypeptide, or a trispecific antibody or antibody fragment of a fusion polypeptide, or a tetraspecific antibody or antibody fragment of a fusion polypeptide.

in one embodiment, the antibody is an IgG class antibody. In one embodiment, the antibody is an antibody of the subclass IgG1, IgG2, IgG3, or IgG 4. In one embodiment, the antibody is an antibody of the subclass IgG1 or IgG 4.

Brief Description of Drawings

Figure 1 calculates the peak definition of relative retention time on the FcRn column.

Figure 2FcRn relative retention is plotted against heparin column relative retention. Cross number: clearance >12 mL/kg/day ("rapid"); solid square: clearance ("margin") between 8 and 12 mL/kg/day; solid circle: a clearance greater than 2.5 mL/kg/day but less than 8 mL/kg/day; solid asterisk: a clearance of 2.5 mL/kg/day or less.

figure 3FcRn relative retention is plotted against heparin column relative retention. Cross number: clearance >12 mL/kg/day ("rapid"); solid square: clearance ("margin") between 8 and 12 mL/kg/day; solid circle: a clearance greater than 2.5 mL/kg/day but less than 8 mL/kg/day; solid asterisk: a clearance of 2.5 mL/kg/day or less; the vertical line marks the retention time range for therapeutically appropriate clearance (lower left quadrant, FcRn < 1.78; heparin: < 0.87).

figure 4 FcRn relative retention of IVIG (intravenous immunoglobulin) is plotted against heparin column relative retention. 2: the fraction with the highest heparin binding; 1: the fraction with the lowest heparin binding.

Figure 5 antibodies No. 5 and variants thereof FcRn relative retention was plotted against heparin column relative retention. 1: a wild-type antibody; 20: HC variants with irregular (patched) negative charges; 27: positively-charged anomalous HC variants; 112: positively-charged anomalous LC variants; 183: positively-charged irregular HC variants.

figure 6 clearance of antibody No. 5 and its variants in FcRn knockout mice. 1: a wild-type antibody; 20: (iii) a negatively-charged irregular HC variant; 27: positively-charged anomalous HC variants; 112: positively-charged anomalous LC variants; 183: positively-charged irregular HC variants.

Figure 7 time-dependent serum concentrations of non-irregular (non-patch) and irregular (patch) variants of antibody No. 5 in FcRn knockout mice.

Figure 8 FcRn relative retention of antibody No. 5 and its histidine variants is plotted against heparin column relative retention.

Figure 9 time-dependent serum concentrations of antibody No. 5 and its HC histidine variant in FcRn knockout mice.

figure 10 clearance of antibody No. 5 and its HC histidine variant in FcRn knockout mice.

The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made to the method described without departing from the spirit of the invention.

Materials and methods

Antibodies

The reference antibodies used in the experiment were anti-pTau antibody having the heavy chain amino acid sequence of SEQ ID NO:01 and the light chain amino acid sequence of SEQ ID NO:02 and anti-Her 3 antibody having the heavy chain amino acid sequence of SEQ ID NO:03 and the light chain amino acid sequence of SEQ ID NO: 04.

Synthetic genes were generated at Geneart (Life technologies GmbH, Carlsbad, Calif., USA).

The monoclonal antibodies used herein were transiently expressed in HEK293 cells (see below) and purified by protein a chromatography using standard procedures (see below).

Biochemical characterization included size exclusion chromatography (Waters Biosuite)^TM2507.8 × 300mm, elution: 200mM KH₂PO₄250mM KCl, pH 7.0) and molecular weight distribution using BioAnalyzer 2100(Agilent technologies, Santa Clara, Calif., USA).

expression plasmid

For expression of the above-mentioned antibodies, expression plasmid variants based on cDNA organization with or without the CMV-intron A promoter or on genomic organization with the CMV promoter are used for transient expression (e.g.in HEK293-F cells).

In addition to the antibody expression cassette, the plasmid also contains:

An origin of replication allowing the plasmid to replicate in E.coli,

-a beta-lactamase gene conferring ampicillin resistance in E.coli, and

the dihydrofolate reductase gene from mice (Mus musculus) as a selection marker in eukaryotic cells.

The transcription unit of the antibody gene consists of the following elements:

-one or more unique restriction sites at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

A subsequent intron A sequence in the case of cDNA organization,

-the 5' untranslated region of a human antibody gene,

An immunoglobulin heavy chain signal sequence,

Human antibody chains organized as cDNA or as genome with immunoglobulin exon-intron organization

-a 3' untranslated region having a polyadenylation signal sequence, and

Unique restriction sites at the 3' end

Fusion genes comprising antibody chains are generated by PCR and/or genetic synthesis and assembled by linking the corresponding nucleic acid segments, for example using unique restriction sites in the corresponding plasmids, by known recombinant methods and techniques. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, plasmids were prepared from transformed E.coli cultures (Nucleobond AX, Macherey-Nagel) and larger quantities of plasmid were made.

Cell culture techniques

Standard Cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, j.s., Dasso, m., Harford, j.b., Lippincott-Schwartz, j. and Yamada, K.M (eds.), John Wiley & Sons, inc.

Transient transfection in the HEK293-F System

Antibodies were generated by transient transfection with the corresponding plasmids (e.g., the plasmids encoding the heavy chain and the corresponding light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, serum-free FreeStyle in shake flasks or stirred fermentors^TMHEK293-F cells (Invitrogen) grown in suspension in 293 expression Medium (Invitrogen) with the corresponding expression plasmids and 293fectin^TMOr transfection with a mixture of fectins (Invitrogen). For 2L shake flasks (Corning), HEK293-F cells were plated at a density of 1 × 10⁶cells/mL were seeded in 600mL and 120 rpm, 8% CO₂And (4) incubation. After that day, about 1.5 x 10 will be added⁶cells at individual cells/mL cell density were transfected with a mixture of approximately 42mL of: A)20mL of Opti-MEM (Invitrogen) with 600. mu.g total plasmid DNA encoding the heavy and corresponding light chains, respectively, in equimolar ratio (1. mu.g/mL) and B)20mL of Opti-MEM +1.2mL 293fectin or fectin (2. mu.L/mL). In the fermentationGlucose solution was added during the process according to glucose consumption. The supernatant containing the secreted antibody is harvested after 5-10 days and the antibody is purified directly from the supernatant or the supernatant is frozen and stored. The following antibodies have therefore been generated:

purification of

By using MabSelectSure-Sepharose^TMAffinity chromatography (GE Healthcare, sweden), hydrophobic interaction chromatography using butyl-sepharose (GE Healthcare, sweden) and Superde x 200 size exclusion (GE Healthcare, sweden) chromatography, antibodies were purified from cell culture supernatants.

briefly, in PBS buffer (10mM Na)₂HPO₄，1mM KH₂PO₄137mM NaCl and 2.7mM KCl, pH 7.4) sterile filtered cell culture supernatant was captured on MabSelectSuRe resin equilibrated, washed with equilibration buffer and eluted with 25mM sodium citrate pH 3.0. Eluted antibody fractions were pooled and neutralized with 2M Tris, pH 9.0. The antibody pool was prepared for hydrophobic interaction chromatography by adding 1.6M ammonium sulfate solution to a final concentration of 0.8M ammonium sulfate and adjusting the pH to pH5.0 using acetic acid. After equilibrating the butyl-sepharose resin with 35mM sodium acetate, 0.8M ammonium sulfate, pH5.0, the antibody was applied to the resin, washed with equilibration buffer and eluted with a linear gradient to 35mM sodium acetate pH 5.0. The antibody-containing fractions were pooled and further purified by size exclusion chromatography using a Superde x 20026/60 GL (GE Healthcare, Sweden) column equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. The antibody-containing fractions were pooled, concentrated to the desired concentration using a Vivaspin ultrafiltration device (Sartorius Stedim Biotech s.a., france) and stored at-80 ℃.

