US20170227547A1 - In vitro prediction of in vivo half-life - Google Patents

In vitro prediction of in vivo half-life Download PDF

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US20170227547A1
US20170227547A1 US15/271,091 US201615271091A US2017227547A1 US 20170227547 A1 US20170227547 A1 US 20170227547A1 US 201615271091 A US201615271091 A US 201615271091A US 2017227547 A1 US2017227547 A1 US 2017227547A1
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antibody
retention time
fcrn
region
value
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Thomas Emrich
Hubert Kettenberger
Tilman Schlothauer
Angela Schoch
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Hoffmann La Roche Inc
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Hoffmann La Roche Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B01D15/168—Fluid composition conditioning, e.g. gradient pH gradient or chromatofocusing, i.e. separation according to the isoelectric point pI
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the current invention is in the field of recombinant antibody technology, especially in the field of tailor made antibodies.
  • Human immunoglobulins of the class G contain two antigen binding (Fab) regions that convey specificity for the target antigen and a constant region (Fc-region) that is responsible for interactions with Fc receptors ([1,2]).
  • Human IgGs of subclasses 1, 2 and 4 have an average serum half-life of 21 days, which is longer than that of any other known serum protein ([3]). This long half-life is predominantly mediated by the interaction between the Fc-region and the neonatal Fc receptor (FcRn) ([4,5]). This is one of the reasons, why IgGs or Fc-containing fusion proteins are used as a widespread class of therapeutics.
  • the neonatal Fc receptor FcRn is a membrane-associated receptor involved in both IgG and albumin homeostasis, in maternal IgG transport across the placenta and in antigen-IgG immune complex phagocytosis ([6,9]).
  • Human FcRn is a heterodimer consisting of the glycosylated class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a ⁇ 2 microglobulin ( ⁇ 2 m) subunit ([10]).
  • ⁇ -FcRn glycosylated class I major histocompatibility complex-like protein
  • ⁇ 2 m microglobulin subunit
  • the affinity between the FcRn and the Fc-region is pH dependent, showing nanomolar affinity at endosomal pH of 5-6 and negligible binding at a physiological pH of 7.4 ([13,17,18]).
  • the underlying mechanism conveying long half-life to IgGs can be explained by three fundamental steps. First, IgGs are subject to unspecific pinocytosis by various cell types ([19,20]). Second, IgGs encounter and bind FcRn in the acidic endosome at a pH of 5-6, thereby protecting IgGs from lysosomal degradation ([11,21]). Finally, IgGs are released in the extracellular space at physiological pH of 7.4 [4]. This strict pH-dependent bind-and-release mechanism is critical for IgG recycling and any deviation of the binding characteristics at different pH values may strongly influence circulation half-life of IgGs ([22]).
  • the Fab regions have also been suggested to contribute to FcRn binding ([23-25]), in addition to the specific interaction of the Fc-region with FcRn.
  • Fab-mediated residual binding at neutral pH was correlated with the pharmacokinetic properties of a set of therapeutic antibodies, indicating that IgGs with excessive binding to FcRn at pH 7.3 suffer from reduced terminal half-life ([24]).
  • Schlothauer et al. [25]
  • Schlothauer et al. [25] have described a novel pH-gradient FcRn affinity chromatography method that closely mimics physiological conditions for the dissociation between FcRn and IgGs.
  • IgGs with identical Fc-regions differ in their dissociation from FcRn, thereby indicating the influence of the Fab region on FcRn binding.
  • the amino acid sequences of the antibody Briakinumab are reported in WO 2013/087911 (SEQ ID NO: 39 and SEQ ID NO: 40), of the antibody Ustekinumab in WO 2013/087911 (SEQ ID NO: 37 and SEQ ID NO: 38) and of the antibody Bevacizumab in Drug Bank entry DB00112.
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction influencing the in vivo half-life of the antibody comprising the following steps:
  • the antibody-Fab-FcRn interaction is an interaction between the Fab-region of an antibody with the FcRn. This interaction occurs, if present at all, after the antibody has been bound by the FcRn. Thus, the establishment of this interaction is a two-step process. In the first step an antibody-FcRn complex, to be more precise an antibody-Fc-FcRn complex, is formed. The second step after the antibody-Fc-FcRn complex has been formed is the establishment of the antibody-Fab-FcRn interaction. As can be seen from this, only with a full-length antibody these two interactions, i.e. the antibody-Fc-FcRn interaction and the antibody-Fab-FcRn interaction, can be established.
  • One aspect as reported herein is a method for determining the presence of Fab-FcRn interaction in an antibody-FcRn complex influencing the in vivo half-life comprising the following steps:
  • Another aspect as reported herein is a method for determining the relative in vivo half-life of an antibody comprising the following steps:
  • the antibody has a relative in vivo half-life that is reduced compared to a standard/natural antibody of the IgG class if the retention time determined in step a) and the retention time determined in step b) are substantially different.
  • the antibody of the IgG class is an antibody of the IgG1, IgG2, IgG3 or IgG4 subclass. In one embodiment the antibody of the IgG class is an antibody of the IgG1, IgG3 or IgG4 subclass. In one embodiment the antibody of the IgG class is an antibody of the IgG1 or IgG4 subclass. In one embodiment the antibody of the IgG class is an antibody of the IgG1 subclass. In one embodiment the antibody of the IgG class is an antibody of the IgG4 subclass.
  • a further aspect as reported herein is a method for determining an increase or a decrease in the vivo half-life of a variant antibody relative to its parent antibody comprising the following steps:
  • the in vivo half-life of the variant antibody relative to its parent antibody is increased if i) the retention time of the variant antibody determined in step a) is longer than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same, whereby the in vivo half-life of the variant antibody relative to its parent antibody is decreased if i) the retention time of the variant antibody determined in step a) is shorter than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same.
  • Another aspect as reported herein is a method for selecting an antibody with increased or decreased in the vivo half-life relative to a reference antibody comprising the following steps:
  • an antibody in case of selecting an antibody with increased in vivo half-life relative to the reference antibody an antibody is selected that has i) a retention time determined in step a) that is longer than the retention time of the reference antibody determined in step a), and ii) a retention time determined in step a) that is substantially the same as the retention time determined in step b),
  • an antibody in case of selecting an antibody with decreased in vivo half-life relative to the reference antibody an antibody is selected that has i) a retention time determined in step a) that is shorter than the retention time of the reference antibody determined in step a), and ii) a retention time determined in step a) that is substantially the same as the retention time determined in step b).
  • Another aspect as reported herein is a method for selecting an antibody without antibody-Fab-FcRn interaction influencing the vivo half-life of the antibody:
  • an antibody is selected that has a retention time determined in step a) that is not substantially different from the retention time determined in step b) and thereby selecting an antibody without antibody-Fab-FcRn interaction influencing the vivo half-life of the antibody.
  • One aspect as reported herein is a method for producing an antibody comprising the following steps:
  • One aspect as reported herein is a method for increasing the in vivo half-life of an antibody comprising the step of:
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction influencing the in vivo half-life of the antibody comprising the following steps:
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction influencing the in vivo half-life of the antibody comprising the following steps:
  • One aspect as reported herein is a method for determining the relative in vivo half-life of an antibody comprising the following steps:
  • the antibody has a relative in vivo half-life that is reduced compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is shorter/smaller than the retention time of its parent antibody, and
  • the antibody has a relative in vivo half-life that is increased compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is longer/bigger than the retention time of its parent antibody.
  • One aspect as reported herein is a method for determining an increase or a decrease of the vivo half-life of an antibody comprising the following steps:
  • the antibody has a decrease of the in vivo half-life compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is shorter/smaller than the retention time of its parent antibody, and
  • the antibody has an increase of the in vivo half-life compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is longer/bigger than the retention time of its parent antibody.
  • the antibody is a full length antibody.
  • the positive linear pH gradient is from about pH 5.5 to about pH 8.8.
  • the salt is selected from sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate.
  • the salt is sodium chloride.
  • the first salt concentration is between 50 mM and 200 mM.
  • the first salt concentration is about 140 mM.
  • the second salt concentration is between 300 mM and 600 mM.
  • the second salt concentration is about 400 mM.
  • step a) and step b) differ by at least 5%.
  • step a) and step b) differ by at least 10%.
  • step a) and step b) differ by at least 15%.
  • step b) the retention time in step a) is bigger/longer than in step b).
  • step b) the retention time in step b) is smaller/shorter than in step a).
  • the retention times are substantially different in step a) and step b) the retention times are proportional to one above the square root of the salt concentration ( ⁇ 1/SQRT(c(salt))).
  • the parent or reference antibody is the anti-IL-1R antibody with SEQ ID NO: 01 (heavy chain) and SEQ ID NO: 02 (light chain) for the subclass IgG1 and the anti-IL-1R antibody with SEQ ID NO: 03 (heavy chain) and SEQ ID NO: 04 (light chain) for the subclass IgG4.
  • the parent or reference antibody is the anti-HER2 antibody with SEQ ID NO: 36 (heavy chain) and SEQ ID NO: 37 (light chain) for the subclass IgG1 and the anti-HER2 antibody with SEQ ID NO: 38 (heavy chain) and SEQ ID NO: 39 (light chain) for the subclass IgG4.
  • the parent or reference antibody is Ustekinumab with light and heavy chain amino acid sequence as depicted in FIG. 5 .
  • the FcRn affinity chromatography column comprises a non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m).
  • the FcRn affinity chromatography column comprises a covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m).
  • the complex of the neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is bound to a solid phase.
  • the solid phase is a chromatography material.
  • the complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) is biotinylated and the solid phase is derivatized with streptavidin.
  • beta-2-microglobulin is from the same species as the neonatal Fc receptor (FcRn).
  • beta-2-microglobulin is from a different species as the FcRn.
  • the FcRn is selected from human FcRn, cynomolgus FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn, pig FcRn, minipig FcRn, and rabbit FcRn.
  • the antibody is a monospecific antibody or antibody fragment of fusion polypeptide, or a bispecific antibody or antibody fragment of fusion polypeptide, or a trispecific antibody or antibody fragment of fusion polypeptide, or a tetraspecific antibody or antibody fragment of fusion polypeptide.
  • the antibody is an antibody of the class IgG. In one embodiment the antibody is an antibody of the subclass IgG1, IgG2, IgG3 or IgG4. In one embodiment the antibody is an antibody of the subclass IgG1 or IgG4.
  • FIGS. 1A-1D are identical to FIGS. 1A-1D.
  • FIG. 1 a Briakinumab.
  • the light chain is shown in light gray, the heavy chain is shown in darker grey.
  • Views of the middle and right images are related to the view in the left panel by a rotation about a vertical and a horizontal axis, respectively.
  • FIG. 1 b Ustekinumab.
