HK1218761B - Fc-receptor binding modified asymmetric antibodies and methods of use - Google Patents

Description

Modified asymmetric antibodies that bind to FC receptors and their usage

Technical Field

This invention relates to antibodies and Fc region fusion peptides that are asymmetrically modified in terms of their Fc-receptor interaction, particularly their FcRn interaction, and methods of using thereof.

Background Technology

Almost all Fc receptors bind asymmetrically to the symmetrical Fc region of the antibody.

For example, human Fcγ-receptor IIIA interacts with different amino acid residues on the two Fc regions of the polypeptide chain. Therefore, asymmetrically introduced mutations (e.g., at residues 233 to 238 in the lower hinge region) are needed to increase or decrease the interaction between the antibody and human Fcγ-receptor IIIA.

However, the interactions between human neonatal Fc receptors (FcRn) are symmetric: two FcRn molecules can bind to a single IgG in a 2:1 stoichiometric ratio (see, for example, Huber, A.W. et al., J. Mol. Biol. 230 (1993) 1077-1083). Therefore, mutations introduced asymmetrically reduce binding to one FcRn/by its binding, but do not reduce binding to both FcRn/by their binding.

Examples of asymmetric IgG-like molecules include, but are not limited to, those obtained using the following techniques or styles: Triomab/Quadroma, Knobs-into-Holes, CrossMab, electrostatically matched antibodies, LUZ-Y, chain-exchange engineered domain bodies, Biclonic, and DuoBody.

In WO 2012/125850, a protein containing an Fc region was reported, which has an asymmetric substitution in its Fc region and has increased binding to human Fcγ-receptor IIIA and enhanced ADCC activity.

In WO 2012/58768, isolated heterodimers containing a heterodimer Fc region were reported, wherein the heterodimer Fc region contains a variant CH3 domain containing amino acid mutations that promote heterodimer formation under conditions of increased stability, and wherein the heterodimer Fc region also contains a variant CH2 domain containing asymmetric amino acid modifications to promote selective binding to the Fcγ receptor.

In WO 2011/131746, it was reported that by introducing asymmetric mutations in the CH3 region of two single-specific initiation proteins, the Fab-arm exchange reaction could be forced to become oriented, thus producing a highly stable heterodimeric protein.

Kim et al. (Kim, H. et al., Invest. Ophthalmol. Vis. Sci. 49 (2008) 2025-2029) reported the presence of FcRn transcripts of predicted size in ocular tissues, including the ciliary body and iris, retina, conjunctiva, cornea, lens, and optic nerve bundle, except for the retinal pigment epithelium and choroidal tissue. The blood-eye barrier shows FcRn receptor expression, suggesting that IgG transport from ocular tissues to the bloodstream may utilize this receptor. Since intraocular tissues such as the retina are isolated from the bloodstream via the blood-eye barrier, it would be expected that full-length antibodies would be undetectable in the bloodstream for only a short time after intravitreal injection. However, recent pharmacokinetic data from monkeys and humans show that bevacizumab appears in the bloodstream within hours after intravitreal injection. Therefore, it is possible that FcRn receptor function in the conjunctival lymphatic vessels will act as an efflux receptor to effectively eliminate antigen-antibody IgG complexes from the conjunctival space. Despite similar molecular weights, IgG (150 kDa) was detected in the aqueous humor, but IgA (160 kDa) was not. The inconsistency between the infiltration of IgG and IgA from serum into the aqueous humor can be explained by the presence of FcRn receptors that are selective for IgG.

Kim et al. further reported (Kim, H. et al., Mol. Vis. 15 (2009) 2803-2812) that direct intravitreal injection has become a common protocol for delivering therapeutic antibodies to the posterior segment of the eye for retinal diseases. Both intravitreal administrations of bevacizumab (IgG) and chicken IgY crossed the internal limiting membrane barrier and diffused into deeper retinal structures. After diffusion across the retina, bevacizumab crossed the blood-retinal barrier and leaked into the systemic circulation. Intraretinal chicken IgY localized only along the luminal side of the blood-retinal barrier. Furthermore, choroidal vessels were negative for chicken IgY. Following intravitreal administration, physiologically relevant serum levels of bevacizumab were found to be up to 30% of the injected dose. This suggests a greater risk of systemic side effects than previously recognized. The blood-eye barrier represents a specific mechanism for transporting full-length IgG and clearing it into the systemic circulation. Kim's study confirms the hypothesis that this mechanism involves nascent Fc receptors.

US 2011/054151 reports compositions and methods for the simultaneous bivalent and monovalent co-binding of antigens.

US 2011/236388 reported a bispecific bivalent anti-VEGF/anti-ANG-2 antibody.

An antibody fusion protein modified with an FcRn binding site was reported in WO 2010/121766.

Kim, J.K. et al. reported the localization of the site on human IgG that binds to the MHC class I-related receptor FcRn (Eur. J. Immunol. 29 (1999) 2819-2825).

Qiao, S.-W. et al. reported antibody-mediated antigen presentation-dependent FcRn (Proc. Natl. Acad. Sci. USA 105 (2008) 9337-9342).

Kuo, T.T. et al. reported on the new Fc receptor: from immunity to therapeutics (J. Clin. Immunol. 30 (2010) 777-789).

Firan, M. et al. reported that the MHC class I-associated receptor FcRn plays an essential role in human maternal-fetal γ-globulin transfer (Int. Immunol. 13(2001) 993-1002).

Vidarsson, G. et al. reported that FcRn is an IgG receptor on phagocytes with a novel role in phagocytosis (Blood 108 (2006) 3573-3579).

Gillies, S.D. et al. reported the effectiveness of antibody-interleukin 2 fusion protein by reducing the interaction between the antibody-interleukin 2 fusion protein and the Fc receptor (Cancer Res. 59 (1999) 2159-2166).

The production of heterodimeric proteins was reported in WO 2013/060867.

Summary of the Invention

It has been found that the FcRn binding of antibodies or Fc region fusion peptides can be modified by altering amino acid residues at non-corresponding positions of the individual Fc region peptides, as these alterations work together to modify FcRn binding. Antibodies and Fc region fusion peptides as reported in this article are useful, for example, in treating diseases where a customized systemic retention time is required.

One aspect reported in this article is a variant (human) IgG Fc region containing a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The first Fc region polypeptide and the second Fc region polypeptide are derived from the same parental (human) IgG Fc region polypeptide, and

b) The first Fc region polypeptide has an amino acid sequence that differs from the second Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system.

Therefore, compared with the (human) IgG Fc region, which has the same amino acid residues (as the (parental) human Fc region polypeptide) at the corresponding positions according to the KabatEU index numbering system in the first and second Fc regions, the variant (human) IgG Fc region has a different affinity for the human Fc receptor.

One aspect reported in this article is a variant (human) IgG Fc region containing a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The first Fc region polypeptide has an amino acid sequence that differs from the second Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system.

Therefore, compared with the IgG-type Fc region which has the same amino acid residues at the corresponding positions in the first and second Fc region polypeptides (as in the corresponding human Fc region), the variant (human) IgG-type Fc region has a different affinity for human Fc receptor.

One aspect reported in this article is a variant (human) IgG Fc region containing a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The amino acid sequence of the first Fc region polypeptide differs from that of the first parent IgG-type Fc region polypeptide in one or more amino acid residues.

and

The amino acid sequence of the second Fc region polypeptide differs from that of the second parent IgG-type Fc region polypeptide in one or more amino acid residues, and

b) The first Fc region polypeptide has an amino acid sequence that differs from the second Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system.

Therefore, compared with the parental IgG Fc region of the first and second parental IgG Fc region polypeptides containing a), the variant (human) IgG Fc region has a different affinity for the human Fc receptor.

One aspect reported in this article is a variant (human) IgG Fc region containing a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The amino acid sequence of the first Fc region polypeptide is derived from the first parental IgG-type Fc region polypeptide, and the amino acid sequence of the second Fc region polypeptide is derived from the second parental IgG-type Fc region polypeptide.

b) Introducing one or more mutations into the first Fc region polypeptide and/or the second Fc region polypeptide, such that the first Fc region polypeptide has an amino acid sequence that differs from the amino acid sequence of the second Fc region polypeptide at at least one corresponding position according to the KabatEU index numbering system.

Therefore, compared with the IgG Fc region of the first and second parental IgG Fc region polypeptides containing a), the variant (human) IgG Fc region has a different affinity for the human Fc receptor.

In one implementation across all aspects, the variant (human) IgG class Fc region is a variant (human) IgG class heterodimeric Fc region.

In one embodiment of all aspects, the first parental IgG Fc region polypeptide and the second parental IgG Fc region polypeptide are non-human IgG Fc region polypeptides.

In one embodiment of all aspects, the first parental IgG Fc region polypeptide and the second parental IgG Fc region polypeptide are the same IgG Fc region polypeptide.

In one embodiment of all aspects, the first Fc region polypeptide and the second Fc region polypeptide pair to form a dimer (functional) Fc region, resulting in the formation of a heterodimer.