Purity and antibody integrity were analyzed by CE-SDS after each purification step using the microfluidic Labchip technique (Caliper Life Science, usa). Using the HT protein expression kit, 5. mu.l of protein solution for CE-SDS analysis was prepared and analyzed on a LabChip GXII system using the HT protein expression chip according to the manufacturer's instructions. Data were analyzed using LabChip GX software.

Mouse

B6.Cg-Fcgrt transgenic for mouse FcRn alpha-chain gene defective but hemizygous for human FcRn alpha-chain gene^tm1DcrTg (fcgrt)276Dcr mice (muFcRn-/-huFcRn tg +/-, strain 276) were used for pharmacokinetic studies. Mouse breeding was performed under specific pathogen-free conditions. Mice were obtained from the Jackson laboratory (Bar Harbor, ME, USA) (female, age 4-10 weeks, body weight 17-22g at dosing). All animal experiments were approved by the Bavaria government in Germany (permit No. 55.2-1-54-2532.2-28-10) and conducted in an AAALAC certified animal facility according to European Union laboratory animal Care and use codes. Animals were housed in standard cages and were free to ingest and drink water throughout the study.

pharmacokinetic Studies

a single dose of antibody was injected intravenously via the lateral tail vein at a dose level of 10 mg/kg. Mice were divided into 3 groups of 6 mice each to cover a total of 9 serum collection time points (0.08, 2, 8, 24, 48, 168, 336, 504 and 672 hours post-dose). Each mouse received 2 retro-orbital bleeds in Isofluran^TM(CP-Pharma GmbH, Burgdorf, Germany) under light anesthesia, a third blood sample was taken at euthanasia, blood was taken to a medium serum tube (Microvette 500Z-Gel, Sarstedt, N ü mbrecht, Germany) after 2 hours incubation, the sample was centrifuged at 9,300g for 3 minutes to obtain serum after centrifugation, the serum samples were cryopreserved at-20 ℃ until analysis.

determination of human antibody serum concentration

The concentrations of Ustekinumab (Ustekinumab), brekinumab (Briakinumab), mAb 8 and mAb 9 in the mouse sera were determined by specific enzyme-linked immunoassay. Biotinylated interleukin 12 and digitonin-labeled anti-human-Fc mice specific for antibodiesMonoclonal antibodies (Roche Diagnostics, pentgegberg, germany) were used for capture and detection, respectively. Streptavidin-coated microtiter plates (Roche Diagnostics, pentgere, germany) were coated for 1 hour with biotinylated capture antibody diluted in assay buffer (Roche Diagnostics, pentgere, germany). After washing, serum samples were added at various dilutions, followed by another incubation step for 1 hour. After repeated washing, bound human antibodies were detected by subsequent incubation with detection antibodies followed by incubation with anti-digitonin antibodies conjugated to horseradish peroxidase (HRP; Roche Diagnostics, Pentgere, Germany). Using ABTS (2,2' -biazo-bis [ 3-ethylbenzothiazoline sulfonic acid)](ii) a Roche Diagnostics, germany) as HRP substrate to form colored reaction products. Using a Tecan sunrise plate reader (Switzerland), the absorbance of the resulting reaction product was read at 405nm using a reference wavelength at 490 nm.

all serum samples, positive control samples and negative control samples were analyzed in duplicate and calibrated against the reference standard provided.

Pharmacokinetic (PK) analysis

pharmacokinetic parameters were calculated by non-compartmental analysis using winnonlintm1.1.1(Pharsight, CA, usa).

Briefly, the area under the curve (AUC) was calculated by the logarithmic trapezoidal method due to the nonlinear reduction of the antibody_0-inf) Values and using the apparent terminal rate constant λ z, the values were extrapolated to infinity, extrapolated from the observed concentration at the last time point.

Plasma clearance was calculated as dose rate (D) divided by AUC_0-inf. From equation T_1/2The apparent terminal half-life (T1/2) was estimated at ln2/λ z.

detailed description of the preferred embodiments

the invention is based, at least in part, on the following results: the selection of antibodies can be improved by using a combination of FcRn affinity chromatography and heparin affinity chromatography relative/based on the clearance of antibodies in cynomolgus monkey Single Dose Pharmacokinetic (SDPK) studies compared to isolated (1-dimensional) FcRn chromatography or heparin chromatography, respectively. The improvement resides in reducing the number of rejected antibodies despite having acceptable PK properties, etc.

The present invention is based, at least in part, on the following results: the combination of FcRn affinity chromatography and heparin affinity chromatography allows to define FcRn affinity chromatography columns and heparin affinity chromatography column retention time thresholds and thus retention time regions where antibodies with slow clearance (i.e. long systemic circulation half-life) can be found. Thus, this combination allows for improved selection of antibodies with long systemic circulation half-life, improved accuracy of pharmacokinetic predictions and reduced number of rejected antibodies despite having long systemic circulation half-life, etc.

The present invention is based, at least in part, on the following results: when the retention times on the FcRn affinity chromatography column and on the heparin affinity chromatography column are normalized based on the retention times of the respective on-column reference antibodies, a relative retention time region is defined in which the advantage comprises antibodies with slow clearance. This region is defined by a relative retention time on the FcRn affinity chromatography column of less than 1.78 (with anti-Her 3 antibody as reference antibody) and a relative retention time on the heparin affinity chromatography column of less than 0.87 (with anti-pTau antibody as reference antibody).

I. definition of

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as "numbering according to (Kabat)". In particular, the Kabat numbering system (see p 647-.

carter p.; ridgway j.b.b.; presta l.g. immunology, volume 2, phase 1, february 1996, pages 73-73(1) describe the conjugated clasp dimerization module and its use in antibody engineering.

General information on the nucleotide Sequences of human immunoglobulin light and heavy chains is given in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

Useful methods and techniques for practicing the present invention are described, for example, in the following documents: ausubel, F.M, (eds.), Current Protocols in Molecular Biology, Vol.I to III (1997); glover, N.D. and Hames, B.D. eds, DNA Cloning A Practical Approach, Vol.I to II (1985), Oxford university Press; freshney, R.I. (eds.), Animal Cell Culture-a practical proproach, IRL pressure Limited (1986); watson, J.D. et al, Recombinant DNA, Second Edition, CHSL Press (1992); winnacker, e.l., From Genes to Clones; n.y., VCH Publishers (1987); celis, J. eds., Cell Biology, 2 nd edition, Academic Press (1998); freshney, R.I., Culture of animal cells: A Manual of Basic Technique, 2 nd edition, Alan R.Liss, Inc., N.Y. (1987).

The use of recombinant DNA technology enables the production of nucleic acid derivatives. Such derivatives may be modified, for example, at individual or several nucleotide positions by means of substitutions, alterations, exchanges, deletions or insertions. Modification or derivatization can be carried out, for example, by means of site-directed mutagenesis. Such modifications can be readily carried out by those skilled in the art (see, e.g., Sambrook, J. et al, Molecular Cloning: A laboratory Manual (1999) Cold Spring Harbor laboratory Press, New York, USA; Hames, B.D. and Higgins, S.G., Nucleic acid hybridization-aprinacular (1985) IRL Press, Oxford, England).