  • Light and heavy chains are colored in light and dark gray, respectively. The views are identical to ( FIG. 1 a ).
  • FIG. 1 c Isopotential surface contoured at 2 k B T/e of a human FcRn homology model in complex with human ⁇ 2 microglobulin ( ⁇ 2 m). The Fc domain is shown for clarity.
  • FIG. 1 d Sequence-based calculated net charge vs. pH of Briakinumab and Ustekinumab. Protein structures were prepared with DiscoveryStudio Pro.
  • FIG. 2 pH-dependent FcRn-IgG interaction.
  • FcRn affinity chromatograms of the eleven IgG variants were intensity-normalized for clarity.
  • the view is identical to the right panel in FIG. 1 a and focuses on the CDR regions.
  • a second horizontal axis indicates the elution pH, interpolated from offline pH measurements.
  • FIG. 3 a Blood level curves of Briakinumab (diamonds, orange), Ustekinumab (squares, green), mAb 8 (triangles, purple) and mAb 9 (circles, blue).
  • FIG. 3 b Correlation between the terminal half-life with the FcRn column elution pH.
  • FIG. 4 a Conformation at the start of the simulation.
  • the dashed line indicates the distance between two example amino acids in the Fv region and in the FcRn, which approach during the MD simulation as shown in panel ( FIG. 4 c ).
  • the colors are identical to FIG. 1 .
  • the box indicates the part of the molecule shown in ( FIG. 4 c ).
  • FIG. 4 c Detailed view of the interaction between FcRn and the Fv domains. Note that the interacting framework, CDR and FcRn residues are different in Briakinumab and Ustekinumab.
  • FIG. 4 d Distance between residues 245 (FcRn) and 100 (Ustekinumab LC) and 29 (Briakinumab LC), respectively during the course of the simulation.
  • FIG. 4 e Interaction energies at the end of the simulation (average and standard deviations of conformations at 96, 97, 98, 99 and 100 ns).
  • VDW and “Electrostatic” denote the van-der-Waals and electrostatic contributions, respectively, to the FcRn-Fab interaction. Protein structures were prepared with PyMolTM (Schrodinger LLC).
  • FIG. 5 Sequence alignment of Briakinumab and Ustekinumab light and heavy chains. VH and VL regions are shown in italics; CDRs are marked with an asterisk (*); a hash (#) denotes amino acids in close proximity ( ⁇ 4 ⁇ ) to the FcRn in the starting structure. A “ ⁇ ” symbol marks the residue mutated to Cys to establish a disulfide bridge to the FcRn for MD purposes.
  • FIG. 6 Salt-dependence of the FcRn affinity column retention times of Briakinumab and Ustekinumab.
  • Briakinumab and Ustekinumab were subjected to FcRn column chromatography with pH gradient elution in the presence of increasing amounts of NaCl. Data are fitted to an inverse square root function to account for the charge shielding effect by dissolved salt.
  • FIG. 7 Linearity of applied antibody and area under the curve of a chromatography using an FcRn column as reported herein.
  • FIG. 8 Chromatogram of anti-IGF-1R antibody wild-type and YTE-mutant on FcRn column as reported herein.
  • FIG. 9 FcRn affinity chromatogram of Avastin-wild-type and the Avastin-mutant.
  • FIGS. 10A-10D are identical to FIGS. 10A-10D.
  • FIG. 10A wild-type-like Fc-region, no antibody-Fab-FcRn interaction
  • FIG. 10B wild-type-like Fc-region, antibody-Fab-FcRn interaction
  • FIG. 10C engineered Fc-region with improved FcRn-binding, no antibody-Fab-FcRn interaction
  • FIG. 10D engineered Fc-region with improved FcRn-binding, antibody-Fab-FcRn interaction.
  • FIG. 11 Scheme showing an engineered antibody with improved FcRn binding, antibody-Fab-FcRn interaction but reduced in vivo half-life as the antibody-FcRn interaction results in an improved clearance (retention time above critical retention time).
  • FIG. 13 Dependence of FcRn affinity chromatography retention time on salt concentration and antibody-Fab-FcRn interaction.
  • FIG. 14 Sequence alignment of Bevacizumab and the Bevacizumab variant light chain variable domains. Identical and similar amino acids are shown in grey; CDRs are marked with an asterisk (*).
  • FIG. 15 IL-12 interaction of Briakinumab, Ustekinumab and mAb 1-6; 1: Briakinumab, 2: Ustekinumab, 3: mAb 1, 4: mAb 2, 5: mAb 3, 6: mAb 4, 7: mAb 5, 8: mAb 6.
  • FIG. 16 IL-12 interaction of Briakinumab, Ustekinumab and mAb 7-10; 1: Briakinumab, 2: Ustekinumab, 3: mAb 7, 4: mAb 8, 5: mAb 9, 6: mAb 10.
  • Antibodies did not show differences in pH 6.0 affinity, therefore the Fab region seems to have no influence on pH 6.0 binding. In contrast, the dissociation between FcRn and the antibodies was influenced by the Fab region.
  • the term “about” denotes a range of +/ ⁇ 20% of the thereafter following numerical value. In one embodiment the term about denotes a range of +/ ⁇ 10% of the thereafter following numerical value. In one embodiment the term about denotes a range of +/ ⁇ 5% of the thereafter following numerical value.
  • alteration denotes the mutation (substitution), insertion (addition), modification (derivatization), or deletion of one or more amino acid residues in a parent antibody or fusion polypeptide, e.g. a fusion polypeptide comprising at least an FcRn binding portion of an Fc-region, to obtain a modified antibody or fusion polypeptide.
  • mutation denotes that the specified amino acid residue is substituted for a different amino acid residue.
  • the mutation L234A denotes that the amino acid residue lysine at position 234 in an antibody Fc-region (polypeptide) is substituted by the amino acid residue alanine (substitution of lysine with alanine) (numbering according to the EU index).
  • the replacing amino acid residue may be a “naturally occurring amino acid residues” and selected from the group consisting of alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr
  • the replacing amino acid residue may be a “non-naturally occurring amino acid residue”. See e.g. U.S. Pat. No. 6,586,207, WO 98/48032, WO 03/073238, US 2004/0214988, WO 2005/35727, WO 2005/74524, Chin, J. W., et al., J. Am. Chem. Soc. 124 (2002) 9026-9027; Chin, J. W. and Schultz, P. G., ChemBioChem 11 (2002) 1135-1137; Chin, J. W., et al., PICAS United States of America 99 (2002) 11020-11024; and, Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (all entirely incorporated by reference herein).
  • amino acid insertion denotes the (additional) incorporation of at least one amino acid residue at a predetermined position in an amino acid sequence. In one embodiment the insertion will be the insertion of one or two amino acid residues.
  • the inserted amino acid residue(s) can be any naturally occurring or non-naturally occurring amino acid residue.
  • amino acid deletion denotes the removal of at least one amino acid residue at a predetermined position in an amino acid sequence.
  • antibody herein is used in a broad sense and encompasses various antibody structures, including but not limited to monoclonal antibodies and multispecific antibodies (e.g. bispecific antibodies, trispecific antibodies) so long as they are full length antibodies and exhibit the desired antigen- and/or FcRn-binding activity.
  • binding denotes the binding of an antibody in an in vitro assay. In one embodiment binding is determined in a binding assay in which the antibody is bound to a surface and binding of the antigen to the antibody is measured by Surface Plasmon Resonance (SPR). Binding means e.g. a binding affinity (KD) of 10 ⁇ 8 M or less, in some embodiments of 10 ⁇ 13 to 10 ⁇ 8 M, in some embodiments of 10 ⁇ 13 to 10 ⁇ 9 M.
  • KD binding affinity
  • Binding can be investigated by a BlAcore assay (GE Healthcare Biosensor AB, Uppsala, Sweden).
  • the affinity of the binding is defined by the terms k a (rate constant for the association of the antibody from the antibody/antigen complex), k d (dissociation constant), and K D (k d /k a ).
  • buffer substance denotes a substance that when in solution can level changes of the pH value of the solution e.g. due to the addition or release of acidic or basic substances.
  • CH2 domain denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 231 to EU position 340 (EU numbering system according to Kabat).
  • a CH2 domain has the amino acid sequence of SEQ ID NO: 05: APELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ E STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.
  • CH3-domain denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 341 to EU position 446.
  • the CH3 domain has the amino acid sequence of SEQ ID NO: 06: GQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG.
  • the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • Fc-fusion polypeptide denotes 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 of a receptor) with an antibody Fc-region that exhibits the desired target- and/or protein A and/or FcRn-binding activity.
  • a binding domain e.g. an antigen binding domain such as a single chain antibody, or a polypeptide such as a ligand of a receptor
  • Fc-region of human origin denotes the C-terminal region of an immunoglobulin heavy chain of human origin that contains at least a part of the hinge region, the CH2 domain and the CH3 domain.
  • a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the Fc-region has the amino acid sequence of SEQ ID NO: 07.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.
  • the Fc-region is composed of two heavy chain Fc-region polypeptides, which can be covalently linked to each other via the hinge region cysteine residues forming inter-polypeptide disulfide bonds.
  • FcRn denotes the human neonatal Fc-receptor. FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life.
  • the FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a 15 kDa ⁇ 2-microglobulin ( ⁇ 2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc-region of IgG.
  • IgG and FcRn The interaction between IgG and FcRn is strictly pH dependent and occurs in a 1:2 stoichiometry, with one IgG binding to two FcRn molecules via its two heavy chains (Huber, A. H., et al., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs in the endosome at acidic pH (pH ⁇ 6.5) and IgG is released at the neutral cell surface (pH of about 7.4).
  • the pH-sensitive nature of the interaction facilitates the FcRn-mediated protection of IgGs pinocytosed into cells from intracellular degradation by binding to the receptor within the acidic environment of endosomes. FcRn then facilitates the recycling of IgG to the cell surface and subsequent release into the blood stream upon exposure of the FcRn-IgG complex to the neutral pH environment outside the cell.
  • FcRn binding portion of an Fc-region denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 243 to EU position 261 and approximately from EU position 275 to EU position 293 and approximately from EU position 302 to EU position 319 and approximately from EU position 336 to EU position 348 and approximately from EU position 367 to EU position 393 and EU position 408 and approximately from EU position 424 to EU position 440.
  • one or more of the following amino acid residues according to the EU numbering of Kabat are altered F243, P244, P245 P, 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, V348, C367, V369,
  • full length antibody denotes 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.
  • a full length antibody may comprise further domains, such as e.g. additional scFv or a scFab conjugated to one or more of the chains of the full length antibody. These conjugates are also encompassed by the term full length antibody.
  • hinge region denotes the part of an antibody heavy chain polypeptide that joins the CH1 domain and the CH2 domain, e. g. from about position 216 to position about 230 according to the EU numbering system of Kabat.