In one embodiment of all aspects, the first and second Fc region polypeptides are independently distinct from each other in at least one amino acid residue from their respective parent IgG class Fc region polypeptides.

In one implementation of all aspects, the IgG class is selected from subclasses IgG1, IgG2, IgG3 and IgG4.

In one embodiment of all aspects, the human Fc-receptor is selected from the human neonatal Fc-receptor and the human Fcγ-receptor.

In one embodiment of all aspects, the first Fc region polypeptide differs from the second Fc region polypeptide in one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid residues at the corresponding position according to the KabatEU index numbering system.

In one implementation, the variant (human) IgG class Fc region has weakened Staphylococcus protein A binding activity compared to the corresponding parental human IgG class Fc region.

In one implementation, the variant (human) IgG class Fc region has the same staphylococcal protein A binding activity as the corresponding parent IgG class Fc region.

In one embodiment of all aspects, the variant (human) IgG class Fc region comprises both a first and a second Fc region polypeptide of human IgG1 or human IgG4 subclass (derived from human origin), wherein the first Fc region polypeptide contains one or two mutations selected from group i) I253A, H310A and H435A, or group ii) H310A, H433A and Y436A, or group iii) L251D, L314D and L432D, or group iv) L251S, L314S and L432S (numbered according to the Kabat EU index numbering system) and in the second Fc region polypeptide. It contains one or two mutations selected from the mutations L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, and Y436A (numbered according to the Kabat EU index numbering system), such that all mutations i) I253A, H310A, and H435A, or ii) H310A, H433A, and Y436A, or iii) L251D, L314D, and L432D, or iv) L251S, L314S, and L432S are contained in the Fc region of the variant (human) IgG.

In one embodiment of all aspects, the variant (human) IgG class Fc region comprises a first or second Fc region polypeptide of a human IgG1 or human IgG4 subclass (derived from human origin), said polypeptide comprising, in the Fc region, a mutant I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or a combination thereof (according to the Kabat EUindex numbering system). (number), therefore i) all mutations are in the first or second Fc region polypeptide, or ii) one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, so that all mutations i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S are all contained in the Fc region.

In one embodiment of all aspects, the variant (human) IgG class Fc region comprises the first and second Fc region polypeptides of the human IgG1 subclass, wherein

a) The first and second Fc region peptides also contain mutants L234A and L235A (numbered according to the Kabat EU index system), or

b) The first and second Fc region polypeptides also contain the mutant P329G (numbered according to the Kabat EU index system), or

c) The first and second Fc region peptides also contain mutations L234A and L235A and P329G (numbered according to the Kabat EUindex numbering system), or

d) Both the first and second Fc region peptides further contain mutations L234A and L235A (numbered according to the Kabat EU index system), and the first Fc region peptide further contains mutations Y349C or S354C and T366W, and the second Fc region peptide further contains mutations Y349C or S354C and mutations T366S, L368A and Y407V, or

e) The first and second Fc region peptides also contain mutations L234A and L235A and P329G (numbered according to the Kabat EUindex numbering system), and the first Fc region peptide also contains mutations Y349C or S354C and mutation T366W, and the second Fc region peptide also contains mutations Y349C or S354C and mutations T366S, L368A and Y407V.

In one embodiment, the variant (human) IgG class Fc region comprises the first and second Fc region polypeptides of the human IgG4 subclass, wherein

a) The first and second Fc region peptides also contain the mutants S228P and L235E (numbered according to the Kabat EU index system), or

b) Both the first and second Fc region polypeptides also contain the mutant P329G (numbered according to the Kabat EU index system), or

c) The first and second Fc region peptides also contain mutants S228P and L235E and P329G (numbered according to the Kabat EUindex numbering system), or

d) Both the first and second Fc region peptides also contain the mutations S228P and L235E (numbered according to the Kabat EU index system), and the first Fc region peptide also contains the mutations Y349C or S354C and T366W, and the second Fc region peptide also contains the mutations Y349C or S354C and the mutations T366S, L368A and Y407V.

e) The first and second Fc region peptides also contain mutations S228P and L235E and P329G (numbered according to the Kabat EUindex numbering system), and the first Fc region peptide also contains mutations Y349C or S354C and mutation T366W, and the second Fc region peptide also contains mutations Y349C or S354C and mutations T366S, L368A and Y407V.

One aspect, as reported in this article, is antibodies or Fc region fusion peptides containing variant (human) IgG Fc regions, as reported in this article.

In one implementation, the antibody is a monoclonal antibody.

In one implementation, the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

One aspect, as reported in this article, is the nucleic acid encoding the Fc region of the variant (human) IgG class as reported in this article.

One aspect, as reported in this article, is the nucleic acid encoding the antibody, as reported in this article.

One aspect, as reported in this article, is the nucleic acid encoding the Fc region fusion polypeptide, as reported in this article.

One aspect, as reported in this article, is the host cell containing nucleic acids as reported in this article.

One aspect, as reported herein, is a method for generating a variant (human) IgG class Fc region as reported herein, the method comprising culturing host cells as reported herein to generate the variant (human) IgG class Fc region.

One aspect, as reported herein, is a method for generating antibodies as reported herein, the method comprising culturing host cells as reported herein, thereby generating antibodies.

One aspect, as reported herein, is a method for generating an Fc region fusion peptide as reported herein, the method comprising culturing host cells as reported herein to generate the Fc region fusion peptide.

One aspect, as reported herein, is a pharmaceutical formulation comprising a variant (human) IgG class Fc region as reported herein, an antibody as reported herein, or an Fc region fusion polypeptide as reported herein.

One aspect reported in this article is the use of variant (human) IgG Fc region as reported in this article, or antibodies or Fc region fusion peptides as reported in this article, as used as pharmaceuticals.

One aspect reported in this article is the use of variant (human) IgG Fc region antibodies or Fc region fusion peptides as reported in this article in the manufacture of pharmaceuticals.

The antibodies reported in this article can be used, for example, as T cell recruiters, as Fcγ receptor conjugates with high biological activity (potency) and rapid clearance from the bloodstream (serum), as antibody-drug conjugates with rapid clearance to reduce systemic side effects, or as pre-targeting antibodies.

Attached Figure Description

Figure 1. A conceptual diagram and advantages of <VEGF-ANG-2>IgG1 or IgG4 antibodies with IHH-AAA mutations (= a combination of mutations I253A, H310A and H435A (according to Kabat's EU index number)).

Figure 2. Viscosity measurements based on small-scale DLS: extrapolated viscosity at 150 mg/mL in 200 mM arginine/succinate, pH 5.5 (comparison of <VEGF-ANG-2> antibody VEGFang2-0016 (with IHH-AAA mutation) with reference antibody VEGFang2-0015 (without this type of IHH-AAA mutation)).

Figure 3. Temperature-dependent DLS aggregation (including DLS aggregation initiation temperature) in 20 mM histidine buffer, 140 mM NaCl, pH 6.0 (compare the <VEGF-ANG-2> antibody VEGFang2-0016 (with IHH-AAA mutation) as reported in this paper with the reference antibody VEGFang2-0015 (without this type of IHH-AAA mutation)).

Figure 4. Storage at 40°C for 7 days at 100 mg/mL (main peak decrease and high molecular weight (HMW) increase) (comparison shows less aggregation of the <VEGF-ANG-2> antibody VEGFang2-0016 (with IHH-AAA mutation) reported in this paper, and the reference antibody VEGFang2-0015 (without this type of IHH-AAA mutation)).

Figure 5A and B. A: FcRn steady-state affinity of VEGFang2-0015 (without IHH-AAA mutation) and B: VEGFang2-0016 (with IHH-AAA mutation).

Figure 6. FcγRIIIa interaction measurements of VEGFang2-0015 without the IHH-AAA mutation and VEGFang2-0016 with the IHH-AAA mutation (both are IgG1 subclasses with the P329GLALA mutation; as controls, anti-digoxin antibody (anti-Dig) and IgG4-based antibody of the IgG1 subclass were used).

Figure 7A illustrates the principle of pharmacokinetic (PK)-ELISA assay for determining the concentration of <VEGF-ANG-2> bispecific antibody in serum and whole-eye lysates.

Figure 7B: Serum concentrations after intravenous (i.v.) administration: Comparison of VEGFang2-0015 without IHH-AAA mutation and VEGFang2-0016 with IHH-AAA mutation.

Figure 7C: Serum concentrations after intravitreal application: Comparison of VEGFang2-0015 without IHH-AAA mutation and VEGFang2-0016 with IHH-AAA mutation.

Figure 7D. Ocular lysate concentrations of VEGFang2-0016 (with IHH-AAA mutation) in the right and left eyes (compared to intravenous application, intravitreal application was only performed in the right eye): After intravitreal application, a significant concentration was detected only in the right eye; after intravenous application, no concentration was detected in the ocular lysate due to the low serum half-life of VEGFang2-0016 (with IHH-AAA mutation).