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a cell, a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

the term "about" refers to a range of +/-20% of the numerical value below. In one embodiment, the term "about" refers to a range of +/-10% of the numerical value below. In one embodiment, the term "about" refers to a range of +/-5% of the numerical value below.

The term "determining" as used herein also encompasses the terms "measuring" and "analyzing".

The term "comprising" also includes the term "consisting of … …".

The term "antibody" is used broadly herein and encompasses a variety of antibody structures, including but not limited to monoclonal full-length antibodies and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies) so long as they have an Fc region.

"multispecific antibody" refers to an antibody having binding specificity for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies may be used as full-length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies) or combinations thereof (e.g., full length antibody plus additional scFv or Fab fragments). Engineered antibodies having two, three, or more (e.g., four) functional antigen binding sites have also been reported (see, e.g., US 2002/0004587a 1).

The term "binding to an antigen" refers to the binding of an antibody in an in vitro assay. In one embodiment, binding is determined in a binding assay in which an antibody binds to a surface and the binding of antigen to the antibody is measured by Surface Plasmon Resonance (SPR). Bonded means, for example, 10^-8M or less, in some embodiments 10^-13To 10^-8M, in some embodiments 10^-13To 10^-9Binding affinity (K) of M_D). The term "binding" also encompasses the term "specific binding".

Binding can be studied by BIAcore assay (GE Healthcare Biosensor AB, uppsala, sweden). Affinity of binding action is termed k_a(association rate constant of antibody from antibody/antigen Complex), k_d(dissociation constant) and K_D(k_d/k_a) And (4) defining.

The term "buffering substance" refers to a substance that, when in solution, can alter the pH level of the solution, for example, as a result of the addition or release of an acidic or basic substance.

The "class" of an antibody refers to the type of constant domain or constant region that is possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁and IgA₂. The constant domains of the heavy chains corresponding to different immunoglobulin classes are called α, δ, ε, γ and μ, respectively.

the term "Fc-fusion polypeptide" refers to a fusion of a binding domain (e.g., an antigen binding domain such as a single chain antibody, or a polypeptide such as a ligand for a receptor) to the Fc region of an antibody.

The term "human Fc region" refers to the C-terminal region of a human immunoglobulin heavy chain that contains at least a portion of the hinge region, CH2 domain, and CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy-terminus of the heavy chain. In one embodiment, the Fc region has the amino acid sequence of SEQ ID NO 05. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. The Fc region consists of two heavy chain Fc region polypeptides that can be covalently linked to each other by hinge region cysteine residues that form interchain disulfide bonds.

The term "FcRn" refers to a human neonatal Fc-receptor. FcRn serves to reuse IgG from lysosomal degradation pathways, resulting in decreased clearance and increased half-life. FcRn is a polypeptide consisting of two polypeptides: a heterodimeric protein consisting of a 50kDa class I major histocompatibility complex-like protein (. alpha. -FcRn) and 15kDa beta 2-microglobulin (. beta.2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH dependent and occurs at 1:2 stoichiometry, i.e., one IgG binds to two FcRn molecules through its two heavy chains (Huber, A.H. et al, J.mol.biol.230(1993) 1077-1083). FcRn binding occurs at acidic pH in endosomes (pH <6.5) and IgG is released on the surface of neutrophils (pH about 7.4). The pH sensitive nature of this interaction promotes FcRn-mediated protection of IgG from intracellular degradation by binding to receptors within the acidic environment of the body. FcRn then facilitates the recycling of IgG to the cell surface and subsequent release into the bloodstream when the FcRn-IgG complex is exposed to the neutral pH environment outside the cell.

The term "FcRn-binding portion of an Fc-region" refers to the portion of an antibody heavy chain polypeptide that extends from about EU position 243 to EU position 261, and from about EU position 275 to EU position 293, and from about EU position 302 to EU position 319, and from about EU position 336 to EU position 348, and from about EU position 367 to EU positions 393 and 408, and from about EU position 424 to EU position 440. In one embodiment, one or more of the following amino acid residues, numbered according to the EU of Kabat, are altered: f243, P244, P245, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, a287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, a339, K340, G341, Q342, P343, R344, E345, P346, Q347, V385, C367, V367, F372, Y375, P375, S375, N374, N187, N18, N187, N293, V427, N293, V336, V703, N336, N440, N336, Q342, P343, P344, N440, N375, N440, N187, N440, P18, P376, P18, N375, N187, P374, N187, P374, N187.

The term "full-length antibody" refers to an antibody having a structure substantially similar to a native antibody structure. A full-length antibody comprises two full-length antibody light chains comprising a light chain variable domain and a light chain constant domain and two full-length antibody heavy chains comprising a heavy chain variable domain, a first constant domain, a hinge region, a second constant domain, and a third constant domain. The full-length antibody may comprise other domains, such as additional scfvs or scfabs conjugated to one or more chains of the full-length antibody. These conjugates are also encompassed by the term full length antibody.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may instead contain mutations. Included herein are mutant progeny that have the same function or biological activity as the progeny screened or selected for in the originally transformed cell.

The term "derived from" refers to an amino acid sequence that is derived from a parent amino acid sequence by introducing an alteration at least one position. Thus, the derived amino acid sequence differs from the corresponding parent amino acid sequence at least one corresponding position (Kabat EU index according to the Fc region of the antibody). In one embodiment, the amino acid sequences derived from the parent amino acid sequence differ in the corresponding position by 1 to 15 amino acid residues. In one embodiment, the amino acid sequences derived from the parent amino acid sequence differ in the corresponding position by 1 to 10 amino acid residues. In one embodiment, the amino acid sequences derived from the parent amino acid sequence differ in the corresponding position by 1 to 6 amino acid residues. Likewise, the derived amino acid sequence has high amino acid sequence identity to its parent amino acid sequence. In one embodiment, the amino acid sequences derived from a parent amino acid sequence have 80% or greater amino acid sequence identity. In one embodiment, the amino acid sequences derived from a parent amino acid sequence have 90% or greater amino acid sequence identity. In one embodiment, the amino acid sequences derived from a parent amino acid sequence have 95% or greater amino acid sequence identity.

The term "human Fc region polypeptide" refers to the same amino acid sequence as a "native" or "wild-type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to an amino acid sequence derived from a "native" or "wild-type" Fc region polypeptide by at least one "amino acid change". The "human Fc region" consists of two human Fc region polypeptides. A "variant (human) Fc region" consists of two Fc region polypeptides, thus both may be variant (human) Fc region polypeptides or one is a human Fc region polypeptide and the other is a variant (human) Fc region polypeptide.