  • the hinge region is a shortened hinge region comprising residues 221 to 230 according to the EU numbering system of Kabat.
  • the hinge region is normally a dimeric molecule consisting of two polypeptides with identical amino acid sequence.
  • the hinge region generally comprises about 25 amino acid residues and is flexible allowing the antigen binding regions to move independently.
  • the hinge region can be subdivided into three domains: the upper, the middle, and the lower hinge domain (Roux, et al., J. Immunol. 161 (1998) 4083).
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • derived from denotes that an amino acid sequence is derived from a parent amino acid sequence by introducing alterations at at least one position.
  • a derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbering according to Kabat EU index for antibody Fc-regions).
  • an amino acid sequence derived from a parent amino acid sequence differs by one to fifteen amino acid residues at corresponding positions.
  • an amino acid sequence derived from a parent amino acid sequence differs by one to ten amino acid residues at corresponding positions.
  • an amino acid sequence derived from a parent amino acid sequence differs by one to six amino acid residues at corresponding positions.
  • a derived amino acid sequence has a high amino acid sequence identity to its parent amino acid sequence. In one embodiment an amino acid sequence derived from a parent amino acid sequence has 80% or more amino acid sequence identity. In one embodiment an amino acid sequence derived from a parent amino acid sequence has 90% or more amino acid sequence identity. In one embodiment an amino acid sequence derived from a parent amino acid sequence has 95% or more amino acid sequence identity.
  • human Fc-region polypeptide denotes an amino acid sequence which is identical to a “native” or “wild-type” human Fc-region polypeptide.
  • variant (human) Fc-region polypeptide denotes an amino acid sequence which derived from a “native” or “wild-type” human Fc-region polypeptide by virtue of at least one “amino acid alteration”.
  • a “human Fc-region” is consisting of two human Fc-region polypeptides.
  • a “variant (human) Fc-region” is consisting of two Fc-region polypeptides, whereby both can be variant (human) Fc-region polypeptides or one is a human Fc-region polypeptide and the other is a variant (human) Fc-region polypeptide.
  • the human Fc-region polypeptide has the amino acid sequence of a human IgG1 Fc-region polypeptide of SEQ ID NO: 07, or of a human IgG2 Fc-region polypeptide of SEQ ID NO: 08, or of a human IgG3 Fc-region polypeptide of SEQ ID NO: 09, or of a human IgG4 Fc-region polypeptide of SEQ ID NO: 10.
  • the Fc-region polypeptide is derived from an Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10 and has at least one amino acid mutation compared to the Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10.
  • the Fc-region polypeptide comprises/has from about one to about ten amino acid mutations, and in one embodiment from about one to about five amino acid mutations. In one embodiment the Fc-region polypeptide has at least about 80% homology with a human Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10. In one embodiment the Fc-region polypeptide has least about 90% homology with a human Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10. In one embodiment the Fc-region polypeptide has at least about 95% homology with a human Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10.
  • the Fc-region polypeptide derived from a human Fc-region polypeptide of SEQ ID NO: 07, or 08 or 09, or 10 is defined by the amino acid alterations that are contained.
  • P329G denotes an Fc-region polypeptide derived human Fc-region polypeptide with the mutation of proline to glycine at amino acid position 329 relative to the human Fc-region polypeptide of SEQ ID NO: 07, or 08, or 09, or 10.
  • EU index or EU index as in Kabat or Kabat EU index or EU numbering scheme refers to the numbering of the EU antibody (Edelman, et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85, hereby entirely incorporated by reference).
  • the numbering of the light chain residues is according to the Kabat nomenclature (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), NIH Publication 91 3242).
  • a human IG1 Fc-region polypeptide has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with the mutations L234A, L235A has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with Y349C, T366S, L368A and Y407V mutations has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with S354C, T366W mutations has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with a L234A, L235A and S354C, T366W mutations has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with a P329G mutation has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A mutations and P329G mutation has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with a P329G mutation and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with a P329G mutation and S354C, T366W mutation has the following amino acid sequence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid seauence:
  • a human IgG1 Fc-region derived Fc-region polypeptide with L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:
  • a human IgG4 Fc-region polypeptide has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with S228P and L235E mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with S228P, L235E mutations and P329G mutation has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with S354C, T366W mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E and S354C, T366W mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a P329G mutation has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a P329G and S354C, T366W mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:
  • a human IgG4 Fc-region derived Fc-region polypeptide with a S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., the CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g. cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC).
  • electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g., size exclusion chromatography or ion exchange or reverse phase HPLC.
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous 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 arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody 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.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain.
  • VH variable heavy domain
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • negative linear pH gradient denotes a pH gradient starting at a high (i.e. neutral or alkaline) pH value and ending at a lower (i.e. neutral or acidic) pH value.
  • the negative linear pH gradient starts at a pH value of about 8.8 and ends at a pH value of about 5.5.
  • non-naturally occurring amino acid residue denotes an amino acid residue, other than the naturally occurring amino acid residues as listed above, which can be covalently bound to the adjacent amino acid residues in a polypeptide chain.
  • non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine. Further examples are listed in Ellman, et al., Meth. Enzym. 202 (1991) 301-336. Exemplary method for the synthesis of non-naturally occurring amino acid residues are reported in, e. g., Noren, et al., Science 244 (1989) 182 and Ellman et al., supra.
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • plasmid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the plasmid as a self-replicating nucleic acid structure as well as the plasmid incorporated into the genome of a host cell into which it has been introduced.
  • Certain plasmids are capable of directing the expression of nucleic acids to which they are operatively linked. Such plasmids are referred to herein as “expression plasmid”.
  • positive linear pH gradient denotes a pH gradient starting at a low (i.e. more acidic) pH value and ending at a higher (i.e. less acidic, neutral or alkaline) pH value.
  • the positive linear pH gradient starts at a pH value of about 5.5 and ends at a pH value of about 8.8.
  • recombinant antibody denotes all antibodies (chimeric, humanized and human) that are prepared, expressed, created or isolated by recombinant means. This includes antibodies isolated from a host cell such as a NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression plasmid transfected into a host cell. Such recombinant antibodies have variable and constant regions in a rearranged form. The recombinant antibodies as reported herein can be subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • a “solid phase” denotes a non-fluid substance, and includes particles (including microparticles and beads) made from materials such as polymer, metal (paramagnetic, ferromagnetic particles), glass, and ceramic; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made of polymer, metal, glass, and/or ceramic; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers.
  • a solid phase component of an assay is distinguished from inert solid surfaces in that a “solid support” contains at least one moiety on its surface, which is intended to interact chemically with a molecule.
  • a solid phase may be a stationary component, such as a chip, tube, strip, cuvette, or microtiter plate, or may be non-stationary components, such as beads and microparticles.
  • Microparticles can also be used as a solid support for homogeneous assay formats. A variety of microparticles that allow both non-covalent or covalent attachment of proteins and other substances may be used. Such particles include polymer particles such as polystyrene and poly (methylmethacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See for example 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 sepharose.
  • substantially the same denotes that two values, e.g. the retention times on an FcRn affinity chromatography column of two different antibodies, are within 5% of each other, i.e. they differ by less than 5%.
  • a first retention time of 80 minutes and a second retention time of 84 minutes are substantially the same, whereas a retention time of 80 minutes and a retention time of 85 minutes are not substantially the same, these retention times are different.
  • substantially the same denotes that two values are within 3.5% of each other, i.e. they differ by 3.5% or less.
  • substantially the same denotes that two values are within 2.5% of each other, i.e. they differ by 2.5% or less. The smaller of the two values is taken as basis for this calculation.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies or Fc-region fusion polypeptides as reported herein are used to delay development of a disease or to slow the progression of a disease.
  • bivalent as used within the current application denotes the presence of a specified number of binding sites in a (antibody) molecule.
  • bivalent tetravalent
  • hexavalent denote the presence of two binding site, four binding sites, and six binding sites, respectively, in a (antibody) molecule.
  • the bispecific antibodies as reported herein as reported herein are in one preferred embodiment “bivalent”.
  • variable region or “variable domain” refer to the domain of an antibody heavy or light chain that is involved in binding of the antibody to its antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of an antibody generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T. J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91).
  • FRs framework regions
  • HVRs hypervariable regions
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library 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) 624-628).
  • variant denotes molecules which have 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.
  • the modified antibody or the modified fusion polypeptide comprises an amino acid sequence comprising at least a portion of an Fc-region which is not naturally occurring. Such molecules have less than 100% sequence identity with the parent antibody or parent fusion polypeptide.
  • the variant antibody or the variant fusion polypeptide has an amino acid sequence that has from about 75% to less than 100% amino acid sequence identity with the amino acid sequence of the parent antibody or parent fusion polypeptide, especially from about 80% to less than 100%, especially from about 85% to less than 100%, especially from about 90% to less than 100%, and especially from about 95% to less than 100%.
  • the parent antibody or the parent fusion polypeptide and the variant antibody or the variant fusion polypeptide differ by one (a single), two or three amino acid residue(s).
  • the invention is based, at least in part, on the finding that the charge distribution in the Fv domain influences antibody-FcRn binding and results in additional interactions between the antibody and the FcRn. This changes the FcRn binding characteristics, especially with respect to the dissociation of the antibody-FcRn complex at pH 7.4, thereby reducing FcRn-dependent terminal half-life of the antibody.
  • the neonatal Fc-receptor (FcRn) is important for the metabolic fate of antibodies of the IgG class in vivo.
  • the FcRn functions to salvage wild-type IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a 15 kDa ⁇ 2-microglobulin ( ⁇ 2m).
  • ⁇ -FcRn major histocompatibility complex-like protein
  • ⁇ 2m microglobulin
  • an antibody of the class IgG and the FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc-region polypeptides (see e.g. [16]).
  • an IgGs in vitro FcRn binding properties/characteristics are indicative of its in vivo pharmacokinetic properties in the blood circulation.
  • the amino acid residues interacting with the FcRn are located approximately between EU position 243 and EU position 261, approximately between EU position 275 and EU position 293, approximately between EU position 302 and EU position 319, approximately between EU position 336 and EU position 348, approximately between EU position 367 and EU position 393, at EU position 408, and approximately between EU position 424 and EU position 440.
  • antibodies with reduced half-life in the blood circulation are desired.
  • drugs for intravitreal application should have a long half-life in the eye and a short half-life in the circulation of the patient.
  • Such antibodies also have the advantage of increased exposure to a disease site, e.g. in the eye.
  • Fc-region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., et al. J. Immunol 169 (2002) 5171-5180).
  • Residues I253, H310, H433, N434, and H435 are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M., et al., Int. Immunol.
  • Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29 (1999) 2819-2825).