Figure 7E shows the concentration of ocular lysates of VEGFang2-0015 (without the IHH-AAA mutation) detected in the right and left eyes (compared to intravenous application, intravitreal application was only performed in the right eye): VEGFang2-0015 concentration was detected in the right eye (and to some extent in the left eye) after intravitreal application; this shows diffusion from the right eye into the serum and from there into the left eye, which can be explained by the long half-life of VEGFang2-0015 (without the IHH-AAA mutation); significant concentrations were detected in ocular lysates in both eyes after intravenous application, due to the diffusion of serum-stable VEGFang2-0015 (without the IHH-AAA mutation) into the eyes.

Figure 8. Engineered antibodies compared to the reference wild-type (wt) antibody in terms of their ability to bind to FcRn, showing prolonged in vivo half-life (YTE mutation) or shortened in vivo half-life (IHH-AAA mutation), enhanced (YTE mutation) or weakened binding (IHH-AAA mutation) in SPR analysis, and enhanced or reduced retention time in FcRn column chromatography; a) PK data after a single intravenous bolus administration of 10 mg/kg to huFcRn transgenic male C57BL/6J mice +/- 276: AUC data for wild-type IgG and YTE and IHH-AAA Fc-modified IgG; b) BIAcore sensor map; c) FcRn affinity column elution; wild-type anti-IGF-1R antibody (reference), YTE-mutant anti-IGF-1R antibody, IHH-AAA-mutant anti-IGF-1R antibody.

Figure 9 shows the variation in retention time in FcRn affinity chromatography depending on the number of mutations introduced into the Fc region.

Figure 10 shows the variation in FcRn binding depending on the asymmetric distribution of mutations introduced into the Fc region.

Figure 11 shows the elution chromatography of a bispecific <VEGF-ANG-2> antibody (VEGF/ANG2-0121) with mutants H310A, H433A, and Y436A in both heavy chains from two consecutive protein A affinity chromatography columns.

Figure 12 shows the elution chromatography of anti-IGF-1R antibody (IGF-1R-0045) with mutations H310A, H433A, and Y436A in both heavy chains from a protein A affinity chromatography column.

Figure 13. IgG Fc region modified <VEGF-ANG-2> antibody binds to immobilized protein A on the CM5 chip.

Figure 14 Elution chromatography of different <VEGF-ANG-2> antibodies on an FcRn affinity column.

Figure 15. Binding of different fusion peptides to Staphylococcus protein A (SPR).

Figure 16. Binding of different <VEGF-ANG-2> antibodies and anti-IGF-1R antibody mutants to immobilized protein A (SPR).

Detailed Implementation

I. Definition

The term "about" refers to a range of +/-20% of the following values. In one embodiment, the term "about" refers to a range of +/-10% of the following values. In another embodiment, the term "about" refers to a range of +/-5% of the following values.

For the purposes of this document, a “receptor human framework” is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common sequence framework, as defined below. A receptor human framework “derived from” a human immunoglobulin framework or a human common framework may contain the same amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL receptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human common framework sequence.

"Affinity-mature" antibodies are those with one or more alterations in one or more hypervariable regions (HVRs), which result in improved affinity of the antibody for the antigen compared to parental antibodies that do not possess these alterations.

The term "modification" refers to the mutation (replacement), insertion (addition), or deletion of one or more amino acid residues in a parent antibody or fusion peptide (e.g., a fusion peptide containing at least the FcRn binding portion of the Fc region) to obtain a modified antibody or fusion peptide. The term "mutation" refers to the replacement of the referred amino acid residue with a different amino acid residue. For example, mutation L234A refers to the replacement of the lysine residue at position 234 in the Fc region (peptide) of the antibody with the amino acid residue alanine (lysine replacing alanine) (according to the EU index number).

The term "amino acid mutation" refers to the substitution of at least one existing amino acid residue for another different amino acid residue (= substitution amino acid residue). The substitution amino acid residue can be a "naturally occurring amino acid residue" and is selected from alanine (three-letter code: alanine (three-letter code: ala, single-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, Y), and valine (val, V). The substitution amino acid residue can also be a "non-naturally occurring amino acid residue." See also For example, US 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 references are incorporated herein by reference in their entirety).

The term "amino acid insertion" refers to the 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 can be any naturally occurring or non-naturally occurring amino acid residue.

The term "amino acid deletion" refers to the removal of at least one amino acid residue at a predetermined position in an amino acid sequence.

As used herein, the term “ANG-2” refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated as ANGPT2 or ANG2) (SEQ ID NO:31), which is described, for example, in Maisonpierre, P.C. et al., Science 277 (1997) 55-60 and Cheung, A.H. et al., Genomics 48 (1998) 389-91. Angiopoietin-1 (SEQ ID NO:32) and -2 have been found to be ligands for Tie, a family of tyrosine kinases selectively expressed in the endothelium of blood vessels (Yancopoulos, G.D. et al., Nature 407 (2000) 242-248). Currently, four members of the angiopoietin family are identified. Angiopoietin-3 and-4 (ANG-3 and ANG-4) can broadly represent divergent counterparts at the same locus in mice and humans (Kim, I. et al., FEBS Let, 443 (1999) 353-356; Kim, I. et al., J. Biol. Chem. 274 (1999) 26523-26528). ANG-1 and ANG-2 were initially identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG-1, see Davis, S. et al., Cell 87 (1996) 1161-1169; and for ANG-2, see Maisonpierre, P.C. et al., Science 277 (1997) 55-60). All known angiopoietins primarily bind to Tie2 (SEQ ID NO:33), and both ANG-1 and -2 bind to Tie2 with an affinity of 3 nM (Kd) (Maisonpierre, P.C. et al., Science 277 (1997) 55-60).

The term “antibody” is used in the broadest sense herein and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments, provided they exhibit the desired antigen- and/or protein A and/or FcRn-binding activity.

"Antibody that binds to the same epitope as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of that antibody to its antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

The term "asymmetric Fc region" refers to a pair of Fc region polypeptides with different amino acid residues at corresponding positions according to the Kabat EU index numbering system.

The term "asymmetric Fc region concerning FcRn binding" refers to an Fc region consisting of two polypeptide chains with different amino acid residues at corresponding positions, where the positions are determined according to the KabatEU index numbering system, and thus different positions affect the binding of the Fc region to the human neonatal Fc-receptor (FcRn). For the purposes of this document, the differences between the two polypeptide chains in an asymmetric Fc region concerning FcRn binding do not include differences that have been introduced to promote the formation of heterodimeric Fc regions (e.g., for the generation of bispecific antibodies). These differences can also be asymmetric, i.e., the two chains differ at non-corresponding amino acid residues according to the KabatEU index numbering system. These differences promote heterodimerization and reduce homodimerization. An example of this type of difference is the so-called "knot-in-knot" substitution (see, for example, US7 695 936 and US 2003/0078385). The following knot and tack substitutions in the Fc region of IgG antibodies in the IgG1 subclass have been found to increase heterodimer formation: 1) Y407T in one chain and T366Y in another; 2) Y407A in one chain and T366W in another; 3) F405A in one chain and T394W in another; 4) F405W in one chain and T394S in another; 5) Y407T in one chain and T394S in another. 66Y; 6) T366Y and F405A in one chain and T394W and Y407T in another chain; 7) T366W and F405W in one chain and T394S and Y407A in another chain; 8) F405W and Y407A in one chain and T366W and T394S in another chain; and 9) T366W in one chain and T366S, L368A, and Y407V in another chain, with the last listed being particularly appropriate. Furthermore, changes in the formation of new disulfide bridges between the two Fc region polypeptide chains promote heterodimer formation (see, for example, US 2003/0078385). The following substitutions have been found to increase heterodimer formation in single polypeptide chains of IgG antibodies Fc regions of IgG1 subclasses, producing appropriately spaced cysteine residues that facilitate the formation of new intrachain disulfide bonds: Y349C in one chain and S354C in another; Y349C in one chain and E356C in another; Y349C in one chain and E357C in another; L351C in one chain and S354C in another; T394C in one chain and E397C in another; or D399C in one chain and K392C in another. Other examples of amino acid changes that promote heterodimerization are so-called “charge-pair substitutions” (see, for example, WO 2009/089004). The following charge pair substitutions in the Fc region of IgG antibodies in the IgG1 subclass have been found to increase heterodimer formation: 1) K409D or K409E in one chain and D399K or D399R in the other chain; 2) K392D or K392E in one chain and D399K or D399R in the other chain; 3) K439D or K439E in one chain and E356K or E356R in the other chain; 4) K370D or K370E in one chain and E357K or E357R in the other chain; 5) K409D and K360D in one chain plus D399K and E356K in the other chain; 6) K409D and K370D in one chain plus D399K and E356K in the other chain. 7K; 7) K409D and K392D in one chain plus D399K, E356K and E357K in another chain; 8) K409D and K392D in one chain and D399K in another chain; 9) K409D and K392D in one chain and D399K and E356K in another chain; 10) K409D and K392D in one chain and D399K and D357K in another chain; 11) K409D and K370D in one chain and D399K and D357K in another chain; 12) D399K in one chain and K409D and K360D in another chain; and 13) K409D and K439D in one chain and D399K and E356K in another chain.