In one embodiment, the human Fc region polypeptide has the amino acid sequence of the human IgG1Fc region polypeptide of SEQ ID NO. 05, or the amino acid sequence of the human IgG 2Fc region polypeptide of SEQ ID NO. 06, or the amino acid sequence of the human IgG 3Fc region polypeptide of SEQ ID NO. 07, or the amino acid sequence of the human IgG4 Fc region polypeptide of SEQ ID NO. 08. In one embodiment, the Fc region polypeptide is derived from the Fc region polypeptide of SEQ ID NO 05, or 06, or 07, or 08 and has at least one amino acid mutation compared to the Fc region polypeptide of SEQ ID NO 05, or 06, or 07, or 08. In one embodiment, the Fc region polypeptide comprises/has from about 1 to about 10 amino acid mutations, and in one embodiment about 1 to about 5 amino acid mutations. In one embodiment, the Fc region polypeptide has at least about 80% homology to the human Fc region polypeptide of SEQ ID NO 05, or 06, or 07, or 08. In one embodiment, the Fc region polypeptide has at least about 90% homology to the human Fc region polypeptide of SEQ ID NO 05, or 06, or 07, or 08. In one embodiment, the Fc region polypeptide has at least about 95% homology to the human Fc region polypeptide of SEQ ID NO 05, or 06, or 07, or 08.

the Fc region polypeptides derived from the human Fc region polypeptides of SEQ ID NOs 05, or 06, or 07, or 08 are further defined by the amino acid changes contained. Thus, for example, the term P329G refers to an Fc region polypeptide derived from a human Fc region polypeptide having a proline to glycine mutation at amino acid position 329 relative to the human Fc region polypeptide of SEQ ID NO:05, or 06, or 07, or 08.

the human IgG1Fc region polypeptide has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:05)。

the Fc region polypeptide derived from the Fc region of human IgG1 with mutations L234A, L235A has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:09)。

the human IgG1Fc region-derived Fc region polypeptide having Y349C, T366S, L368A and Y407V mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)。

the human IgG1Fc region-derived Fc region polypeptide having the S354C, T366W mutations has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

The Fc region polypeptide derived from the Fc region of human IgG1 with mutations L234A, L235A and Y349C, T366S, L368A, Y407V has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12)。

The human IgG1Fc region-derived Fc region polypeptide having L234A, L235A and S354C, T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:13)。

The human IgG1Fc region-derived Fc region polypeptide having the P329G mutation has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:14)。

the Fc region polypeptide derived from the Fc region of human IgG1 with the L234A, L235A and P329G mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:15)。

The Fc region polypeptide derived from the Fc region of human IgG1 with the P329G mutation and the Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:16)。

The Fc region polypeptide derived from the Fc region of human IgG1 having the P329G mutation and the S354C and T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:17)。

human IgG1Fc region-derived Fc region polypeptides having L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations have the following amino acid sequences:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:18)。

The human IgG1Fc region-derived Fc region polypeptide having L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19)。

The human IgG4 Fc region polypeptide has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:08)。

The human IgG4 Fc region-derived Fc region polypeptide having S228P and L235E mutations has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20).

The Fc region polypeptide derived from the Fc region of human IgG4 with the S228P, L235E and P329G mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:21)。

the human IgG4 Fc region-derived Fc region polypeptide having the S354C, T366W mutations has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 22).

the human IgG4 Fc region-derived Fc region polypeptide having Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:23)。

The human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E and S354C, T366W mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:24)。

the Fc region polypeptides derived from the Fc region of human IgG4 with the mutations S228P, L235E and Y349C, T366S, L368A, Y407V have the following amino acid sequences:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:25)。

the human IgG4 Fc region-derived Fc region polypeptide having the P329G mutation has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:26)。

The Fc region polypeptide derived from the Fc region of human IgG4 with mutations P329G and Y349C, T366S, L368A, Y407V has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:27)。

The Fc region polypeptide derived from the Fc region of human IgG4 with mutations P329G and S354C, T366W has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:28)。

The human IgG4 Fc region-derived Fc region polypeptides with S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations have the following amino acid sequences:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:29)。

the human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:30)。

a "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least 1, and typically 2, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FR regions correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of an antibody, e.g., a non-human antibody, refer to an antibody that has undergone humanization.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis), or chromatography (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, s. et al, j.chrom.b 848(2007) 79-87.

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation), which are typically present in minute amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for producing monoclonal antibodies being described herein.

"native antibody" refers to a naturally occurring immunoglobulin molecule having an indeterminate structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has one variable region (VH), also known as variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also known as a variable light domain or light chain variable domain, followed by a constant light Chain (CL) domain. The light chains of antibodies can be divided into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

The term "pharmaceutical formulation" refers to a preparation in such a form as to allow the biological activity of the active ingredient contained therein to be effective, and not containing additional components that are unacceptably toxic to a subject to whom the formulation will be administered.

"pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical formulation that is not toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "plasmid" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes plasmids which are self-replicating nucleic acid structures as well as plasmids which are incorporated into the genome of a host cell into which the vector has been introduced. Certain plasmids are capable of directing the expression of nucleic acids to which they are operably linked. Such plasmids are referred to herein as "expression plasmids".

the term "positive linear pH gradient" refers to a pH gradient that begins at a low (i.e., more acidic) pH value and ends at a higher (i.e., less acidic, neutral, or basic) pH value. In one embodiment, a positive linear pH gradient begins at a pH of about 5.5 and ends at a pH of about 8.8.

The term "recombinant antibody" as used herein refers to all antibodies (chimeric, humanized and human) prepared, expressed, produced or isolated by recombinant means. This includes antibodies isolated from host cells such as NS0, HEK, BHK or CHO cells or from animals that are transgenic for human immunoglobulin genes (e.g., mice) or antibodies expressed using recombinant expression plasmids transfected into host cells. Such recombinant antibodies have rearranged forms of variable and constant regions. Recombinant antibodies as reported herein can undergo somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

"solid phase" refers to a non-fluid substance and includes particles (including microparticles and beads) made from a variety of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glasses, and ceramics; gel substances such as silica, alumina and polymer gels; capillaries that may be constructed of polymers, metals, glass, and/or ceramics; zeolites and other porous materials; an electrode; a microtiter plate; a solid state strip; and cuvettes, tubes or other spectrometer sample containers. The solid phase component of the assay is distinguished from an inert solid surface in that the "solid support" contains on its surface at least one moiety intended to chemically interact with a molecule. The solid phase may be a fixed component, such as a chip, tube, strip, cuvette or microtiter plate, or may be a non-fixed component, such as beads and microparticles. Microparticles may also be used as solid supports in homogeneous assay formats. A variety of microparticles may be used that allow non-covalent or covalent attachment to proteins and other substances. Such particles include polymeric particles such as polystyrene and poly (methyl methacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as quartz glass, and metal oxide particles. See, e.g., Martin, C.R. et al, Analytical Chemistry-News & Features, May 1(1998)322A-327A, which is incorporated herein by reference. In one embodiment, the solid support is an agarose gel.

As used herein, the term "valency" refers to the presence of a specified number of binding sites in an (antibody) molecule. As such, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of 2 binding sites, 4 binding sites and 6 binding sites, respectively, in the (antibody) molecule. In a preferred embodiment, the bispecific antibody as reported herein is "bivalent".

the term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. The variable domains of antibody heavy and light chains (VH and VL, respectively) typically have similar structures, each domain comprising four Framework Regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, t.j. et al, Kuby Immunology, 6 th edition, w.h.freeman and co., n.y. (2007), page 91). A single VH domain or VL domain may be sufficient to confer antigen binding specificity. Alternatively, antibodies that bind a particular antigen can be isolated using VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al, J.Immunol.150(1993)880- "887; clackson, T.et al, Nature 352(1991) 624-.