  • Residues M252Y, S254T, T256E have been described by Dall'Acqua et al. to improve FcRn binding by protein-protein interaction studies (Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006) 23514-23524).
  • FcRn human neonatal Fc receptor
  • IgG-FcRn interactions can be analyzed using plasmon surface resonance (SPR) assays (Wang, W., et al., Drug Metab. Disp. 39 (2011) 1469-1477; Datta-Mannan, A., et al., Drug Metab. Disp. 40 (2012) 1545-1555; Vaughn, D. E. and Bjorkman, P. J., Biochemistry 36 (1997) 9374-9380; Raghavan, M., et al., Proc. Natl. Acad. Sci. USA 92 (1995) 11200-11204; Martin, W. L. and Bjorkman, P. J., Biochemistry 38 (1999) 12639-12647).
  • SPR plasmon surface resonance
  • SPR analysis of the IgG-FcRn interaction provides a qualitative result indicating expected or aberrant binding properties of a sample but does neither give a hint for the cause of aberrant binding nor a quantitative estimation of the amount of antibody with aberrant binding.
  • Briakinumab (OzespaTM) and Ustekinumab (StelaraTM) were used as a model system. Both Briakinumab and Ustekinumab are fully human monoclonal IgG1 antibodies. They bind to the same human p40-subunit of interleukin 12 (IL-12) and interleukin 23 (IL-23) [26] and they are not cross-reactive to the corresponding mouse IL-12 and IL-23 [27,28].
  • IL-12 interleukin 12
  • IL-23) interleukin 23
  • Briakinumab and Ustekinumab are an IgG1 ⁇ antibody with variable heavy and light chain domains of the V H 5 and V ⁇ 1D germline families and an IgG1 ⁇ antibody with variable heavy and light chain domains of the V H 3 and V ⁇ 1 germline families, respectively.
  • Briakinumab and Ustekinumab show differences in several allotype-specific amino acids in the constant domains (see FIG. 5 ). However, these amino acid residues are outside of the (cognate) FcRn binding regions and can therefore be considered to play no role in FcRn-dependent PK [11].
  • Ustekinumab has a (reported) median terminal half-life of 22 days [29]
  • Briakinumab has a terminal half-life of only 8-9 days [26,30,31].
  • Briakinumab exhibits a non-uniform charge distribution at physiological pH of 7.4 (see e.g. the published crystal structure of Ustekinumab [27] and a homology model of Briakinumab).
  • Briakinumab shows a large positively charged region on the Fv domain (see FIG. 1 a ) which is absent in Ustekinumab (see FIG. 1 b ).
  • FcRn possesses a strong and extended negatively charged region (see FIG. 1 c ) which is however not involved in cognate Fc-region binding.
  • Briakinumab and Ustekinumab have calculated isoelectric points of 9.7 and 9.4, respectively.
  • the net charge of Briakinumab is slightly more positive over the entire pH range (see FIG. 1 d ).
  • FcRn binding affinity of Briakinumab and Ustekinumab at pH 6.0 is comparable, i.e. both values differ at most by one order or magnitude, in one embodiment at most 5-fold, whereas the dissociation from the FcRn is very different.
  • Briakinumab and Ustekinumab it could be shown that the interaction is predominantly electrostatic and correlates with the extent of a positively charged region (see below).
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction in an antibody-Fc-FcRn complex influencing the in vivo half-life of the antibody comprising the following steps:
  • One aspect as reported herein is a method for determining the relative in vivo half-life of an antibody comprising the following steps:
  • the antibody has a relative in vivo half-life that is reduced compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is shorter/smaller than the retention time of its parent antibody, and
  • the antibody has a relative in vivo half-life that is increased compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is longer/bigger than the retention time of its parent antibody.
  • One aspect as reported herein is a method for determining an increase or a decrease of the vivo half-life of an antibody comprising the following steps:
  • the antibody has a decrease of the vivo half-life compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is shorter/smaller than the retention time of its parent antibody, and
  • the antibody has an increase of the in vivo half-life compared to its parent antibody if the K D values differ by at most a factor of 10 and the retention time determined in step b) of the variant antibody is longer/bigger than the retention time of its parent antibody.
  • Variant antibodies mAb 5 and mAb 6 bear Ustekinumab CDRs (heavy and light chain parts) on the Briakinumab framework and vice versa. Grafting Ustekinumab CDRs on Briakinumab (mAb 5) shifted the retention time of mAb 5 close to that of Ustekinumab. Grafting Briakinumab CDRs on Ustekinumab (mAb 6) described/presented an elution profile which was still close to Ustekinumab.
  • MAb 3 comprising the HCs of Ustekinumab and the LCs of Briakinumab, as well as mAb 2 containing the Fv domain of Briakinumab on the Ustekinumab constant domains both eluted close to Briakinumab.
  • the FcRn column retention times were aligned with isoelectric points and net charges of the antibodies. No correlation between the FcRn column retention times and the isoelectric points or the net charges of the Fv domains at lysosomal pH 6.0 or physiological pH 7.4 can be seen (see Table 3). However, the measured FcRn column retention times increased with the extent of positively charged regions, especially around the light chain variable domains (see FIG. 2 ).
  • One aspect as reported herein is a method for increasing the in vivo half-life of an antibody comprising the step of:
  • Amino acids may be grouped according to common side-chain properties:
  • FcRn column retention times were also determined in a different set up with increased ionic strength in the mobile phase, i.e. in the presence of increased salt concentrations.
  • Charge-mediated interactions are known to be weakened under high ionic strength conditions, whereas hydrophobic interactions are typically strengthened by salt. It has been found that the FcRn column retention time of Briakinumab was shortened in the presence of salt and was proportional to the inverse square root of the ionic strength as suggested by the Debye-Huckel law of charge screening [32]. The retention time of Ustekinumab remained essentially unaffected (see FIG. 6 ). Thus, a significant part of the excessive FcRn-Briakinumab interaction is charge-mediated.
  • FcRn affinity chromatography of the engineered variants showed that antibodies with the same Fv domain (mAb 1 & mAb 2) and the same LC (mAb 3 & mAb 4) elute at nearly identical FcRn column retention times. Furthermore, grafting Ustekinumab CDRs on Briakinumab (mAb 5) shifts the elution pH close to that of Ustekinumab. Thus, the light chain CDRs provide the main influence on Briakinumab's FcRn binding.
  • Briakinumab and Ustekinumab together with two variants of Briakinumab (mAb 8 and mAb 9), which had FcRn column retention times between Briakinumab and Ustekinumab, were tested.
  • the distribution and elimination processes of the four antibodies are in agreement with other IgG PK studies (see FIG. 3 a ). Briakinumab showed a faster decrease in the ⁇ -phase than the other antibodies.
  • the terminal half-life was examined, which is exclusively calculated in the elimination phase where FcRn recycling dominates [39].
  • the terminal half-life of the four antibodies correlate linearly with the in vitro FcRn column elution pH: The higher the FcRn column elution pH the shorter the terminal half-life, thereby demonstrating that the FcRn column is a predictive/sensitive tool for in vitro FcRn dissociation.
  • the correlation between terminal half-life and the FcRn column elution pH confirms the importance of the fast FcRn-IgG dissociation at physiological pH.
  • the FcRn-IgG complex is built in the endosomes at pH 6.0, therefore, less binding results in less IgG-recycling and faster clearance.
  • the FcRn-IgG complex is released to the plasma membrane, where the dissociation of the IgG and the FcRn has to take place at a physiological pH of 7.4 within a short period of time [40]. Consequently, dissociation at physiological pH is also important for a prolonged half-life [22,40].
  • a homology model of a human FcRn-Fc complex was generated using the published rat FcRn structure as a template.
  • the position of the Fv domains of Briakinumab and Ustekinumab was modeled based on the crystal structure of a complete IgG1 (PDB code 1HZH).
  • These homology models contain two copies of FcRn ( ⁇ -FcRn with ⁇ 2 m) on one complete IgG molecule (see FIG. 4 a ).
  • the distance between FcRn and the Fv domains is >40 ⁇ in the starting structure and exceeds the Debye length of approx. 8 ⁇ under physiological conditions [32].
  • the dynamics of the FcRn-IgG complexes were simulated by molecular dynamics simulation over a period of 100 ns in explicit water and physiological ionic strength. During the course of the simulation, one of the two Fab regions approached the tip of FcRn and persisted in this conformation for the rest of the simulation time (see FIG. 4 b, c, d ). The region on FcRn found to interact with the Fv domain had hitherto not been described as being involved in IgG binding. Surprisingly, in the MD simulations not only Briakinumab but also Ustekinumab assumed a conformation with Fv and FcRn interacting with one another (see FIG. 4 b, c ).
  • the second salt concentration is generally higher/bigger than the first salt concentration, so that these concentrations are not about identical, i.e. they differ by at least 10%, in one embodiment by at least 20%.
  • the antibody can be a variant antibody of a parent antibody in which case the reference antibody is the parent antibody.
  • the reference antibody is an antibody that has substantially the same retention time as its Fc-region after IdeS cleavage or papain cleavage.
  • antibodies with extended half-life are desired.
  • drugs with an extended half-life in the circulation of a patient in need of a treatment require decreased dosing or increased dosing intervals.
  • Such antibodies also have the advantage of increased exposure to a disease site, e. g. a tumor.
  • the retention time is substantially not affected by the change from low to high salt concentration or by the cleavage of the Fc-region then no antibody-Fab-FcRn interaction is present and a higher retention time on the FcRn affinity chromatography column correlates with an increased half-life in vivo. But if the retention time is affected, especially if it is reduced, by a change from low to high salt concentrations or by cleavage of the Fc-region then the in vivo half-life correlates differently to the retention time on the FcRn affinity chromatography column, i.e.
  • a longer retention time on the FcRn affinity chromatography column correlates to a shorter in vivo half-life due to reduced antibody-FcRn dissociation at physiological pH and, without being bound by this theory, an increased lysosomal degradation of the antibody.
  • the herein used FcRn affinity chromatography column comprises a matrix and matrix bound chromatographical functional groups, wherein the matrix bound chromatographical functional group comprises a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin.
  • starting point for the method as reported herein is a parent or reference antibody that is characterized by its binding to the FcRn.
  • One aspect as reported herein is the use of a method as reported herein for determining the presence of antibody-Fab-FcRn interaction in an antibody-Fc-FcRn complex influencing the in vivo half-life comprising the following steps:
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction in an antibody-Fc-FcRn complex influencing the in vivo half-life comprising the following steps:
  • Variant antibodies show either increased or decreased binding to FcRn when compared to a parent antibody polypeptide or compared to a reference antibody, and, thus, have a modified half-life compared to the parent/reference antibody in serum.