The term "(antigen) binding" refers to the binding of an antibody to its antigen in an in vitro assay, in one embodiment of which the antibody binds to a surface and the binding is measured by surface plasmon resonance (SPR). Binding means binding to an affinity ( _KD ) of ^10⁻⁸ M or less, ^10⁻¹³ to ^10⁻⁸ M in some embodiments, and ^10⁻¹³ to ^10⁻⁹ M in some embodiments.

Binding can be studied using the BIAcore assay (GE Healthcare Biosensor AB, Uppsala, Sweden). The affinity of binding is defined by the terms k _{_a} (association rate constant of the antibody from the antibody/antigen complex), k_{_D} (dissociation constant), and K_{_D}(k_{_D}/k_{_a} ).

The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and/or light chain originates from a specific source or species, while the remainder of the heavy chain and/or light chain originates from a different source or species.

The term "CH2 domain" refers to the portion of the antibody heavy chain polypeptide extending from EU position 231 to EU position 340 (according to Kabat's EU numbering system). In one embodiment, the CH2 domain has the amino acid sequence of SEQ ID NO:09: APELLGG PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQ ESTYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.

The term "CH3 domain" refers to the portion of the antibody heavy chain polypeptide extending from EU position 341 to EU position 446. In one embodiment, the CH3 domain has the amino acid sequence of SEQ ID NO:10: GQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG.

An antibody "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as _IgG1 , _IgG2 , _IgG3 , _IgG4 , _IgA1 , and _IgA2 . The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The term "comparable length" refers to two polypeptides containing the same number of amino acid residues or differing in length by one or more, up to a maximum of 10 amino acid residues. In one embodiment, the Fc region polypeptide contains the same number of amino acid residues or may differ by 1 to 10 amino acid residues. In one embodiment, the Fc region polypeptide contains the same number of amino acid residues or may differ by 1 to 5 amino acid residues. In one embodiment, the Fc region polypeptide contains the same number of amino acid residues or may differ by 1 to 3 amino acid residues.

The term "derived from" refers to an amino acid sequence that is derived from a parent amino acid sequence by introducing a change at at least one position. Therefore, the derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbered according to the Kabat EU index numbering system for the antibody Fc region). In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 15 amino acid residues. In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 10 amino acid residues. In one embodiment, the amino acid sequence derived from the parent amino acid sequence differs from the corresponding position by 1 to 6 amino acid residues. Similarly, the derived amino acid sequence has high amino acid sequence identity with its parent amino acid sequence. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 80% or greater amino acid sequence identity. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 90% or greater amino acid sequence identity. In one embodiment, the amino acid sequence derived from the parent amino acid sequence has 95% or greater amino acid sequence identity.

"Effective functions" refer to those biological activities attributable to the Fc region of an antibody that vary with antibody class. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The “effective amount” of an active agent (e.g., a pharmaceutical preparation) refers to the amount that, at the necessary dosage and time period, effectively achieves the desired therapeutic or preventative outcome.

The term "Fc-fusion polypeptide" refers to a fusion of a binding domain (such as an antigen-binding domain like a single-chain antibody, or a polypeptide like a receptor ligand) with the Fc region of an antibody that exhibits desired target binding and/or protein A binding and/or FcRn-binding activity.

The term "human Fc region" refers to the C-terminal region of the human immunoglobulin heavy chain, which contains at least a portion of the hinge region, CH2 domain, and CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. In one embodiment, the Fc region has the amino acid sequence of SEQ ID NO: 60. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interests, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 913242, also known as the EU index. The Fc region consists of two heavy-chain Fc region polypeptides, which can be covalently linked to each other by hinge region cysteine residues that form disulfide bonds between polypeptides.

The term "FcRn" refers to the human neonatal Fc receptor. FcRn rescues IgG from lysosomal degradation, leading to reduced clearance and increased half-life. FcRn is a heterodimer composed of two polypeptides: a 50 kDa major histocompatibility complex-like protein class I (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 region of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a 1:2 stoichiometric ratio, meaning that one IgG molecule binds 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 endosomes at acidic pH (pH < 6.5) and IgG is released at neutral cell surfaces (pH approximately 7.4). This pH-sensitive interaction facilitates FcRn-mediated protection of IgG from intracellular degradation by binding to receptors within the acidic environment of the endosome. FcRn then assists in the recycling of IgG to the cell surface and its subsequent release into the bloodstream when the FcRn-IgG complex is exposed to a neutral pH environment outside the cell.

The term "Fc region FcRn binding portion" refers to a portion of the antibody heavy chain polypeptide that extends from approximately EU position 243 to EU position 261, from approximately EU position 275 to EU position 293, from approximately EU position 302 to EU position 319, from approximately EU position 336 to EU position 348, from approximately EU position 367 to EU position 393 and EU position 408, and from approximately EU position 424 to EU position 440. In one implementation, one or more of the following amino acid residues, according to Kabat EU numbers, are changed: F243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W27. 7. Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E 293, 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, P3 46. Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU designations).

"Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The term "full-length antibody" refers to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein. Full-length antibodies may contain other domains, such as scFv or scFab conjugated to one or more chains of the full-length antibody. These conjugates are also covered by the term full-length antibody.

The term “heterodimer” or “heterodimeric” refers to a molecule containing two polypeptide chains (e.g., of comparable length) having an amino acid sequence in which at least one distinct amino acid residue is present at a corresponding position, which is determined according to the Kabat EU index.

The terms “homodimer” and “homodimeric” refer to molecules containing two polypeptide chains of comparable length, wherein the two polypeptide chains have the same amino acid sequence at corresponding positions, which are determined according to Kabat’s EU index.

As reported in this paper, antibodies or Fc region fusion peptides may be homodimers or heterodimers with respect to their Fc regions, depending on the mutation or property of interest. For example, with respect to FcRn and/or protein A binding (i.e., property of interest), the Fc region (antibody) is homodimer with respect to the mutations H310A, H433A, and Y436A (i.e., both heavy chain Fc region peptides contain these mutations) (these mutations are of interest with respect to the FcRn and/or protein A binding properties of the Fc region fusion peptide or antibody), but is heterodimer with respect to the mutations Y349C, T366S, L368A, and Y407V (these mutations are not of interest because they involve heavy chain heterodimerization and not FcRn/protein A binding properties) and with respect to the mutations S354C and T366W (the first group is contained only in the first Fc region peptide, and the second group is contained only in the second Fc region peptide). Furthermore, for example, the Fc region fusion peptides or antibodies reported in this paper can be heterodimeric with respect to the mutations I253A, H310A, H433A, H435A, and Y436A (i.e., these mutations all involve the FcRn and/or protein A binding properties of the dimer peptides), meaning that one Fc region peptide contains the mutations I253A, H310A, and H435A, while another Fc region peptide contains the mutations H310A, H433A, and Y436A.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. Progeny may not be identical to parental cells in terms of nucleic acid content and may contain mutations. Mutant progeny are included herein, which have the same function or biological activity as progeny screened or selected from the initially transformed cells.

A "human antibody" is an antibody that possesses an amino acid sequence corresponding to antibodies produced by humans or human cells, or is derived from a non-human source using a human antibody library or other sequences encoding human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

The “human common framework” is a framework that represents the most frequently occurring amino acid residues in the selection of the VL or VH framework sequence of human immunoglobulins. Typically, the selection of the VL or VH framework sequence of human immunoglobulins comes from a subgroup of variable domain sequences. Typically, the sequence subgroup is as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th Edition, Bethesda MD (1991), NIH Publication 91-3242, Volumes 1-3. In one embodiment, for VL, this subgroup is subgroup κI as described above by Kabat et al. In one embodiment, for VH, this subgroup is subgroup III as described above by Kabat et al.

The term "human Fc region polypeptide" refers to an amino acid sequence identical to that of a "natural" or "wild-type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to an amino acid sequence derived from a "natural" or "wild-type" human Fc region polypeptide due to at least one "amino acid alteration." A "human Fc region" consists of two human Fc region polypeptides. A "variant (human) Fc region" consists of two Fc region polypeptides, where both can be variant (human) Fc region polypeptides, or one can be a human Fc region polypeptide and the other a variant (human) Fc region polypeptide.

In one embodiment, the human Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide of SEQ ID NO: 60, or the amino acid sequence of the human IgG2 Fc region polypeptide of SEQ ID NO: 61, or the amino acid sequence of the human IgG3 Fc region polypeptide of SEQ ID NO: 62, or the amino acid sequence of the human IgG4 Fc region polypeptide of SEQ ID NO: 63. In one embodiment, the variant (human) Fc region polypeptide is derived from the Fc region polypeptide of SEQ ID NO: 60, or 61, or 62, or 63 and has at least one amino acid mutation compared to the human Fc region polypeptide of SEQ ID NO: 60, or 61, or 62, or 63. In one embodiment, the variant (human) Fc region polypeptide contains/has from about 1 to about 12 amino acid mutations, and in one embodiment has from about 1 to about 8 amino acid mutations. In one embodiment, the variant (human) Fc region polypeptide has at least about 80% homology to the human Fc region polypeptide of SEQ ID NO: 60, or 61, or 62, or 63. In one embodiment, the variant (human) Fc region polypeptide has at least about 90% homology with the human Fc region polypeptide of SEQ ID NO: 60, 61, 62, or 63. In another embodiment, the variant (human) Fc region polypeptide has at least about 95% homology with the human Fc region polypeptide of SEQ ID NO: 60, 61, 62, or 63.