The terms "variant", "modified antibody" and "modified fusion polypeptide" refer to a molecule having an amino acid sequence that differs from the amino acid sequence of a parent molecule. Typically, such molecules have one or more alterations, insertions, or deletions. In one embodiment, the modified antibody or modified fusion polypeptide comprises an amino acid sequence comprising at least a portion of a non-naturally occurring Fc region. Such molecules have less than 100% identity to the parent antibody or parent fusion polypeptide sequence. In one embodiment, the variant antibody or variant fusion polypeptide has an amino acid sequence having from about 75% to less than 100%, particularly from about 80% to less than 100%, particularly from about 85% to less than 100%, particularly from about 90% to less than 100%, and particularly from about 95% to less than 100% amino acid sequence identity to the amino acid sequence of the parent antibody or parent fusion polypeptide. In one embodiment, the parent antibody or parent fusion polypeptide differs from the variant antibody or variant fusion polypeptide by one (single), two or three amino acid residues.

methods as reported herein

The clearance rate of antibodies with the same Fc region covers a wide range. There is the effect of the corresponding Fv region, i.e., the biophysical property distinguishes rapidly clearing antibodies from slowly clearing antibodies.

Without being bound by this theory, it is hypothesized that direct, charge-mediated Fv and FcRn interactions impair the release of antibody from FcRn at pH 7.4.

It has now been found that a combination of swallow and FcRn binding assessment can be used to predict/more accurately assess the pharmacokinetics of antibodies. This has been achieved by a combination of FcRn affinity chromatography (pH gradient elution) and heparin affinity chromatography (salt gradient elution).

Separation of intravenous immunoglobulin (IVIG, IgG polyclonal mixture isolated from pooled human donor serum (>1000 donors) on a heparin affinity chromatography column) yielded several broad peaks covering a broad retention time range. On the FcRn column, the same material showed a narrow elution peak, suggesting similar binding affinity of FcRn for the antibody contained therein (see figure 4).

Heparin is the main component of highly negatively charged glycosaminoglycans (polysaccharides) and sugar cups (glycocalix) that cover endothelial cells.

Fractions with the highest (figure 4: 2) and lowest (figure 4: 1) heparin retention times were tested in FcRn wild-type mice and FcRn knockout mice. Observed clearance rates are shown in the following table:

Clearance rate [ mL/day/kg]	FcRn-wild type mice	FcRn-knockout mice
			IVIG heparin fraction 1	4	32
IVIG heparin fraction 2	5	76

The clearance rate of the hepatinin conjugate is higher under two environments, which indicates that the swallowing rate is higher.

In a mouse model expressing murine FcRn with significantly higher affinity for huIgG than endogenous mIgG, FcRn recycling leverages pharmacokinetics (without being bound by this theory, competition of endogenous mIgG with injected huIgG is strongly diminished compared to the scenario in patients). In FcRn knockout mice, FcRn recycling does not contribute to pharmacokinetics and each swallowing event leads to degradation of the ingested IgG. Thus, the clearance rate thus determined should be directly proportional to the swallowing rate of the injected IgG sample, resulting in a significantly more pronounced clearance rate. These data are well correlated with the predicted value of heparin columns, thus increasing evidence of the effectiveness of heparin columns as predictors of antibody pharmacokinetics by swallowing.

The following 35 antibodies with different patterns and different specificities have been generated and analyzed with the method as reported in the current example:

a bivalent, monospecific IgG1 antibody having a wild-type-Fc region is an antibody comprising two antibody light chains (each comprising a light chain variable domain and a light chain constant domain) and two antibody heavy chains (each comprising a heavy chain variable domain, a hinge region and a heavy chain constant domain CH1, CH2 and CH3), whereby the Fc region is a human IgG1Fc region, whereby the Fc region C-terminal amino acid residues K or GK may be present or absent independently of each other in the two antibody heavy chains. In one embodiment, the human IgG1Fc region has the amino acid sequence of SEQ ID NO. 05.

A bivalent, bispecific IgG1 antibody having a KiH-Fc region is an antibody comprising two antibody light chains (each comprising a light chain variable domain and a light chain constant domain) and two antibody heavy chains (each comprising a heavy chain variable domain, a hinge region and a heavy chain constant domain CH1, CH2 and CH3), whereby the Fc region is a human IgG1Fc region, whereby the Fc region C-terminal amino acid residues K or GK may be present or absent independently of each other in the two antibody heavy chains, whereby one of the heavy chains comprises a buckle mutation and the corresponding other heavy chain comprises a knot mutation. In one embodiment, the heavy chain Fc region has the amino acid sequences of SEQ ID NO 09 and 10, respectively.

The CH3 domain in the heavy chain Fc-region of bivalent bispecific antibodies can be altered by the "knob-in-hole" technique, which is described in detail in several examples, e.g.WO 96/027011, Ridgway J.B. et al, Protein Eng.9(1996) 617. 621 and Merchant, A.M. et al, nat. Biotechnol.16(1998) 677. 681. In this approach, the interaction surface of the two CH3 domains is altered to increase heterodimerization of the two heavy chains containing the two CH3 domains. One of the two CH3 domains (of both heavy chains) may be a "knot" and the other a "knot". Introduction of disulfide bonds further stabilized the heterodimer (Merchant, A.M et al, Nature Biotech.16(1998) 677-681; Atwell, S. et al, J.mol.biol.270(1997)26-35) and increased yield.

The mutation T366W in the CH3 domain of the antibody heavy chain is referred to as a "knot mutation" and the mutations T366S, L368A, Y407V in the CH3 domain of the antibody heavy chain are referred to as "knot mutations" (numbering according to the Kabat EU index). Additional interchain disulfide bonds between the CH3 domains may also be exploited, for example, by Merchant, A.M et al, Nature biotech.16(1998) 677-: the Y349C mutation was introduced into the CH3 domain of the heavy chain with the "knot mutation" and the E356C mutation or S354C mutation was introduced into the CH3 domain of the heavy chain with the "knot mutation", or vice versa.

a bivalent, monospecific IgG1 antibody cytokine fusion with a KiH lalagg-Fc region is an antibody comprising two antibody light chains (each comprising a light chain variable domain and a light chain constant domain) and two antibody heavy chains (each comprising a heavy chain variable domain, a hinge region and a heavy chain constant domain CH1, CH2 and CH3), whereby the Fc region is a human IgG1Fc region, whereby the Fc region C-terminal amino acid residues K or GK may be present or absent independently of each other in the two antibody heavy chains, whereby one of the heavy chains comprises a buckle mutation and the corresponding other heavy chain comprises a knot mutation, whereby the two heavy chains further comprise the amino acid mutations L234A, L235A and P329G. In one embodiment, the heavy chain Fc region has the amino acid sequences of SEQ ID NOs 18 and 19, respectively.

bivalent, bispecific CrossMab with a KiH-Fc region are antibodies comprising

a) A first light chain and a first heavy chain of an antibody specifically binding to a first antigen, and a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, or

b) A first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other,

The Fc region is thus a human IgG1Fc region, and thus the C-terminal amino acid residues K or GK of the Fc region may or may not be present independently of each other in the two antibody heavy chains, whereby one of the heavy chains comprises a buckle mutation and the corresponding other heavy chain comprises a knot mutation. In one embodiment, the heavy chain Fc region has the amino acid sequences of SEQ ID NO 09 and 10, respectively.