  • Fc-region variants with increased affinity for the FcRn are predicted at first to have longer serum half-life compared to those with decreased affinity for the FcRn (i.e. with reduced retention time on an FcRn column compared to a parent antibody or reference antibody).
  • Antibody variants with increased in vivo half-life have applications in methods of treating mammals, especially humans, where long half-life of the administered antibody is desired, such as in the treatment of a chronic disease or disorder.
  • Antibody variants with decreased affinity for the FcRn have applications in methods of treating mammals, especially humans, where a short half-life of the administered antibody or fusion polypeptide is desired, such as in vivo diagnostic imaging.
  • One aspect as reported herein is the use of a method as reported herein for identifying antibodies that exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature.
  • One aspect as reported herein is the use of a method as reported herein for identifying antibodies that exhibit reduced transport across the blood brain barrier from the brain into the vascular space.
  • the FcRn is selected from human FcRn, cynomolgus FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn, pig FcRn, minipig FcRn, and rabbit FcRn.
  • beta-2-microglobulin is from the same species as the FcRn.
  • beta-2-microglobulin is from a different species as the FcRn.
  • parent antibody comprises at least one binding domain and at least one Fc-region. In one embodiment the parent antibody comprises two binding domains and two Fc-regions.
  • the parent antibody comprises at least one binding domain that specifically binds to a target which mediates a biological effect (in one embodiment a ligand capable of binding to a cell surface receptor or a cell surface receptor capable of binding a ligand) and mediates transmission of a negative or positive signal to a cell.
  • the parent antibody comprises at least one binding domain specific for an antigen targeted for reduction or elimination (in one embodiment a cell surface antigen or a soluble antigen) and at least one Fc-region.
  • Antibodies specifically binding to a target can be raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen (e.g. purified antigen, cells or cellular extracts comprising such antigens, or DNA encoding for such antigen) and optionally an adjuvant.
  • the relevant antigen e.g. purified antigen, cells or cellular extracts comprising such antigens, or DNA encoding for such antigen
  • an adjuvant e.g. purified antigen, cells or cellular extracts comprising such antigens, or DNA encoding for such antigen
  • the antibody is a full length antibody.
  • the antibody is a monoclonal antibody.
  • the parent antibody is a bispecific antibody.
  • the parent antibody is a chimeric antibody.
  • the pH is a gradient from about pH 5.5 to about pH 8.8.
  • the non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin is bound to a solid phase.
  • the conjugation of the non-covalent complex to the solid phase is performed by chemically binding via N-terminal and/or c-amino groups (lysine), ⁇ -amino groups of different lysins, carboxy-, sulfhydryl-, hydroxyl-, and/or phenolic functional groups of the amino acid backbone of the antibody, and/or sugar alcohol groups of the carbohydrate structure of the antibody.
  • lysine c-amino groups
  • ⁇ -amino groups of different lysins carboxy-, sulfhydryl-, hydroxyl-, and/or phenolic functional groups of the amino acid backbone of the antibody
  • sugar alcohol groups of the carbohydrate structure of the antibody.
  • non-covalent complex is conjugated to the solid phase via a specific binding pair. In one embodiment the non-covalent complex is conjugated to biotin and immobilization to a solid support is performed via solid support immobilized avidin or streptavidin.
  • a specific binding pair is in one embodiment selected from streptavidin or avidin/biotin, antibody/antigen (see, for example, Hermanson, G. T., et al., Bioconjugate Techniques, Academic Press (1996)), lectin/polysaccharide, steroid/steroid binding protein, hormone/hormone receptor, enzyme/substrate, IgG/Protein A and/or G, etc.
  • Fc residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall'Acqua, W. F., et al. J. Immunol 169 (2002) 5171-5180).
  • Residues I253, H310, H433, N434, and H435 (EU numbering according to Kabat) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, J. K., et al., Eur. J. Immunol. 24 (1994) 542).
  • Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J. K., et al., Eur. J. Immunol. 29 (1999) 2819).
  • Residues M252Y, S254T, T256E have been described by Dall'Acqua et al. to improve FcRn binding by protein-protein interaction studies (Dall'Acqua, W. F., et al. J. Biol. Chem. 281 (2006) 23514-23524).
  • a pharmaceutically acceptable buffer substance such as e.g. phosphoric acid or salts thereof, acetic acid or salts thereof, citric acid or salts thereof, morpholine or salts thereof, 2-(N-morpholino) ethanesulfonic acid (MES) or salts thereof, histidine or salts thereof, glycine or salts thereof, tris (hydroxymethyl) aminomethane (TRIS) or salts thereof, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or salts thereof.
  • phosphoric acid or salts thereof such as e.g. phosphoric acid or salts thereof, acetic acid or salts thereof, citric acid or salts thereof, morpholine or salts thereof, 2-(N-morpholino) ethanesulfonic acid (MES) or salts thereof, histidine or salts thereof, glycine or salts thereof, tris (hydroxymethyl) aminomethane (TRIS) or salts
  • the buffer substance is selected from phosphoric acid or salts thereof, or acetic acid or salts thereof, or citric acid or salts thereof, or histidine or salts thereof.
  • the buffer substance has a concentration of from 10 mM to 500 mM.
  • the buffer substance has a concentration of from 10 mM to 300 mM.
  • the buffer substance has a concentration of from 10 mM to 250 mM.
  • the buffer substance has a concentration of from 10 mM to 100 mM.
  • the buffer substance has a concentration of from 15 mM to 50 mM.
  • the buffer substance has a concentration of about 20 mM.
  • An exemplary starting solution for the positive linear pH gradient comprises in one embodiment 20 mM MES and 140 mM NaCl, adjusted to pH 5.5.
  • An exemplary final solution for the positive linear pH gradient comprises in one embodiment 20 mM TRIS and 140 mM NaCl, adjusted to pH 8.8.
  • the fraction of the starting solution is reduced from 100% to 0% and the fraction from the final solution is increased from 0% to 100% so that after the positive linear pH gradient 100% of the final solution is applied to the column.
  • the starting and the final solution comprises an additional salt.
  • the additional salt is selected from sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate.
  • the solutions comprise of from 50 mM to 1000 mM of the additional salt. In one embodiment the solutions comprise of from 50 mM to 750 mM of the additional salt. In one embodiment the solutions comprise of from 50 mM to 500 mM of the additional salt. In one embodiment the solution comprise of from 50 mM to 750 mM of the additional salt. In one embodiment the solution comprise about 140 mM to about 400 mM of the additional salt.
  • the starting and the final solution comprises sodium chloride. In one embodiment the starting and the final solution comprises of about 140 mM to about 400 mM sodium chloride.
  • An optimal salt concentration for binding of antibodies to FcRn can be determined (140 mM NaCl). If the salt concentration is higher (400 mM) binding to FcRn is reduced due to interference with the charge interactions by the increase of the ionic strength of the solution and a shorter retention time is obtained.
  • the FcRn affinity chromatography in step a) and step b) are performed under identical conditions except for the concentration of the salt, which is different between step a) and step b).
  • the second salt concentration is bigger than the first salt concentration.
  • the second salt concentration is at least twice the first salt concentration.
  • the amount of applied antibody shows a linear correlation to the area under the curve of the eluted peak.
  • the retention time of antibodies having a wild-type Fc-region (IgG1 or IgG2 or IgG4) varies between 45 and 49 min. (tested with 35 therapeutic antibodies against 36 antigens, data not shown) under the conditions of Example 5. If the conditions of Example 2 are used the retention time is increased to about 85 min as the gradient is longer.
  • the anti-IGF-1R antibody mutant YTE shows an increased retention time (see FIG. 8 ).
  • One aspect as reported herein is a method for determining the relative in vivo half-life of an antibody comprising the following steps:
  • the antibody has a relative in vivo half-life that is reduced compared to a standard/natural antibody of the IgG1, IgG3 or IgG4 subclass if the retention time determined in step a) and the retention time determined in step b) are substantially different.
  • One aspect as reported herein is the use of the method as reported herein for determining the relative in vivo half-life of an antibody wherein the method comprises the following steps:
  • the antibody has a relative in vivo half-life that is reduced compared to a standard/natural antibody of the IgG1, IgG3 or IgG4 subclass if the retention time determined in step a) and the retention time determined in step b) are substantially different.
  • One aspect as reported herein is a method for determining an increase or a decrease in the vivo half-life of a variant antibody relative to its parent antibody comprising the following steps:
  • the in vivo half-life of the variant antibody relative to its parent antibody is increased if i) the retention time of the variant antibody determined in step a) is bigger/longer than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same,
  • the in vivo half-life of the variant antibody relative to its parent antibody is decreased if i) the retention time of the variant antibody determined in step a) is smaller/shorter than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same.
  • One aspect as reported herein is the use of a method as reported herein for determining an increase or a decrease in the vivo half-life of a variant antibody relative to its parent antibody wherein the method comprises the following steps:
  • the in vivo half-life of the variant antibody relative to its parent antibody is increased if i) the retention time of the variant antibody determined in step a) is bigger/longer than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same,
  • the in vivo half-life of the variant antibody relative to its parent antibody is decreased if i) the retention time of the variant antibody determined in step a) is smaller/shorter than the retention time of its parent antibody determined in step a), and ii) the retention time of the variant antibody determined in step a) and the retention time of the variant antibody determined in step b) are substantially the same.
  • One aspect as reported herein is the use of a method as reported herein for determining the in vivo half-life of an antibody.
  • the longer in vivo half-life corresponded to a longer retention time in the FcRn chromatography.
  • An extended half-life of an Fc-engineered trastuzumab variant recently was shown to have enhanced in vitro binding to FcRn as measured by flow cytometry (Petkova, S. B., et al., Int. Immunol. 18 (2006) 1759-1769).
  • a variant of the anti-VEGF IgG1 antibody Bevacizumab with 11-fold improved FcRn affinity was shown to have a five-fold extended half-life in human FcRn transgenic mice and a three-fold longer half-life in cynomolgus monkeys (Zalevsky, J., et al., Nat. Biotechnol. 28 (2010) 157-159).
  • the complex is mono-biotinylated.
  • the chromatography material comprising a non-covalent complex of neonatal Fc receptor (FcRn) and beta-2-microglobulin as ligand has a stability of at least 100 cycles in the methods and uses as reported herein.
  • a cycle is a pH gradient from the first pH value to the second pH value of the respective method or use whereby for regeneration of the material no further change of conditions is required than the final conditions of the method or use.
  • a cycle is a pH gradient from about pH value pH 5.5 to about pH value pH 8.8.
  • the Fc-region e.g. can be obtained by enzymatic cleavage with the enzyme IdeS or papain, or can be produced recombinantly. This is important as antibody-Fab-FcRn interactions influence the in vivo half-life of the antibody.