Variant (human) Fc region polypeptides derived from human Fc region polypeptides of SEQ ID NO: 60, 61, 62, or 63 are defined by changes in the amino acids contained therein. Thus, for example, the term P329G refers to a variant (human) Fc region polypeptide derived from a human Fc region polypeptide having a proline-to-glycine mutation at amino acid position 329 relative to human Fc region polypeptides of SEQ ID NO: 60, 61, 62, or 63.

As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and referred to herein as “according to (Kabat) numbering.” Specifically, the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647–660) is used for the constant domains CL of the κ and λ isoforms of the light chain, and the Kabat EU index numbering system (see pages 661–723) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3).

The human IgG1 Fc region polypeptide has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 60).

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

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 64).

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

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 65).

The Fc region polypeptide derived from the human IgG1 Fc region with S354C and T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 66).

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

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 67).

The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, S354C, and T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 68).

The Fc region polypeptide derived from the Fc region of human IgG1 with the P329G mutation has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 69).

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

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 70).

The Fc region polypeptide derived from the human IgG1 Fc region with P239G mutation and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 71).

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

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 72).

The Fc region polypeptide derived from the human IgG1 Fc region with mutations L234A, L235A, P329G and Y349C, T366S, L368A, Y407V has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 73).

The Fc region polypeptide derived from the human IgG1 Fc region with L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 74).

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

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 63).

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

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 75).

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

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 76).

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

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:77).

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

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 78).

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

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 79).

The Fc region polypeptide derived from the human IgG4 Fc region with S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 80).

The Fc region polypeptide derived from the Fc region of human IgG4 with the P329G mutation has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 81).

The Fc region polypeptide derived from the human IgG4 Fc region with P239G and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 82).

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

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 83).

The Fc region polypeptide derived from the human IgG4 Fc region with mutations S228P, L235E, P329G and Y349C, T366S, L368A, Y407V has the following amino acid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 84).

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

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 85).

The following shows the alignment results (EU number) of the Fc region from different individuals:

"Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody will comprise at least one, and generally substantially all of two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those HVRs of the non-human antibody, and all or substantially all of the FRs correspond to those FRs of the human antibody. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody, for example, a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term “hypervariant region” or “HVR” refers to each of the following regions of an antibody variable domain that is sequence-hypervariant (“complementarity-determining region” or “CDR”) and forms a structurally defined loop (“hypervariant loop”), and/or contains antigen contact residues (“antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs as mentioned herein include…

(a) Hypervariable rings appearing at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901-917);

(b) CDRs appearing at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

(c) Antigenic contacts appearing at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

(d) Combinations of (a), (b) and/or (c), including HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3) and 94-102(H3).

In one implementation, the HVR residues include those identified elsewhere in this specification.

Unless otherwise stated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered in this document according to the Kabat EU index numbering system (Kabat et al., above).

As used herein, the term “IGF-1R” refers to any naturally occurring IGF-1R from any vertebrate source, wherein, unless otherwise stated, said vertebrate source includes mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term covers “full-length”, unprocessed IGF-1R, and any form of IGF-1R produced by cellular processing. This term also covers naturally occurring IGF-1R variants, such as splice variants or allelic variants. The amino acid sequence of human IGF-1R is shown in SEQ ID NO:11.

"Individual" or "object" is a mammal. 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). In some embodiments, the individual or object is a human.

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

"Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained within cells, which typically contain such molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

"Isolated nucleic acid encoding anti-IGF-1R antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, including such nucleic acid molecules in a single vector or in separate vectors and such nucleic acid molecules present at one or more locations in the host cell.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical and/or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or those that appear during the production of the monoclonal antibody preparation), which are typically present in minute quantities. In contrast to polyclonal antibody preparations, which generally comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies intended for use according to the invention can be produced by a variety of techniques, including but not limited to hybridoma methods, 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 producing monoclonal antibodies are described herein.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be classified into one of two types based on the amino acid sequence of their constant domains, called κ (κ) and λ (λ).

The term "instructions for use" is used to refer to the instructions for use customarily included in the commercial packaging of a therapeutic product, which contain information about indications, usage, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.

The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after the aligned sequence has been fitted with vacancies as needed to achieve the maximum percentage of sequence homology and no conserved substitutions are considered as part of the sequence identity. Alignment to determine the percentage of amino acid sequence identity can be performed in a variety of ways within the capabilities of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for the aligned sequences, including any algorithm required to achieve the maximum alignment across the full-length sequence being compared. However, for the purposes of this document, the amino acid sequence identity % value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is licensed by Genentech, Inc., and the source code has been submitted with user documentation to the U.S. Copyright Office at 20559, Washington, D.C., where it is registered under U.S. Copyright Registry No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be assembled from source code. The ALIGN-2 program should be assembled for use on a UNIX operating system (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

When using ALIGN-2 to compare amino acid sequences, the amino acid sequence identity percentage of a given amino acid sequence A with a given amino acid sequence B, the same as given amino acid sequence B, or for given amino acid sequence B (which can alternatively be described as a given amino acid sequence A having or containing a certain amino acid sequence identity % with given amino acid sequence B, the same as given amino acid sequence B, or for given amino acid sequence B) is calculated as follows:

100 multiplied by the fraction X/Y

Let X be the number of amino acid residues that are identified as identical matches in the alignment results of A and B by the sequence alignment program ALIGN-2, and let Y be the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A relative to B will not be equal to the amino acid sequence identity % of B relative to A. Unless otherwise stated, all amino acid sequence identity values % used herein were obtained using the ALIGN-2 computer program as described immediately preceding the paragraph.

The term "pharmaceutical preparation" refers to a preparation in such a form that the bioactivity of the active ingredient contained therein is effective, and that it does not contain any additional components that would be unacceptably toxic to the subject to whom the preparation will be administered.

"Pharmaceutical-grade carriers" refer to components in a pharmaceutical preparation that are non-toxic to the target drug, excluding the active ingredient. Pharmaceutical-grade carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, the term "peptide linker" refers to a peptide having an amino acid sequence, said peptide being of synthetic origin in one embodiment. In one embodiment, a peptide linker is a peptide having an amino acid sequence of at least 30 amino acids, and in another embodiment, a peptide having an amino acid sequence of 32 to 50 amino acids. In one embodiment, a peptide linker is a peptide having an amino acid sequence of 32 to 40 amino acids. In one embodiment, the peptide linker is (GxS)n, where G = glycine, S = serine, (x = 3, n = 8, 9, or 10) or (x = 4 and n = 6, 7, or 8), in one embodiment, x = 4, n = 6, or 7, and in another embodiment, x = 4, n = 7. In one embodiment, _the peptide linker is (G4S) _6G2 .

The term "recombinant antibody" refers to all antibodies (chimeric, humanized, and human antibodies) prepared, expressed, produced, or isolated through recombinant means. This includes antibodies isolated from host cells such as NSO or CHO cells, or from animals transgenic for human immunoglobulin genes (e.g., mice), or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant antibodies have variable and constant regions in rearranged form. Recombinant antibodies, as reported herein, can undergo in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies, although derived from and associated with human germline VH and VL sequences, may not naturally exist within the in vivo human antibody germline library.

As used herein, the term “treatment” (and its grammatical variations such as “treatment” or “curative therapy”) refers to a clinical intervention intended to alter the natural processes of an individual receiving treatment, and may be intended to prevent or be implemented during a clinicopathological process. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improved prognosis. In some implementations, antibodies or Fc-region fusion peptides, as reported herein, are used to delay disease onset or to slow disease progression.

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

The term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in antibody binding to its antigen. The variable domains (VH and VL, respectively) of the antibody heavy and light chains typically have similar structures, each containing 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), p. 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to specific antigens can be isolated using the VH or VL domains of the antigen-binding antibody to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

The term "ocular vascular disease" includes, but is not limited to, intraocular neovascular syndromes such as diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, neovascular glaucoma, retinal vein occlusion, central retinal vein occlusion, macular degeneration, age-related macular degeneration, retinitis pigmentosa, retinal angiomatous proliferation, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal angiogenesis, retinal angiogenesis, choroidal angiogenesis, and retinal degeneration (see, for example, Garner, A., Vascular diseases, cited in: Pathobiology of ocular disease, A dynamic approach, Garner, A. and Klintworth, G.K., eds., 2nd ed., Marcel Dekker, New York (1994), pp. 1625-1710).

As used herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors incorporated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids linked to them. Such vectors are referred to herein as "expression vectors."