2:1 heterodimeric T cell bispecific antibodies with KiH-LALALAPG-Fc region are antibodies comprising

a) a first Fab fragment that specifically binds to a first antigen;

b) A second Fab fragment which specifically binds to a second antigen and in which the variable domains VL and VH or constant domains CL and CH1 of the Fab light and Fab heavy chains are replaced with each other;

c) A third Fab fragment that specifically binds to the first antigen; and

d) An Fc region consisting of first and second heavy chain Fc regions;

Wherein

(iii) The first Fab molecule under a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule under b), and the second Fab molecule under b) and the third Fab molecule under C) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the heavy chain Fc regions under d),

the Fc region is thus a human IgG1Fc region, and thus the C-terminal amino acid residues K or GK of the Fc region may or may not be present independently of each other in the two antibody heavy chain Fc regions, whereby one of the heavy chain Fc regions comprises a buckle mutation and the respective other heavy chain Fc region comprises a knot mutation, whereby both heavy chain Fc regions further comprise the amino acid mutations L234A, L235A and P329G. In one embodiment, the heavy chain Fc region has the amino acid sequences of SEQ ID NOs 18 and 19, respectively.

A trivalent, bispecific IgG1-Fab fusion with a KiH-LALALAPG-Fc region comprising

a) Two light chains and two heavy chains of an antibody that specifically binds to a first antigen (and comprises two Fab fragments),

b) An additional Fab fragment of the antibody that specifically binds to the second antigen, wherein the additional Fab fragment is fused to the C-terminus of one of the heavy chains of a) via a peptide linker,

wherein in the additional Fab fragment the variable domains VL and VH are replaced by each other, and/or the constant domains CL and CH1 are replaced by each other,

The Fc region is thus a human IgG1Fc region, and thus the C-terminal amino acid residues K or GK of the Fc region may or may not be present independently of each other in the two antibody heavy chains, whereby one of the heavy chains comprises the buckle mutation and the corresponding other heavy chain comprises the knot mutation, whereby both heavy chains further comprise the amino acid mutations L234A, L235A and P329G. In one embodiment, the heavy chain Fc region has the amino acid sequences of SEQ ID NOs 18 and 19, respectively.

An Fc region-cytokine fusion with a lalapc-Fc region is an antibody Fc region fusion comprising two antibody heavy chain Fc region fragments (each comprising at least a fragment of the hinge region and heavy chain constant domains CH1, CH2 and CH3), whereby the Fc region is a human IgG1Fc region, whereby the C-terminal amino acid residues K or GK of the Fc region may be present or absent independently of each other in the two antibody heavy chain Fc region fragments, whereby the two antibody heavy chain Fc region fragments comprise the amino acid mutations L234A, L235A and P329G. In one embodiment, the heavy chain Fc region has the amino acid sequence of SEQ ID NO 15.

To smooth out diurnal, interhuman and inter-laboratory variations, retention times have been normalized to the reference antibody on each affinity column.

for the heparin affinity chromatography column, an anti-pTau antibody having the heavy chain of SEQ ID NO:01 and the light chain of SEQ ID NO:02 was selected. This antibody showed relatively long retention times on heparin affinity chromatography columns, resulting in robust relative retention time calculations.

For the FcRn affinity chromatography column, a preparation was selected comprising oxidized variants of the anti-Her 3 antibody having the heavy chain of SEQ ID NO 03 and the light chain of SEQ ID NO 04. This antibody was selected because it had a comparable AUC profile to the antibody used in Stracke, j. et al (mAbs 6(2014) 1229-1242). The retention times of 35 antibodies on FcRn and heparin affinity chromatography columns have been determined. In addition, single dose pharmacokinetics were determined in cynomolgus monkeys. The results (same antibody sequences as previously described) are shown in the table below.

The clearance rates are ranked as follows:

And (3) fast: 12 mL/kg/day;

Boundary value: 8-12 mL/kg/day;

Acceptable: x is more than or equal to 2.5 and less than 8 mL/kg/day;

Very well: <2.5 mL/kg/day.

The following table shows that the antibodies in brackets have been eliminated based on the isolated, isolated results of the two affinity chromatographs; based on pharmacokinetic determinations, the antibodies in parentheses in the column "clearance" were eliminated.

In the table below, the results are given based on the relative retention times on the respective columns. Rejected antibodies are shown in parentheses.

The correlation between retention time and pharmacokinetic behavior is shown in figure 2. It can be seen that there is a hitherto unknown correlation between the relative retention times and the pharmacokinetic behavior on the two affinity chromatography columns. It has been found that a region comprising an antibody with a slow clearance is predominantly bounded by a relative retention time on an FcRn affinity chromatography column of less than 1.78 (with anti-Her 3 antibody as reference antibody) and a relative retention time on a heparin affinity chromatography column of less than 0.87 (with anti-pTau antibody as reference antibody). The respective correlations with the plotted thresholds are shown in fig. 3.

In the following table, antibodies eliminated (in parentheses) by four methods are shown, respectively: based on isolated heparin affinity chromatography (column 1), on isolated FcRn affinity chromatography (column 2), on SDPK clearance (column 3) and on the relative retention times on two orthogonal affinity columns in the inventive method as reported herein.

Thus, with the method of the present invention, improvement in the selection of antibodies with long systemic circulation half-life, improvement in the accuracy of pharmacokinetic prediction, and reduction in the number of rejected antibodies despite having long systemic circulation half-life can be achieved.

Only about 50% of the antibodies are correctly predicted when using baculovirus-based assays known from the prior art.

Thus, the methods of the invention can be used to identify antibodies, including Fc region fusion polypeptides, with good pharmacokinetic properties without the need for animal pharmacokinetic studies.

accordingly, the invention includes a method of selecting an antibody (in cynomolgus monkeys) having systemic clearance suitable for use (in humans) as a therapeutic agent, comprising the steps of:

a) Optionally providing a sample comprising the antibody,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient, and

c)

i) If the relative retention time of the antibody on the FcRn affinity chromatography column is less than a first threshold relative to the retention time of a first reference antibody on the FcRn affinity chromatography column, and,

ii) if the ratio of the retention time of the antibody on the heparin affinity chromatography column to the retention time of a second reference antibody on the heparin affinity chromatography column is less than a second threshold value,

Antibodies were selected.

The invention also comprises a method of selecting an antibody (specifically) binding to at least one antigen (in cynomolgus monkeys) having systemic clearance suitable for use (in humans) as a therapeutic agent, comprising the steps of:

a) Providing the antibodies in a different pattern,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using each different antibody pattern of a), and

c) selecting an antibody pattern having

i) A relative retention time on the FcRn affinity chromatography column that is less than a first threshold relative to the retention time of the first reference antibody on the FcRn affinity chromatography column, and

ii) a ratio of the retention time on the heparin affinity chromatography column to the retention time of a second reference antibody on the heparin affinity chromatography column that is less than a second threshold.

the invention also comprises a method of selecting an antibody (specifically) binding to at least one antigen (in cynomolgus monkeys) having systemic clearance suitable for use (in humans) as a therapeutic agent, comprising the steps of:

a) Providing at least two antibodies that bind to at least one antigen, said antibodies

i) Have different CDR sequences, or

ii) have the same CDR sequences and different variable domain sequences, or

iii) have the same CDR sequences in different patterns,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient with each different antibody of a), and

c) Selecting an antibody having

i) The relative retention time on the FcRn affinity chromatography column relative to the retention time of the first reference antibody on the FcRn affinity chromatography column is less than a first threshold, and

ii) the ratio of the retention time on the heparin affinity chromatography column to the retention time of the second reference antibody on the heparin affinity chromatography column is less than a second threshold.

in one embodiment, the first reference antibody is an oxidized antibody preparation. In one embodiment, the oxidized antibody preparation is a preparation comprising an antibody in a non-oxidized form, in a mono-oxidized form (only one of the two methionines at position 252 is oxidized), and in a di-oxidized form (both methionine at position 252 are oxidized) with respect to the methionine residue at position 252 in the heavy chain CH2 domain (numbering according to Kabat). In one embodiment, the relative retention time is calculated based on the following equation