  • the antibody in question is a variant antibody further aspects have to be considered: in the variant antibody the antibody-Fc-FcRn interaction as well as the antibody-Fab-FcRn interaction can be changed due to the introduced modifications with respect to the parent antibody.
  • the reference antibody is an antibody that has substantially the same retention time as its Fc-region after IdeS cleavage or papain cleavage.
  • the antibody-Fab-FcRn interaction can have an influence on the in vivo half-life of the antibody. Also as outlined above the antibody-Fc-FcRn interaction can have an influence on the in vivo half-life of the antibody. Thus, both interactions have to be accounted for.
  • One aspect of the current invention is a method for selecting an antibody comprising the following steps (see FIG. 12 ):
  • the elution is by a positive linear pH gradient at a constant salt concentration or by using a linear salt gradient at a constant pH value.
  • the antibody is a variant antibody of a parent antibody and the reference antibody is the parent antibody.
  • the variant antibody has amino acid alterations in the antibody-Fab or/and in the antibody-Fc-region.
  • the antibody-Fab-FcRn interaction is a secondary interaction that is established, if present at all, after an antibody-Fc-FcRn complex has been formed.
  • Both interactions i.e. the antibody-Fc-FcRn and the antibody-Fab-FcRn interaction, are charge mediated non-covalent interactions.
  • One aspect as reported herein is a method for determining the presence of antibody-Fab-FcRn interaction influencing the in vivo half-life of the antibody comprising the following steps:
  • the first pH value is 5.5. In one embodiment the second pH value is 8.8.
  • step a) and step b) are identical.
  • the salt gradient is a sodium chloride gradient.
  • the salt gradient is from 0 mM to 250 mM salt.
  • Another molecule without charge patch in the CDRs is chosen to create a positive charge patch in the LC-CDRs to verify the findings reported above that positive charge at this position influences FcRn binding affinity of antibodies in general.
  • Bevacizumab was chosen because it had only little charge in the LC-CDRs.
  • the three basic amino acid residues that were identified using Briakinumab are the arginine residues R27, R55 and R94.
  • Aspartic acid D27, leucine L54 and threonine T93 are exchanged into lysine residues to create a positive charge patch (see FIG. 14 ).
  • Bevacizumab-wild-type has a retention time of 84.7 minutes, whereas Bevacizumab-mutant elutes after 86.9 minutes.
  • the positive charge patch in the Fv of Bevacizumab causes a retention time shift of 2.2 minutes.
  • the results indicate that charge in the Fv of an IgG1 influences FcRn binding affinity in general, especially the dissociation from the FcRn is influenced.
  • Fc-region by at most a factor of 10 and that has a retention time that is substantially the same as the retention time of its Fc-region.
  • the antibodies used in the experiments were Ustekinumab (CNTO 1275, StelaraTM), CAS Registry Number 815610-63-0, variable domains in SEQ ID NO: 42 and 43), Briakinumab (ABT 874, J 695, OzespaTM, variable domains in SEQ ID NO: 40 and 41) as well as ten variants and mutants of Ustekinumab and Briakinumab, hereafter referred to as mAb 1 to mAb 10, respectively.
  • mAb 1 to mAb 10 ten variants and mutants of Ustekinumab and Briakinumab
  • Synthetic genes were produced for Ustekinumab, Briakinumab, mAb 5 and mAb 6 at Geneart (Life technologies GmbH, Carlsbad, Calif., USA). Site-directed mutagenesis was used to exchange specific amino acids to produce mAb 1, mAb 2, mAb 7, mAb 8 and mAb 9.
  • MAb 3 was transfected with plasmids encoding Ustekinumab heavy chains and Briakinumab light chains and mAb 4 vice versa.
  • the monoclonal antibodies used herein were transiently expressed in HEK293 cells (see below) and purification was performed by protein A chromatography using standard procedures (see below).
  • the biochemical characterization included size exclusion chromatography (Waters BioSuiteTM 250 7.8 ⁇ 300 mm, eluent: 200 mM KH 2 PO 4 , 250 mM KCl, pH 7.0) and analysis of the molecular weight distribution using the BioAnalyzer 2100 (Agilent technologies, Santa Clara, Calif., USA).
  • Fc fragments were obtained by IdeS digestion of antibodies within 30 minutes at 37° C. using the FabRICATOR-Kit (GENOVIS, Lund, Sweden).
  • variants of expression plasmids for transient expression e.g. in HEK293-F cells based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were applied.
  • the transcription unit of the antibody gene was composed of the following elements:
  • the fusion genes comprising the antibody chains were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective plasmids.
  • the subcloned nucleic acid sequences were verified by DNA sequencing.
  • larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).
  • the antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy chain, as well as the corresponding light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyleTM 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectinTM or fectin (Invitrogen).
  • HEK293-F cells Invitrogen growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyleTM 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectinTM or fectin (Invitrogen).
  • HEK293-F cells were seeded at a density of 1*10 6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO 2 .
  • the antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-SepharoseTM (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.
  • sterile filtered cell culture supernatants were captured on a MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0.
  • the eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0.
  • the antibody pools were prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution to a final concentration of 0.8 M ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid.
  • the antibodies were applied to the resin, washed with equilibration buffer and eluted with a linear gradient to 35 mM sodium acetate pH 5.0.
  • the antibody containing fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0.
  • the antibody containing fractions were pooled, concentrated to the required concentration using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France) and stored at ⁇ 80° C.
  • the functional characterization includes analysis of the interaction with the target (human IL-12) to test if Briakinumab and Ustekinumab were produced correctly and the target binding is still functional.
  • the mAb variants were modified in the Fab region and it is tested if these modifications alter the target binding.
  • the interaction of the antibodies used in the mouse PK study with mouse IL-12/-23 are analyzed to exclude target-mediated clearance effects in the mouse study.
  • binding levels to mouse Fc ⁇ receptor I are measured because stronger binding to mouse Fc ⁇ RI could lead to a faster decrease in a PK study due to faster uptake into antigen presenting cells.
  • the absorbance-concentration curves of the variants with cross-over exchanges (mAb 1-6) and with modified charge distribution (mAb 7-10) are shown in FIGS. 15 and 16 , respectively.
  • Briakinumab, Ustekinumab and mAbs with exchanged Fv domains (mAb 1 and mAb 2) show similar IL-12 binding profiles.
  • the binding of Briakinumab, Ustekinumab and mAb 2 ranges in a 20% window, mAb 3 in a 30% window.
  • MAbs with exchanged LCs (mAb 3and mAb 4) and mAbs with exchanged CDRs (mAb 5 and mAb 6) do not bind to IL-12.
  • Briakinumab variants with modified charge distribution (mAb 7-9) bind IL-12 in a range of 30% relative to Briakinumab indicating similar IL-12 binding. Only mAb 10 shows reduced IL-12 binding with 63% binding compared to Briakinumab.
  • K D (Briakinumab) 0.2 nM vs.
  • K D (Ustekinumab) 0.07 nM).
  • the high affinity of Ustekinumab to IL-12 is also described in literature ([52]).
  • Table 12 summarizes the calculated kinetic parameters of the target interaction.
  • Monoclonal antibodies with exchanged Fv domains (mAb 1and mAb 2) and mAbs with modified charge distributions (mAb 7-10) have affinities to IL-12 similar to Briakinumab and Ustekinumab.
  • MAb 3 and mAb 5 do not bind to IL-12 and mAb 4 and mAb 6 show very weak binding to IL-12. The data is in agreement with the ELISA results.
  • the affinity of the mAbs and FcRn was assessed to be similar to the Ustekinumab-FcRn affinity if differences were smaller than one decimal power to the Ustekinumab-FcRn K D .
  • K D s were assessed to be different if K D differences were bigger than one decimal power to the Ustekinumab-FcRn K D .
  • the FcRn affinities at pH 6.0 fell in a narrow range for all mAbs.
  • Briakinumab had a relative K D of 0.2 and the variants ranged between Briakinumab and Ustekinumab except for mAb 10 that had a relative K D of 1.1.
  • K D values below 1 ⁇ M were assessed to show moderate affinity, between 1-5 ⁇ M to show weak affinity and above 5 ⁇ M to show no binding to FcRn.
  • Briakinumab and Ustekinumab showed similar affinities at pH 6.0.
  • Ustekinumab showed very weak affinity at pH 6.6 and no affinity at pH 6.8.
  • Briakinumab showed a moderate affinity up to pH 6.8, weak affinity at pH 7.0 and no binding at pH 7.2.
  • the F(ab′) 2 fragment and the Fc-region fragment were prepared by incubation for 30 min. at 37° C. using the FabRICATOR-Kit (GENOVIS, Lund, Sweden).
  • the resulting cleavage products F(ab′) 2 and Fc-region were separated on a size exclusion chromatography (SEC) column (Superdex 200, GE Healthcare, Zurich, Switzerland) using an ⁇ KTA Explorer chromatography system (GE Healthcare, Uppsala, Sweden) and the peak fractions were pooled.
  • SEC size exclusion chromatography
  • ⁇ KTA Explorer chromatography system GE Healthcare, Uppsala, Sweden
  • the binding properties of the antibodies to FcRn were analyzed by surface plasmon resonance (SPR) technology using a BlAcore T100 instrument (BIAcore AB, Uppsala, Sweden).
  • SPR surface plasmon resonance
  • BlAcore T100 instrument BIOS AB, Uppsala, Sweden
  • This system is well established for the study of molecular interactions. It allows a continuous real-time monitoring of ligand/analyte bindings and thus the determination of kinetic parameters in various assay settings.
  • SPR-technology is based on the measurement of the refractive index close to the surface of a gold coated biosensor chip. Changes in the refractive index indicate mass changes on the surface caused by the interaction of immobilized ligand with analyte injected in solution.
  • the FcRn receptor was immobilized onto a BIAcore CM5-biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) via amine coupling to a level of 400 Response units (RU).
  • the assay was carried out at room temperature with PBS, 0.05% Tween20 pH 6.0 (GE Healthcare Bioscience) as running and dilution buffer. 200 nM of native or oxidized antibody samples were injected at a flow rate of 50 ⁇ L/min at room temperature. Association time was 180 s, dissociation phase took 360 s.
  • Regeneration of the chip surface was reached by a short injection of HBS-P, pH 8.0.
  • Evaluation of SPR-data was performed by comparison of the biological response signal height at 180 s after injection and at 300 s after injection.
  • the corresponding parameters are the RU max level (180 s after injection) and late stability (300 s after end of injection).