As used herein, the term “VEGF” refers to human vascular endothelial growth factor (VEGF/VEGF-A), 165 amino acid human vascular endothelial cell growth factor (amino acids 27-191 of the precursor sequence of human VEGF165: SEQ ID NO: 30; amino acids 1-26 represent the signal peptide), and related 121, 189, and 206 vascular endothelial cell growth factor isoforms, as described in Leung, D.W. et al., Science 246 (1989) 1306-1309; Houck et al., Mol. Endocrin. 5 (1991) 1806-1814; Keck, P.J. et al., Science 246 (1989) 1309-1312 and Connolly, D.T. et al., J. Biol. Chem. 264 (1989) 20017-20024; along with the naturally occurring alleles and processed forms of these growth factors. VEGF is involved in regulating normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular diseases (Ferrara, N. et al., Endocrin. Rev. 18 (1997) 4-25; Berkman, R.A. et al., J. Clin. Invest. 91 (1993) 153-159; Brown, L.F. et al., Human Pathol. 26 (1995) 86-91; Brown, L.F. et al., Cancer Res. 53 (1993) 4727-4735; Matttern, J. et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, H.F. et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a glycoprotein that has been isolated from several sources and includes several isotypes of homodimers. VEGF exhibits highly specific mitotic-promoting activity against endothelial cells.

As used herein, the term “having a mutation IHH-AAA” refers to a combination of mutations I253A (Ile253Ala), H310A (His310Ala), and H435A (His435Ala) in the constant heavy chain region of IgG1 or IgG4 subclasses, and as used herein, the term “having a mutation HHY-AAA” refers to a combination of mutations H310A (His310Ala), H433A (His433Ala), and Y436A (Tyr436Ala) in the constant heavy chain region of IgG1 or IgG4 subclasses, and as used herein, the term “having a mutation YTE” refers to a combination of mutations M252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) in the constant heavy chain region of IgG1 or IgG4 subclasses, where the numbering is based on Kabat’s EU index.

As used herein, the term "mutant P329G LALA" refers to a combination of mutations L234A (Leu234Ala), L235A (Leu235Ala), and P329G (Pro329Gly) in the constant heavy chain region of the IgG1 subclass, with the numbering based on the Kabat EU index. Similarly, the term "mutant SPLE" refers to a combination of mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region of the IgG4 subclass, with the numbering based on the Kabat EU index. Likewise, the term "mutant SPLE and P239G" refers to a combination of mutations S228P (Ser228Pro), L235E (Leu235Glu), and P329G (Pro329Gly) in the constant heavy chain region of the IgG4 subclass, with the numbering based on the Kabat EU index.

II. This invention

This invention is based, at least in part, on the discovery that the FcRn binding of antibodies or Fc region fusion peptides can be modulated by altering amino acid residues at non-corresponding positions of the individual Fc region peptides, because these alterations work together to modulate FcRn binding. Antibodies and Fc region fusion peptides as reported herein are useful, for example, for treating diseases in which a customized systemic retention time is required.

A. Newly formed Fc receptor (FcRn)

The nascent Fc receptor (FcRn) is crucial for the metabolic fate of IgG antibodies in vivo. FcRn acts as a rescue agent for wild-type IgG from lysosomal degradation, leading to reduced clearance and increased half-life. It is a heterodimer composed of two polypeptides: a 50 kDa major histocompatibility complex-like protein class I (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 region of the Fc region of IgG antibodies. The interaction between IgG antibodies and FcRn is strictly pH-dependent and occurs in a 1:2 stoichiometric ratio, meaning that one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc region polypeptides (see, for example, Huber, A.H. et al., J. Mol. Biol. 230 (1993) 1077-1083).

Therefore, the in vitro FcRn binding properties/characteristics of IgG indicate its in vivo pharmacokinetic properties in blood circulation.

Different amino acid residues in the CH2 and CH3 domains of the heavy chain participate in the interaction between FcRn and the Fc region of IgG antibodies. The amino acid residues that interact with FcRn are located approximately between EU positions 243 and 261, approximately between EU positions 275 and 293, approximately between EU positions 302 and 319, approximately between EU positions 336 and 348, approximately between EU positions 367 and 393, at EU position 408, and approximately between EU positions 424 and 440. More specifically, the following amino acid residues, according to Kabat EU numbers, are involved in the interaction between the Fc region and FcRn: F243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N2 76. 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, L31 4. N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E 345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380 W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440.

Site-directed mutagenesis studies have demonstrated that the key binding sites for FcRn in the Fc region of IgG are histidine 310, histidine 435, and isoleucine 253, and to a lesser extent histidine 433 and tyrosine 436 (see, for example, Kim, J.K. et al., Eur. J. Immunol. 29 (1999) 2819-2825; Raghavan, M. et al., Biochem. 34 (1995) 14649-14657; Medesan, C. et al., J. Immunol. 158 (1997) 2211-2217).

Methods to increase IgG binding to FcRn have been implemented by mutating IgG at the following amino acid residues: threonine 250, methionine 252, serine 254, threonine 256, threonine 307, glutamic acid 380, methionine 428, histidine 433, and asparagine 434 (see Kuo, T.T. et al., J. Clin. Immunol. 30 (2010) 777-789).

In some cases, antibodies with a reduced half-life in the bloodstream are required. For example, intravitreal drugs should have a long half-life in the eye and a short half-life in the patient's circulation. These antibodies also have the advantage of increased exposure at disease sites such as the eye.

Different mutations affecting FcRn binding and half-life in the bloodstream are known. Fc region residues crucial for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall’Acqua, W.F. et al., J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (according to Kabat’s EU designation) are involved in this interaction (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533-2536; Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Kim, J.K. et al., Eur. J. Immunol. 24 (1994) 542-548). Residues I253, H310, and H435 were found to be crucial for the interaction between human Fc and mouse FcRn (Kim, J.K. et al., Eur. J. Immunol. 29(1999) 2819-2825). Through protein-protein interaction studies, Dall’Acqua et al. have described residues M252Y, S254T, and T256E as improving FcRn binding (Dall’Acqua, W.F. et al., J. Biol. Chem. 281(2006) 23514-23524). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for this interaction (Firan, M. et al., Int. Immunol. 13(2001) 993-1002; Shields, R.L. et al., J. Biol. Chem. 276(2001) 6591-6604). Various mutants of residues 248–259 and 301–317, and 376–382 and 424–437 have been reported and investigated in Yeung, Y.A. et al. (J. Immunol. 182(2009) 7667-7671). Exemplary mutations and their effects on FcRn binding are listed in the table below.

surface

It has been found that a single mutation on one side of an Fc region polypeptide is sufficient to significantly weaken its binding to the Fc receptor. Introducing more mutations into the Fc region further weakens the binding to FcRn. However, unilateral asymmetric mutations are insufficient to completely inhibit FcRn binding. Bilateral mutations are necessary for complete inhibition of FcRn binding.

Therefore, the Fc region of the variant (human) IgG is a heterodimer and the pairing of the first (heavy chain) Fc region polypeptide and the second (heavy chain) Fc region polypeptide to form a functional Fc region leads to the formation of the heterodimer.

The table below shows the results of symmetry engineering of the IgG1 Fc region affecting FcRn binding (comparison of retention time on FcRn-affinity chromatography column between mutations).

surface

Retention times of less than 3 minutes correspond to non-binding because the substance is in the flow (void peak).

The single mutation H310A is the most silent symmetric mutation that eliminates any FcRn binding.

Symmetrical single mutations I253A and H435A resulted in a relative shift in retention time of 0.3–0.4 minutes. This can generally be considered an undetectable binding interaction.

The single mutation Y436A results in a detectable interaction strength with the FcRn affinity column. Without this theoretical constraint, such a mutation may have an FcRn-mediated half-life that differs from zero-interaction mutations such as the I253A, H310A, and H435A combination (the IHH-AAA mutation).

The table below shows the results obtained using symmetrically modified anti-HER2 antibodies (for reference, see WO 2006/031370).

surface

The effect of introducing asymmetric mutations into the Fc region that affect FcRn binding has been illustrated with bispecific anti-VEGF/ANG2 antibodies (see below), which are assembled using a knots-into-holes technique (see, for example, US 7,695,936, US 2003/0078385; "knobs-into-holes" mutation: S354C/T366W, "holes-into-holes" mutation: Y349C/T366S/L368A/Y407V). The effect of asymmetrically introduced mutations on FcRn binding can be readily determined using FcRn affinity chromatography (see Figure 9 and the table below). Antibodies that elute slightly later from the FcRn affinity column, i.e., those with a longer retention time on the FcRn affinity column, have a longer in vivo half-life, and vice versa.

surface

Asymmetric IHH-AAA- and LLL-DDD- mutations (LLL-DDD- mutations = L251D, L314D, and L432D) showed weaker binding than the corresponding parental or wild-type antibodies.

Symmetrical HHY-AAA mutations (= mutation combinations H310A, H433A, and Y436A) produce an Fc region that no longer binds to human FcRn, while protein A binding is maintained (see Figures 11, 12, 13, and 14).