Wherein t is_rel，iRelative retention time of the antibody; t is t_iRetention time of antibody. In one embodiment, the first reference antibody is an anti-Her 3 antibody having a heavy chain with the amino acid sequence of SEQ ID NO. 03 and a light chain with the amino acid sequence of SEQ ID NO. 04. In one embodiment, the first threshold is 2. In one embodiment, the first threshold is 1.8. In one embodiment, the first threshold is 1.78.

in one embodiment, the second reference antibody is an anti-pTau antibody having a heavy chain having the amino acid sequence of SEQ ID NO. 01 and a light chain having the amino acid sequence of SEQ ID NO. 02. In one embodiment, the second threshold is 1. In one embodiment, the second threshold is 0.8. In one embodiment, the second threshold is 0.78.

in one embodiment, the systemic clearance rate in cynomolgus monkeys suitable for therapeutic use (i.e., the antibody may be used as a therapeutic agent) is 8 mL/kg/day or less. In one embodiment, the systemic clearance is less than 8 mL/kg/day. In one embodiment, the systemic clearance is less than 6 mL/kg/day.

in one embodiment, the method for selecting an antibody having a systemic clearance in cynomolgus monkeys of less than 8 mL/kg/day comprises the steps of:

a) optionally providing a sample comprising the antibody,

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient, and

c)

i) If the relative retention time on the FcRn affinity chromatography column is less than 1.78 times the difference in retention time between peak 2 and peak 3 of the oxidized anti-Her 3 antibody preparations of SEQ ID NO:03 and 04, and

ii) if the relative retention time on the heparin affinity chromatography column is less than 0.87 times the retention time of the anti-pTau antibodies of SEQ ID NO:01 and 02,

Antibodies were selected.

In one embodiment, the relative retention time in step c) i) is calculated according to the following equation:

Based on the peak definition (t) according to FIG. 1_rel，i: relative retention time of peak i; t is t_i: retention time peak of (a); t is t_{Peak 2}: retention time of peak 2 of partially oxidized anti Her3 antibody according to figure 1; t is t_{peak 3}: retention time of peak 3 of anti Her3 antibody according to figure 1).

in one embodiment, the relative retention time in step c) ii) is calculated according to the following formula:

(t_rel，i: relative retention time of peak i; t is t_i: retention time of peak i; t is t_pTau: retention time of anti-pTau antibody peak).

In one embodiment, an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is used as an affinity chromatography ligand in FcRn affinity chromatography with a positive linear pH gradient,

wherein a non-covalent complex of a neonatal Fc receptor and beta-2-microglobulin is bound to a chromatographic material and the non-covalent complex is conjugated to a solid phase via a specific binding pair,

Wherein the pH gradient is from a first pH to a second pH, wherein the first pH is pH 3.5 to pH 6.4 and the second pH is pH7.4 to pH 9.5, and

Wherein the non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is monobiotinylated and the solid phase is derivatized with streptavidin.

In one embodiment, the pH gradient is from a first pH value to a second pH value, whereby the first pH value is pH 5.5 and the second pH value is pH 8.8.

In one embodiment, the antibody binds to two antigens.

In one embodiment, the method further comprises the steps of:

d) if none of the provided antibodies or antibody patterns meet the criteria of step c), providing at least one other antibody pattern or antibody and repeating steps b) and c).

In one embodiment, FcRn affinity chromatography is performed with an FcRn affinity chromatography column comprising a streptavidin sepharose conjugated to FcRn β -2-microglobulin complex (3mg complex/1 ml sepharose) and having a length of about 50mm and an inner diameter of about 4.6 mm. In one embodiment, FcRn affinity chromatography is performed as follows: i) mu.g of the sample was applied to an FcRn affinity column equilibrated with 20mM MES buffer, supplemented with 140mM NaCl, adjusted to pH 5.5; ii) washing the column with a buffer comprising (v/v) 80% 20mM MES buffer supplemented with 140mM NaCl, adjusted to pH 5.5 and 20% 20mM Tris/HCl adjusted to pH 8.8, at a flow rate of 0.5 mL/min for 10 min; iii) elution with such a linear gradient from the buffer of step ii) to a buffer comprising (v/v) 30% 20mM MES buffer supplemented with 140mM NaCl, adjusted to pH 5.5, and 70% 20mM Tris/HCl, adjusted to pH 8.8, at a flow rate of 0.5 mL/min and measuring the retention time.

In one embodiment, an oxidized anti-Her 3 antibody preparation is obtained by incubating an anti-Her 3 antibody with a 0.02% hydrogen peroxide solution for 18 hours at room temperature.

In one embodiment, heparin affinity chromatography is performed with a heparin affinity chromatography column comprising heparin conjugated with sulfated glycosaminoglycans on hydroxylated methacrylic acid polymers and having a length of about 50mm and an inner diameter of about 5 mm. In one embodiment, heparin affinity chromatography is performed as follows: i) applying 20 to 50 μ g protein sample in low salt buffer (≦ 25mM ionic strength) to a heparin affinity chromatography column pre-equilibrated at room temperature with 50mM TRIS buffer adjusted to pH 7.4; ii) elution with a linear gradient of 0-100% 50mM Tris buffer supplemented with 1000mM NaCl and adjusted to pH7.4 over 32 minutes at a flow rate of 0.8 mg/mL.

in one embodiment, the beta-2-microglobulin is from the same species as FcRn.

An FcRn affinity chromatography column as used herein comprises a matrix and a matrix-bound chromatography functionality, wherein the matrix-bound chromatography functionality comprises a non-covalent complex of a neonatal Fc receptor (FcRn) and β -2-microglobulin.

In one embodiment herein, the FcRn is selected from the group consisting of human FcRn, cynomolgus monkey FcRn, mouse FcRn, rat FcRn, sheep FcRn, canine FcRn, pig FcRn, mini-pig FcRn, and rabbit FcRn.

In one embodiment, the beta-2-microglobulin is from the same species as FcRn.

in one embodiment, the beta-2-microglobulin is from a different species than FcRn.

In one embodiment, the antibody is selected from the group consisting of a full length antibody, a CrossMab, a 2:1 heterodimeric T cell bispecific antibody, an antibody-cytokine fusion polypeptide, an Fc region-cytokine fusion polypeptide, and an antibody-Fab fusion polypeptide.

In one embodiment, the antibody comprises an Fc region selected from the group consisting of a human IgG1Fc region, a human IgG1Fc region having mutations L234A, L235A, and P329G, a human IgG1Fc region having a binding clip mutation, and combinations thereof.

in one embodiment, the antibody format is selected from the group consisting of full length antibody, CrossMab, 2:1 heterodimeric T cell bispecific antibody and any of the foregoing fused to one, two or three additional Fab, scFv, scFab, CrossFab molecules, either directly or via a peptide linker.

In one embodiment, the antibody pattern comprises an Fc region selected from the group consisting of a human IgG1Fc region, a human IgG1Fc region having mutations L234A, L235A, and P329G, a human IgG1Fc region having a binding clip mutation, and combinations thereof.

In one embodiment, the antibody is a monoclonal antibody.

In one embodiment, the antibody is a bispecific antibody.

in one embodiment, the antibody is a chimeric antibody.