  • the steady state binding levels and the equilibrium dissociation constants (K D ) for huFcRn and the IgGs were determined at pH 6.0 using a BIAcore T100 SPR instrument (GE Healthcare, Little Chalfont, United Kingdom). Human FcRn was immobilized on a BIAcore CM5-biosensor chip (GE Healthcare Bioscience) via amine-coupling to a level of 50 response units (RU). For mAb 5 and mAb 6, a CM4-biosensor chip was used. The assay was performed using PBS with 0.05% Tween20 (both from Roche Diagnostics, Mannheim, Germany) adjusted to pH 6.0 as running and dilution buffer at room temperature.
  • Tween20 both from Roche Diagnostics, Mannheim, Germany
  • a concentration series of the samples was prepared in a range of 1500 nM to 23 nM and each sample was injected at a flow rate of 5 ⁇ L/min. Association and dissociation times of 600 and 360 seconds were used, respectively.
  • the chip was regenerated by injection of PBS containing 0.05% Tween20 at pH 7.5.
  • the equilibrium dissociation constant K D was calculated as steady state affinity and normalized to the K D of Ustekinumab.
  • Mouse husbandry was carried out under specific pathogen free conditions. Mice were obtained from the Jackson Laboratory (Bar Harbor, Me., USA) (female, age 4-10 weeks, weight 17-22 g at time of dosing).
  • a single dose of antibody was injected i.v. via the lateral tail vein at a dose level of 10 mg/kg.
  • the mice were divided into 3 groups of 6 mice each to cover 9 serum collection time points in total (at 0.08, 2, 8, 24, 48, 168, 336, 504 and 672 hours post dose).
  • Each mouse was subjected twice to retro-orbital bleeding, performed under light anesthesia with IsofluraneTM (CP-Pharma GmbH, Burgdorf, Germany); a third blood sample was collected at the time of euthanasia. Blood was collected into serum tubes (Microvette 500Z-Gel, Sarstedt, Numbrecht, Germany). After 2 h incubation, samples were centrifuged for 3 min at 9.300 g to obtain serum. After centrifugation, serum samples were stored frozen at ⁇ 20° C. until analysis.
  • HRP horseradish peroxidase
  • ABTS 2,2′ Azino-di [3-ethylbenzthiazoline sulfonate]; Roche Diagnostics, Germany
  • HRP substrate was used as HRP substrate to form a colored reaction product.
  • Absorbance of the resulting reaction product was read at 405 nm with a reference wavelength at 490 nm using a Tecan sunrise plate reader (Mannedorf, Switzerland).
  • area under the curve (AUC 0-inf ) values were calculated by logarithmic trapezoidal method due to non-linear decrease of the antibodies and extrapolated to infinity using the apparent terminal rate constant ⁇ z, with extrapolation from the observed concentration at the last time point.
  • Plasma clearance was calculated as Dose rate (D) divided by AUC 0-inf .
  • Tukey's HSD test was used as statistical test for analysis of statistically significant differences in the terminal half-life.
  • pH dependent net charge were calculated with the open-source program EMBOSS iep assuming all cysteines involved in disulfide bridges.
  • a homology model for the Briakinumab Fab fragment was generated using modeller 9v7 using PDB structure 1 AQK [41] as a template.
  • the isopotential surfaces for Briakinumab and Ustekinumab Fabs were calculated from this model (Briakinumab) or the crystal structure of Ustekinumab (PDB ID 3HMX), respectively. Structures were protonated using the “prepare protein” protocol with CHARMm force field in DiscoveryStudio Pro, Version 3.5 (Accelrys Inc., San Diego, USA) at pH 7.4 and an ionic strength of 0.145 M. The electrostatic potential was calculated with the “electrostatic potential” protocol in DiscoveryStudio Pro, which invokes the DelPhi program [42].
  • Electrostatic interactions were calculated using PME summation with real-space electrostatic cut-off of 1.0 nm.
  • the Lennard-Jones potential was cut off at 1.0 nm.
  • LINCS was used to constrain all protein bond lengths, allowing a time-step of 2 fs.
  • the temperature was kept constant at 300 K using the V-rescale algorithm. Following energy minimization (target: maximum force ⁇ 1000 kJ/mol/nm), a 30 ps equilibration was performed before a trajectory was simulated over a length of 100 ns.
  • the electrostatic contribution to the non-bonded interactions between the FcRn and the Fab domain which approaches the FcRn in the MD trajectory was calculated with DiscoveryStudio Pro.
  • the protein was protonated at pH 7.4, an ionic strength of 145 mM and a temperature of 37° C. with same settings as described above. Structures were minimized with a maximum of 1000 steps of the “smart minimizer” protocol before interaction energies were calculated using the “calculate interaction energy” protocol with the CHARMm force field in DiscoveryStudio Pro. Implicit waters and the GBMV electrostatics model were used. This calculation was performed at the beginning of the trajectory (0 ns) and at 96 to 100 ns in 1 ns intervals.
  • FcRn was transiently expressed by transfection of HEK293 cells with two plasmids containing the coding sequence of FcRn and of beta-2-microglobulin.
  • the transfected cells were cultured in shaker flasks at 36.5° C., 120 rpm (shaker amplitude 5 cm), 80% humidity and 7% CO 2 .
  • the cells were diluted every 2-3 days to a density of 3 to 4*10 5 cells/ml.
  • a 14 l stainless steel bioreactor was started with a culture volume of 8 l at 36.5° C., pH 7.0 ⁇ 0.2, pO 2 35% (gassing with N 2 and air, total gas flow 200 ml min ⁇ 1 ) and a stirrer speed of 100-400 rpm.
  • 10 mg plasmid DNA (equimolar amounts of both plasmids) was diluted in 400 ml Opti-MEM (Invitrogen). 20 ml of 293fectin (Invitrogen) was added to this mixture, which was then incubated for 15 minutes at room temperature and subsequently transferred into the fermenter.
  • the cells were supplied with nutrients in continuous mode: a feed solution was added at a rate of 500 ml per day and glucose as needed to keep the level above 2 g/l.
  • the supernatant was harvested 7 days after transfection using a swing head centrifuge with 1 l buckets: 4000 rpm for 90 minutes.
  • the supernatant (13 L) was cleared by a Sartobran P filter (0.45 ⁇ m+0.2 ⁇ m, Sartorius) and the FcRn beta-2-microglobulin complex was purified therefrom.
  • FcRn 3 mg FcRn were solved/diluted in 5.3 mL 20 mM sodium dihydrogenphosphate buffer containing 150 mM sodium chloride and added to 250 ⁇ l PBS and 1 tablet complete protease inhibitor (complete ULTRA Tablets, Roche Diagnostics GmbH).
  • FcRn was biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC). The biotinylation reaction was done at room temperature overnight.
  • the biotinylated FcRn was dialyzed against 20 mM sodium dihydrogen phosphate buffer comprising 150 mM NaCl, pH 7.5 at 4° C. overnight to remove excess of biotin.
  • streptavidin sepharose For coupling to streptavidin sepharose, one gram streptavidin sepharose (GE Healthcare, United Kingdom) was added to the biotinylated and dialyzed FcRn and incubated at 4° C. overnight.
  • the FcRn derivatized sepharose was filled in a 1 ml XK column (GE Healthcare, United Kingdom) and the FcRn column then was equilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt buffer containing 140 mM sodium chloride, pH 5.5.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • the receptor derivatized sepharose was filled in a 1 ml XK column (GE Healthcare) and the FcRn column then was equilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) buffer containing 140 mM NaCl, pH 5.5.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • the samples were prepared in 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, 140 mM sodium chloride, pH 5.5. Each sample contained 30 ⁇ g mAb per injection. Antibodies were eluted by a linear pH gradient from pH 5.5 to 8.8 within 120 minutes using 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, 140 mM sodium chloride, pH 5.5 and 20 mM tris(hydroxymethyl)aminomethane TRIS, 140 mM sodium chloride, pH 8.8 as eluents and a flow rate of 0.5 ml/min.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • FcRn column chromatography shows binding at acidic pH (pH 5.5-6.0) and release at higher pH values.
  • pH is increased in the gradient up to pH 8.8.
  • the chromatograms were integrated manually by using the Chromeleon software (Dionex, Germany). The experiments were performed at room temperature. The elution profile was obtained by continuous measurement of the absorbance at 280 nm. To determine the elution pH at particular retention times, samples were collected every 5 minutes and the pH was measured offline.
  • the receptor derivatized sepharose was filled in a 1 ml XK column (GE Healthcare) and the FcRn column then was equilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) buffer containing 400 mM NaCl, pH 5.5.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • the samples were prepared in 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, 400 mM sodium chloride, pH 5.5. Each sample contained 30 ⁇ g mAb per injection. Antibodies were eluted by a linear pH gradient from pH 5.5 to 8.8 within 120 minutes using 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, 400 mM sodium chloride, pH 5.5 and 20 mM tris(hydroxymethyl)aminomethane TRIS, 400 mM sodium chloride, pH 8.8 as eluents and a flow rate of 0.5 ml/min.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • FcRn column chromatography shows binding at acidic pH (pH 5.5-6.0) and release at higher pH values.
  • pH is increased in the gradient up to pH 8.8.
  • the chromatograms were integrated manually by using the Chromeleon software (Dionex, Germany). The experiments were performed at room temperature. The elution profile was obtained by continuous measurement of the absorbance at 280 nm. To determine the elution pH at particular retention times, samples were collected every 5 minutes and the pH was measured offline.
  • the receptor derivatized sepharose was filled in a 1 ml XK column (GE Healthcare) and the FcRn column then was equilibrated with 10 mM 2-(N-morpholine)-ethanesulfonic acid (MES) buffer, pH 7.8.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • the samples were prepared in 10 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, pH 7.8. Each sample contained 30 ⁇ g mAb per injection. Antibodies were eluted by a linear salt gradient from 0 nM to 250 nM sodium chloride within 60 minutes using 10 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, pH 7.8 and 10 mM 2-(N-morpholine)-ethanesulfonic acid (MES) sodium salt, 250 mM sodium chloride, pH 7.8 as eluents and a flow rate of 0.5 ml/min. The experiments were performed at room temperature. The elution profile was obtained by continuous measurement of the absorbance at 280 nm. The chromatograms were integrated manually by using the Chromeleon software (Dionex, Germany).
  • the receptor derivatized sepharose was filled in a 1 ml XK column (GE Healthcare) and the FcRn column then was equilibrated with 20 mM 2-(N-morpholine)-ethanesulfonic acid (MES) buffer containing 150 mM NaCl, pH 5.5.