The effects of introducing asymmetric mutations into the Fc region that affect FcRn binding have been further illustrated using bispecific anti-VEGF/ANG2 antibodies (VEGF/ANG2), monospecific anti-IGF-1R antibodies (IGF-1R), and full-length antibodies fused to the C-terminus of both heavy chains (fusions), wherein these antibodies are assembled using a spike-in-pore technique to allow the introduction of asymmetric mutations (see, for example, US 7,695,936, US 2003/0078385; “spiking chain” mutation: S354C/T366W, “pore chain” mutation: Y349C/T366S/L368A/Y407V). The effects of asymmetrically introduced mutations on FcRn binding and protein A binding can be readily determined using FcRn affinity chromatography, protein A affinity chromatography, and SPR-based methods (see the table below).

Mutant combinations of I253A, H310A, H435A, or L251D, L314D, L432D, or L251S, L314S, L432S result in loss of binding to protein A, while mutant combinations of I253A, H310A, H435A, or H310A, H433A, Y436A, or L251D, L314D, L432D result in loss of binding to human neonatal Fc receptor.

One aspect, as reported in this article, is antibodies or Fc region fusion peptides containing variant human IgG Fc regions, as reported in this article.

The Fc region of the fusion polypeptide imparts the aforementioned characteristics to its fusion partner. The fusion partner can be any biologically active molecule, wherein the in vivo half-life of the molecule should be reduced or increased, i.e., its in vivo half-life should be explicitly defined or customized for the intended application of the molecule.

Fc fusion peptides may, for example, comprise a variant (human) IgG Fc region as reported herein and a receptor protein that binds to a target (including a ligand), such as, for example, a TNFR-Fc fusion peptide (TNFR = human tumor necrosis factor receptor), or an IL-1R-Fc fusion peptide (IL-1R = human interleukin-1 receptor), or a VEGFR-Fc fusion peptide (VEGFR = human vascular endothelial growth factor receptor), or an ANG2R-Fc fusion peptide (ANG2R = human angiopoietin 2 receptor).

Fc region fusion peptides may include, for example, variant (human) IgG class Fc regions as reported herein and antibody fragments that bind to targets (including ligands), such as antibody Fab fragments, scFv (see, for example, Nat. Biotechnol. 23(2005) 1126-1136), or domain antibodies (dAbs) (see, for example, WO 2004/058821, WO 2003/002609).

Fc region fusion peptides may, for example, include a variant (human) IgG-like Fc region and a (naturally occurring or artificial) receptor ligand as reported herein.

B. Exemplary Antibodies

In one aspect, the present invention provides isolated antibodies with regulated FcRn binding activity, i.e., these antibodies bind to human FcRn with higher or lower affinity compared to antibodies without mutations affecting FcRn binding activity.

In one embodiment, the antibody comprises an Fc region, the Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The first Fc region polypeptide and the second Fc region polypeptide are derived from the same human Fc region polypeptide, and

b) The first Fc region polypeptide is modified in that its amino acid sequence differs from that of the second Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system, and the second Fc region polypeptide is modified in that its amino acid sequence differs from that of the first Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system. Therefore, the modification positions in the first Fc region polypeptide and the modification positions in the second Fc region polypeptide are different.

c) The Fc region of the human Fc region polypeptide containing a) (i.e., the polypeptide having the same amino acid residues as the human Fc region polypeptide in the corresponding position according to the KabatEU index numbering system) as the first and second Fc region polypeptides has a different affinity for the human Fc receptor.

An exemplary antibody reported herein, and in one aspect of the invention, is a bispecific bivalent antibody with FcRn binding elimination, said antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2.

in

i) The first antigen-binding site that specifically binds to VEGF includes the CDR3H region of SEQ ID NO:14, the CDR2H region of SEQ ID NO:15, and the CDR1H region of SEQ ID NO:16 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:17, the CDR2L region of SEQ ID NO:18, and the CDR1L region of SEQ ID NO:19 in the light chain variable domain.

ii) The second antigen-binding site that specifically binds to ANG-2 includes the CDR3H region of SEQ ID NO:22, the CDR2H region of SEQ ID NO:23, and the CDR1H region of SEQ ID NO:24 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:25, the CDR2L region of SEQ ID NO:26, and the CDR1L region of SEQ ID NO:27 in the light chain variable domain.

iii) The bispecific antibody contains an Fc region, wherein the Fc region comprises a first Fc region polypeptide and a second Fc region polypeptide.

in

a) The first Fc region polypeptide and the second Fc region polypeptide are derived from the same human Fc region polypeptide, and

b) The first Fc region polypeptide is modified in that its amino acid sequence differs from that of the second Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system, and the second Fc region polypeptide is modified in that its amino acid sequence differs from that of the first Fc region polypeptide at at least one corresponding position according to the Kabat EU index numbering system. Therefore, the modification positions in the first Fc region polypeptide and the modification positions in the second Fc region polypeptide are different.

c) The Fc region of the human Fc region polypeptide containing a) (i.e., the polypeptide having the same amino acid residues as the human Fc region polypeptide in the corresponding position according to the KabatEU index numbering system) as the first and second Fc region polypeptides has a different affinity for the human Fc receptor.

In one embodiment of all aspects, the Fc region is a variant (human) IgG class Fc region. In another embodiment, the variant (human) IgG class Fc region is an IgG class heterodimeric Fc region.

In one embodiment of all aspects, the first Fc region polypeptide and the second Fc region polypeptide pair to form a (functional) Fc region, resulting in the formation of a heterodimer.

In one implementation, the human Fc region polypeptide is an IgG1 subclass or an IgG4 subclass human Fc region polypeptide.

In one embodiment, the human Fc region polypeptide is a human Fc region polypeptide of the IgG1 subclass, which also includes the mutants L234A, L235A, and P329G.

In one embodiment, the human Fc region polypeptide is a human Fc region polypeptide of the IgG4 subclass, which also contains the mutants S228P and L235E.

In one embodiment, the first Fc region polypeptide further comprises the mutations S354C and T366W, and the second Fc region polypeptide further comprises the mutations Y349C, T366S, L368A, and Y407V.

In one implementation scheme, the bispecific antibody is characterized by:

i) The first antigen-binding site that specifically binds to VEGF contains the amino acid sequence of SEQ ID NO:20 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:21 as the light chain variable domain VL, and

ii) The second antigen binding site that specifically binds to ANG-2 contains the amino acid sequence of SEQ ID NO:28 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:29 as the light chain variable domain VL.

In one embodiment, the bispecific antibody is characterized in that the Fc region of iii) belongs to the human IgG1 subclass. In another embodiment, the bispecific antibody is characterized in that the Fc region of the human IgG1 subclass also contains mutations L234A, L235A, and P329G (according to Kabat's EU index number).

In one embodiment, the bispecific antibody is characterized in that the Fc region of iii) belongs to the human IgG4 subclass. In one embodiment, the bispecific antibody is characterized in that the Fc region of the human IgG4 subclass further includes the mutations S228P and L235E (according to Kabat's EU index number). In one embodiment, the bispecific antibody is characterized in that the Fc region of the human IgG4 subclass further includes the mutations S228P, L235E, and P329G (according to Kabat's EU index number).

In one embodiment, the bispecific antibody comprises an Fc region containing a first and second Fc region polypeptide of human IgG1 or human IgG4 subclass (i.e., derived from human sources). The first Fc region polypeptide contains one or two mutations selected from group i) I253A, H310A, H435A, or group ii) H310A, H433A, Y436A, or group iii) L251D, L314D, L432D, or group iv) L251S, L314S, L432S (numbered according to the Kabat EU index numbering system) and in the second Fc region. The polypeptide contains one or two mutations selected from the mutations L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, and Y436A (numbered according to the Kabat EU index system), so that all mutations i) I253A, H310A, H435A, or ii) H310A, H433A, Y436A, or iii) L251D, L314D, L432D, or iv) L251S, L314S, and L432S are contained in the Fc region.

In one embodiment, the bispecific antibody comprises an Fc region containing first and second Fc region peptides of human IgG1 or human IgG4 subclass (derived from human origin), wherein the peptides contain mutant I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or combinations thereof (according to Kabat EUindex). (System number), therefore all mutations are in the first or second Fc region polypeptide, or one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, thus all mutations i) I253A, H310A, H435A, or ii) H310A, H433A, Y436A, or iii) L251D, L314D, L432D, or iv) L251S, L314S and L432S are contained in this Fc region.

Other aspects, as reported herein, include pharmaceutical formulations containing bispecific antibodies, pharmaceutical formulations for treating ocular vascular diseases, the use of bispecific antibodies in the manufacture of pharmaceuticals for treating ocular vascular diseases, and methods of treating patients with ocular vascular diseases by administering bispecific antibodies to patients requiring such treatment. In one embodiment, the bispecific antibody or a pharmaceutical formulation containing a bispecific antibody is administered via intravitreal application.