Typically, the soluble extracellular domain of FcRn (SEQ ID NO:31 for human FcRn) with a C-terminal His-Avi Tag (SEQ ID NO:32) is co-expressed with β 2-microglobulin (SEQ ID NO:33 for human β -2-microglobulin) in mammalian cells. The non-covalent FcRn-microglobulin complex was biotinylated and loaded on streptavidin-derivatized agarose gel.

In principle, any buffer substance may be used in the method as reported herein.

to further elucidate the charge-to-heparin/FcRn-binding and thus pharmacokinetics, variants of antibody No. 5 were synthesized that cover the biophysical space of charge and hydrophobicity normally seen in antibody Fv.

variants carrying positive charge patches show strong heparin and FcRn pillars retention (relative retention of FcRn pillars 0.5 and greater, and relative retention on heparin pillars 0.8 or greater) predictive of rapid clearance.

Variants carrying negative charge blocks (negatively charged patch) showed weak heparin and FcRn pillars retention predicted for slow clearance (relative retention of FcRn pillars 0.25 and less and relative retention on heparin pillars 0.6 or less).

when combining negative and positive charge blocks, the resulting antibody variant behaves as if it carries only positive charge blocks.

Clearance was determined in FcRn knockout mice for wild type antibody No. 5 and its four variants (fig. 5 and 6). In FcRn knockout mice, all three tested variants carrying a positive charge block showed very high clearance compared to wild-type antibody No. 5, correlating well with column retention. In this mouse model, variants carrying negatively charged blocks showed a significant decrease in clearance, also correlated well with column retention.

To determine the effect of "irregularity" on clearance (i.e., the effect of the concentration of surface charges forming the charge patch as opposed to a uniform charge distribution), five variants of antibody No. 5 were generated in which the total number of positive and negative charges remained constant while the charge distribution on the Fab surface changed from "uniform" incrementally to "irregular".

Although, positive and negative charge masses were produced, heparin column retention increased with increasing irregularity. This suggests that the influence of positive charge blocks is greater or even dominant compared to negative charge blocks, as already seen above. The calculated pI of these variants was hardly changed, while the clearance of the most irregular variants in FcRn knockout mice was significantly higher than those with the most uniform charge distribution (almost doubled; fig. 7). These data suggest, without being bound by this theory, that the effect of "irregularities" or thus positive charge blocks determines clearance compared to the pI which is a fairly broad and therefore poor predictor of pharmacokinetics.

To determine the effect of replacing the permanently positively charged amino acids lysine and arginine with pH-dependent charged histidines, a variant of antibody No. 5 was generated in which all of the HC Fv positively charged amino acid residues were replaced with histidines (7 changes total). This variant was compared to the wild type antibody No. 5, based on the relative retention times of the heparin and FcRn columns and the pharmacokinetics in vivo.

Without being bound by this theory, it is assumed that at neutral pH in serum, the charge of histidine is mostly neutral, thus reducing binding to negatively charged cups and subsequently reducing the rate of swallowing. Inside the acidic endosome, histidine carries the most positive charge and thus contributes to binding to FcRn by virtue of Fv-FcRn affinity. While recycling, histidine-mutated IgG is brought to the cell surface where good pharmacokinetics requires efficient dissociation from FcRn. Histidine, now exposed to the neutral pH of serum, becomes deprotonated and thus impairs avidity interactions with FcRn, allowing improved release to serum.

It was found that histidine mutants showed reduced heparin retention, whereas FcRn retention remained largely unchanged (fig. 8). Without being bound by this theory, this reflects pH dependence.

In vivo, clearance of histidine mutants was significantly reduced compared to wild-type antibody in FcRn knockout mice (fig. 9 and clearance 10).

The invention also comprises a method for selecting a variant antibody of a parent antibody that (specifically) binds to the same antigen as the parent antibody, has a systemic clearance rate that is different from the systemic clearance rate of the parent antibody and is suitable (in humans) as a therapeutic agent based on its pharmacokinetic properties, comprising the steps of:

a) Providing at least one variant antibody of a parent antibody, wherein the charge distribution in the Fv fragment has been altered by

i) At least one (permanently) negatively charged or uncharged amino acid residue is changed into a (permanently) positively charged amino acid residue, or

ii) at least one (permanently) positively charged or uncharged amino acid residue is changed to a (permanently) negatively charged amino acid residue, or

iii) changing at least one (permanently) charged amino acid residue into an amino acid residue with an opposite charge, or

iv) changing at least one permanently charged amino acid residue into a pH-dependent charged amino acid residue, or

v) a combination of i) to iv),

b) Selecting a variant antibody that has a systemic clearance rate that is different from the systemic clearance rate of the parent antibody and is suitable (in humans) as a therapeutic agent.

In one embodiment, the method comprises the additional steps of:

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using a parent antibody and at least one variant antibody, and

c) selecting a variant antibody having

i) Relative retention time on an FcRn affinity chromatography column that is less than the retention time of the parent antibody on the (same) FcRn affinity chromatography column (under the same elution conditions), or

i) Relative retention time on heparin affinity chromatography column that is less than the retention time of the parent antibody on (same) heparin affinity chromatography column under (same elution conditions), or

iii) both i) and ii).

In one embodiment, the method comprises the additional steps of:

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using a parent antibody and at least one variant antibody, and

c) Selecting a variant antibody having

i) A relative retention time on the FcRn affinity chromatography column that is greater than the retention time of the parent antibody on the (same) FcRn affinity chromatography column (under the same elution conditions), or

i) Relative retention time on heparin affinity chromatography column that is greater than the retention time of the parent antibody on (same) heparin affinity chromatography column under (same elution conditions), or

iii) both i) and ii).

In one embodiment, the method comprises the additional steps of:

b) FcRn affinity chromatography with a positive linear pH gradient and heparin affinity chromatography with a positive linear conductivity/salt gradient using a parent antibody and at least one variant antibody, and

c) selecting a variant antibody having

i) relative retention time on an FcRn affinity chromatography column that is less than or greater than the retention time of a parent antibody on an (same) FcRn affinity chromatography column (under the same elution conditions), or

i) Relative retention time on heparin affinity chromatography column that is less than or greater than (under the same elution conditions) the retention time of the parent antibody on heparin affinity chromatography column, or

iii) both i) and ii), wherein in i) the relative retention time is smaller and in ii) it is larger, or vice versa.

in one embodiment, the variant antibody has at least one additional negatively charged moiety on its (solvent-exposed) surface.

Different methods and tools for determining charged patches on the (solvent exposed) surface of an antibody are known to the person skilled in the art. There are tools offered by different suppliers or academic teams. For example, a computer calculation method based on an X-ray structure or homology model is used herein, followed by pH protonation of acidic and basic amino acid side chains and calculation of 3D charge distribution using software CHARMM and Delphi as performed in the software suite Discovery Studio (supplier: Dassault System).

In one embodiment, the variant antibody has at least one additional positively charged moiety on its (solvent exposed) surface.

in one embodiment, the variant antibody has the same (surface) net charge as the parent antibody.

In one embodiment the (permanently) negatively charged amino acid residue is selected from glutamic acid and aspartic acid.

In one embodiment the (permanently) positively charged amino acid residue is selected from arginine and lysine.

In one embodiment, the pH-dependent charged amino acid residue is histidine.

in one embodiment, the permanently charged amino acid residues have the same (net) charge over a pH range of pH 6 to pH 8.

in one embodiment, the pH-dependent charged amino acid residue has a first (net) charge at pH 6 and an opposite second (net) charge at pH 8.