  • MES 2-(N-morpholine)-ethanesulfonic acid
  • Antibody or fusion protein samples containing 50 to 100 ⁇ g of protein were adjusted to pH 5.5 and applied to the FcRn column using AKTA explorer 10 XT or Dionex Summit (Dionex, Idstein, Germany). The column with 5 cm bed height was then washed with 5-10 column volumes of equilibration buffer 20 mM MES, 150 mM NaCl, pH 5.5. The affinity-bound Fc-containing proteins were eluted with a pH gradient to 20 mM Tris/HCl, 150 mM NaCl, pH 8.8, in 30 column volumes. For complete elution of modified antibodies, the pH was increased in the gradient up to pH 8.8. The experiments were carried out at room temperature. The elution profile was obtained by continuous measurement of the absorbance at 280 nm. The time taken for an analyte peak, X, to reach the detector after sample injection was called the retention time.
  • the clarified supernatants containing hexahis-tagged proteins were loaded on a Ni-NTA affinity chromatography resin (Qiagen, Hanbrechtikon, Switzerland) at 4° C. After wash steps with 20 mM sodium phosphate buffer comprising 500 mM NaCl at pH 7.4 and containing 20 mM respectively 100 mM imidazole, proteins were eluted at a flow rate of 2 ml/min using batch elution with the same buffer containing 300 mM imidazole on an AKTA Prime chromatography system (Amersham Pharmacia Biotech, Uppsala, Sweden).
  • Fractions were pooled and further purified in sodium phosphate buffer containing 500 mM NaCl on size exclusion chromatography (SuperdexTM 200, GE Healthcare, Zurich, Switzerland). Purified proteins were quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, Del.) and analyzed by SDS PAGE on NuPAGE 4-12% Bis-Tris gels in MES buffer under denaturing and reducing conditions.
  • Cynomolgus FcRn affinity column behaves similar as human FcRn affinity column concerning binding of humanized antibodies. On the other hand binding of humanized antibodies to murine FcRn column is stronger than to human FcRn affinity column as can be seen by later retention.
  • the F(ab′) 2 fragment and the Fc-region fragment were prepared by cleavage of the full-length antibody 1:1 diluted with 100 mM Tris, pH 8.0, by adding 1 ⁇ g IdeS cysteine protease per 50 ⁇ g antibody and incubation for 2 h at 37° C.
  • the resulting cleavage products F(ab′) 2 and Fc were separated on a size exclusion chromatography (SEC) column (Superdex 200, GE Healthcare, Zurich, Switzerland) using an ⁇ KTA Explorer chromatography system (GE Healthcare, Uppsala, Sweden) and the peak fractions were pooled.
  • SEC size exclusion chromatography
  • mice Male and female C57BL/6J mice (background); mouse FcRn deficient, but hemizygous transgenic for human FcRn (huFcRn (276) ⁇ /tg (30, 31) were used throughout the pharmacokinetic study.
  • the animals weighed between 17 and 25 g.
  • the respective antibody was given as a single intravenous bolus injection via the tail vein. Due to limited blood volume of mice, three groups of four male and four female animals each were required to cover nine sampling time points, i.e. three sampling time points per animal. Blood samples were taken in group 1 at 5 min, 24 hours and 336 hours, in group 2 at 2 hours, 168 hours and 504 hours and in group 3 at 8 hours, 48 hours and 672 hours after administration. Blood samples of about 100 ⁇ L were obtained by retrobulbar puncture and stored at room temperature for 60 min. to allow clotting. Serum samples of at least 40 ⁇ L were obtained by centrifugation at 9,300 ⁇ g at 4° C. for 3 min and immediately frozen and stored at ⁇ 20° C. until assayed.
  • Serum concentrations of the human therapeutic antibodies in murine serum were determined by an antigen-captured enzyme linked immunosorbent assay (ELISA) specific for the antigen binding region (Fab) of the administered antibody and its variants. All reagents or samples were incubated at room temperature on a shaker at 400 rpm. Each washing step included three cycles. Briefly, streptavidin-coated microtiter plates were coated with biotinylated antibody diluted in assay buffer. After washing with phosphate-buffered saline-polysorbate 20 (Tween20), serum samples in various dilutions were added and incubated for 1 h.
  • ELISA antigen-captured enzyme linked immunosorbent assay
  • human therapeutic antibodies were detected by subsequent incubation with human Fcy-specific monoclonal antibody Fab fragments conjugated with digoxigenin that do not cross react with mouse IgG.
  • an anti-digoxigenin antibody conjugated with horseradish peroxidase (HRP) was added and incubated for 1 h.
  • HRP substrate was added as HRP substrate to form a colored reaction product. Absorbance of the resulting reaction product was read at 405 nm with a reference wavelength at 490 nm. All serum samples and positive or negative control samples were analyzed in replicates and calibrated against reference standard.
  • AUC(0-672) clearance terminal half-life antibody [h*[g/ml] [ml/min/kg] [h] wild-type antibody 15.693 ⁇ 1.879 0.0107 ⁇ 0.0013 96.8 ⁇ 8.9 YTE-mutant 27.359 ⁇ 2.731 0.0055 ⁇ 0.0006 211.4 ⁇ 40.6
  • mice Male and female C57BL/6J mice (background); mouse FcRn deficient, but hemizygous transgenic for human FcRn (huFcRn (276) ⁇ /tg (30, 31) were used throughout the pharmacokinetic study.
  • the respective antibody was given as a single intravenous bolus injection (10 mg/kg). Due to limited blood volume of mice, three groups of six animals each were required to cover nine sampling time points. The last sampling point was four weeks after administration.
  • PK parameters were calculated and summarized: AUC 0-inf , Cl, V SS and T 1/2 .
  • PK parameters in FcRn knockout mice were calculated after administration of 10 mg/kg to 6 animals per group.
  • PK data represent the mean ⁇ standard deviation.
  • AUC 0-inf Cl V SS T 1/2 sample [h*mg/mL] [mL/min/kg] [L/kg] [h] Briakinumab 1.0 ⁇ 0.1 0.163 ⁇ 0.008 0.113 ⁇ 0.004 10.6 ⁇ 0.6
  • Ustekinumab and mAb 9 are comparable regarding AUC 0-inf , Cl, V SS and T 1/2 .
  • Briakinumab has a smaller AUC 0-inf , faster Cl and smaller T 1/2 than Ustekinumab and mAb 9.
  • the calculation of the T 1/2 might differ from the actual value, because time points after 3 and 4 days would have been needed to calculate the terminal half-lives more precisely.
  • the statistical analysis of the terminal half-lives was calculated using the Tukey HSD Test. A statistical significance could be detected between the terminal half-lives of Briakinumab and Ustekinumab and of Briakinumab and mAb 9.
  • ADAs The formation of ADAs was tested by detection of drug/ADA immune complexes.
  • FcRn knockout mice administration of 10 mg/kg Briakinumab resulted in formation of Briakinumab/ADA immune complexes after about 168-192 hours (7-8 days).
  • ADA-positive samples after Briakinumab administration in FcRn knockout mice Serum concentrations of each sampling time point after i.v. administration of 10 mg/kg Briakinumab in FcRn knockout mice.
  • ADA- positive samples are illustrated as * and ** describing formation of moderate and severe drug/ADA immune complexes, respectively.
  • ** b.l.q. * b.l.q. ** b.l.q. ** 216/9 b.l.q. * b.l.q. b.l.q. ** b.l.q. ** b.l.q. ** 336/14 b.l.q. b.l.q. ** b.l.q. * b.l.q. ** b.l.q. ** b.l.q. * b.l.q. * b.l.q. * b.l.q. * b.l.q. * b.l.q. * b.l.q. below limit of quantification
  • Serum concentrations of Briakinumab in human FcRn transgenic mice Serum concentrations are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group. ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.
  • Serum concentrations of Ustekinumab in human FcRn transgenic mice Serum concentrations are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group. ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.
  • Serum concentrations of mAb 8 in human FcRn transgenic mice are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group. ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.
  • Serum concentrations of mAb 9 in human FcRn transgenic mice are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group.
  • ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.
  • Serum concentrations of Briakinumab in FcRn knockout mice Serum concentrations are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group. ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.
  • Serum concentrations of Ustekinumab in FcRn knockout mice are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group.
  • time M 1 M 2 M 3 M 4 M 5 M 6 Mean SD [h/d] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] [ ⁇ g/mL] 0.08 209 221 229 228 220 219 221 7.4 2 153 164 158 155 157 149 156 4.8 8 80 95 88 96 104 95 93 8.0 24/1 50 47 37 44 38 37 42 5.5 48/2 16 16 17 13 11 14 15 2.2 168/7 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.3 0.6 0.4 0.5 0.1 192/8 0.5 0.2 0.1 0.1 0.2 0.4 0.2 216/9 b.l.q.
  • Serum concentrations of mAb 9 in FcRn knockout mice Serum concentrations are determined after administration of a 10 mg/kg single dose i.v. injection to 6 animals per group. ADA-positive samples are illustrated as * and ** for formation of moderate and severe drug/ADA immune complexes, respectively.

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EP3608674A1 (en) * 2018-08-09 2020-02-12 Regeneron Pharmaceuticals, Inc. Methods for assessing binding affinity of an antibody variant to the neonatal fc receptor
WO2020084032A1 (en) 2018-10-25 2020-04-30 F. Hoffmann-La Roche Ag Modification of antibody fcrn binding
CN113308476A (zh) * 2021-05-12 2021-08-27 广东药康生物科技有限公司 一种FcRn基因人源化动物模型的构建方法
US11440971B2 (en) 2014-11-06 2022-09-13 Hoffmann-La Roche Inc. Fc-region variants with modified FcRn-binding and methods of use
EP3903102B1 (en) 2018-12-30 2023-04-12 F. Hoffmann-La Roche AG Ph-gradient spr-based binding assay
US11668722B2 (en) * 2017-04-28 2023-06-06 Hoffmann-La Roche Inc. Antibody selection method
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US20160275238A1 (en) * 2013-09-06 2016-09-22 Hoffmann-La Roche Inc. Method for improving antibody stability
US10418126B2 (en) * 2013-09-06 2019-09-17 Hoffmann-La Roche Inc. Method for improving antibody stability
US11440971B2 (en) 2014-11-06 2022-09-13 Hoffmann-La Roche Inc. Fc-region variants with modified FcRn-binding and methods of use
US11668722B2 (en) * 2017-04-28 2023-06-06 Hoffmann-La Roche Inc. Antibody selection method
EP3608674A1 (en) * 2018-08-09 2020-02-12 Regeneron Pharmaceuticals, Inc. Methods for assessing binding affinity of an antibody variant to the neonatal fc receptor
US12172106B2 (en) 2018-08-09 2024-12-24 Regeneron Pharmaceuticals, Inc. Methods for assessing binding affinity of an antibody variant to the neonatal Fc receptor
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US12351622B2 (en) 2018-12-05 2025-07-08 Bica Therapeutics Inc. Modified product of Fc domain of antibody
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CN113308476A (zh) * 2021-05-12 2021-08-27 广东药康生物科技有限公司 一种FcRn基因人源化动物模型的构建方法

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