Another aspect of the invention is a nucleic acid molecule encoding the heavy chain and/or light chain of a bispecific antibody as reported herein.

The present invention also provides expression vectors containing nucleic acids as reported herein (the expression vectors are capable of expressing the nucleic acids in prokaryotic or eukaryotic host cells), and host cells containing such vectors for recombinant production of bispecific antibodies as reported herein.

The present invention also includes a prokaryotic or eukaryotic host cell, said host cell containing a vector as reported herein.

The present invention also includes a method for generating bispecific antibodies as reported herein, the method being characterized by: expressing a nucleic acid as reported herein in prokaryotic or eukaryotic host cells and recovering the bispecific antibody from the cells or cell culture supernatant. One embodiment is a method for preparing bispecific antibodies as reported herein, the method comprising the steps of...

a) Transform host cells using a vector containing nucleic acid molecules encoding antibodies;

b) Culture host cells under conditions that allow for antibody synthesis; and

c) Recover antibodies from the culture.

The present invention also includes antibodies obtained by this method of generating bispecific antibodies.

The antibodies reported herein possess highly valuable properties due to specific modifications in their Fc regions, which are beneficial to patients with ocular vascular diseases. They exhibit high stability in the vitreous environment and diffuse slowly from the eye (compared to smaller antibody fragments without a constant heavy chain region), where actual disease is present and treated (thus potentially improving treatment regimens compared to non-IgG-like antibodies such as Fab fragments and (Fab) ₂ fragments). On the other hand, the antibodies reported herein rapidly clear from serum (which is urgently needed to reduce potential side effects from systemic exposure). Surprisingly, they also exhibit lower viscosity (see Figure 2) (compared to the non-mutated combinations of I253A, H310A, and H435A in the constant region) and are therefore particularly suitable for intravitreal administration via fine needles during the treatment of ocular diseases (for such applications, fine needles are generally used and high viscosity makes proper administration quite difficult). The lower viscosity also allows for higher concentrations of formulations.

Surprisingly, the antibodies reported in this paper showed a lower tendency to aggregate during storage (see Figure 4) (compared to the unmutated combination of I253A, H310A and H435A in the Fc region), which is crucial for intravitreal application in the eye (since aggregation in the eye can lead to complications during this type of treatment).

The bispecific antibodies reported in this article have shown good efficacy in inhibiting vascular diseases.

In some implementations, bispecific antibodies, such as those reported herein, exhibit valuable properties due to specific modifications in their constant regions (e.g., P329GLALA), such as non-binding to Fcγ receptors/no Fcγ receptor binding activity, which reduces the risk of side effects such as thrombosis and/or unwanted cell death (due to, for example, ADCC).

In one embodiment as reported herein, the bispecific antibody as reported herein is bivalent.

In one aspect of the invention, the bispecific bivalent antibody as reported herein is characterized by comprising

a) The heavy and light chains of the first full-length antibody that specifically binds to VEGF, and

b) Modified heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged.

This bispecific bivalent antibody pattern, which specifically binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), is described in WO 2009/080253 (including a CH3 domain with a protrusion-entry-pore modification). The antibody based on this bispecific bivalent antibody pattern is named CrossMab.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:38 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:40 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:39 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:41 is used as the modified light chain of the second full-length antibody.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:34 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:36 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:35 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:37 is used as the modified light chain of the second full-length antibody.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:42 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:44 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:43 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:45 is used as the modified light chain of the second full-length antibody.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:90 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:40 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:91 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:41 is used as the modified light chain of the second full-length antibody.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:88 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:36 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:89 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:37 is used as the modified light chain of the second full-length antibody.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:92 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:44 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:93 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:45 is used as the modified light chain of the second full-length antibody.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:40 and SEQ ID NO:41.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:36 and SEQ ID NO:37.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:44 and SEQ ID NO:45.

In another aspect, as reported in this article, bispecific antibodies are characterized by containing

a) The heavy and light chains of the first full-length antibody that specifically binds to VEGF, and

b) The heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker.

This bispecific bivalent antibody pattern, which specifically binds to human vascular endothelial growth factor (VEGF) and human angiopoietin-2 (ANG-2), is described in WO 2011/117330 (including a CH3 domain with a protrusion-in-pore modification). Antibodies based on this bispecific bivalent antibody pattern are named the single-arm single-chain pattern Fab (OAscFab).

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:46 is used as the heavy chain of the first full-length antibody and the amino acid sequence of SEQ ID NO:48 is used as the light chain of the first full-length antibody, and

b) The amino acid sequence of SEQ ID NO:47 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:49 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:51 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:50 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:94 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:48 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:95 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

In one implementation, this type of bispecific bivalent antibody is characterized by containing

a) The amino acid sequence of SEQ ID NO:96 is used as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:51 is used as the light chain of the first full-length antibody.

b) The amino acid sequence of SEQ ID NO:97 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

In one embodiment, the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) of the heavy and light chains of the second full-length antibody are further stabilized by introducing disulfide bonds between the following positions: heavy chain variable domain position 44 and light chain variable domain position 100 (according to Kabat number (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health). (Tutes of Health, Bethesda, MD (1991)). Techniques for introducing disulfide bonds to provide stabilization are described, for example, in WO 94/029350; Rajagopal, V. et al., Prot. Eng. 10 (1997) 1453-1459; Kobayashi et al., Nuclear Medicine & Biology 25 (1998) 387-393; and Schmidt, M. et al., Oncogene 18 (1999) 1711-1721.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:94, SEQ ID NO:95 and SEQ ID NO:48.

Therefore, one aspect reported herein is a bispecific bivalent antibody comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that it comprises the amino acid sequences of SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:51.

In one implementation, the CH3 domain of a bispecific bivalent antibody, as reported herein, is altered using a "knob-into-hole" technique, detailed in several examples such as WO 96/027011, Ridgway J.B. et al., Protein Eng. 9 (1996) 617-621, and Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-681. In this approach, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two heavy chains containing these two CH3 domains. One of the two CH3 domains (of the two heavy chains) can be a "knob," and the other a "hole." The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

In a preferred embodiment as described in all aspects herein, the bispecific antibody is characterized by:

The CH3 domains of one heavy chain and the CH3 domain of another heavy chain each converge at the interface containing the initial interface between the antibody CH3 domains.

The interface is altered to promote the formation of bispecific antibodies, wherein the alteration is characterized by:

a) This alters the CH3 domain of a heavy chain.

Thus, within the bispecific antibody, at the initial interface of the CH3 domain of one heavy chain, it meets with the initial interface of the CH3 domain of another heavy chain.

Replacing amino acid residues with amino acid residues having larger side chain volume creates a protrusion within the interface of a CH3 domain of one heavy chain, which can then be placed in a cavity within the interface of a CH3 domain of another heavy chain.

and

b) This alters the CH3 domain of the other heavy chain.

Thus, within the bispecific antibody, at the initial interface of the second CH3 domain, which meets the initial interface of the first CH3 domain,

By replacing amino acid residues with amino acid residues having smaller side chain volumes, a cavity is created inside the interface of the second CH3 domain, within which a protrusion from the interface of the first CH3 domain can be placed.

Therefore, the antibody of the present invention is characterized in a preferred embodiment as follows:

The CH3 domains of the heavy chain of the full-length antibody in a) and the CH3 domains of the heavy chain of the full-length antibody in b) each converge at the altered interface in the initial interface between the CH3 domains containing the antibody.

in

i) In a CH3 domain of a heavy chain

Replacing amino acid residues with amino acid residues having larger side chain volume creates a protrusion within the interface of a CH3 domain of one heavy chain, which can then be placed in a cavity within the interface of a CH3 domain of another heavy chain.

And among them

ii) In the CH3 domain of another heavy chain

By replacing amino acid residues with amino acid residues having smaller side chain volumes, a cavity is created inside the interface of the second CH3 domain, and the protruding portion inside the interface of the first CH3 domain can be placed inside the cavity.

In a preferred embodiment, the amino acid residues with larger side chain volume are selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

In a preferred embodiment, the amino acid residues with smaller side chain volume are selected from alanine (A), serine (S), threonine (T), and valine (V).

In one implementation, the two CH3 domains are further modified by introducing cysteine (C) residues at the corresponding positions in each CH3 domain, thereby allowing disulfide bonds to form between the two CH3 domains.

In one implementation, the bispecific antibody contains a T366W mutation in the CH3 domain of the "protrusion chain" and T366S, L368A, and Y407V mutations in the CH3 domain of the "pore chain." Additional interchain disulfide bonds between the CH3 domains can also be used (Merchant, A.M. et al., Nature Biotech 16 (1998) 677-681), for example, by introducing a Y349C or S354C mutation into the CH3 domain of the "protrusion chain" and a Y439C, E356C, or S354C mutation into the CH3 domain of the "pore chain."

In one implementation, the bispecific antibody, as reported herein, contains a mutant Y349C or S354C and a mutant T366W in one of the two CH3 domains and a mutant S354C or E356C or Y349C and mutants T366S, L368A and Y407V in the other of the two CH3 domains. In a preferred embodiment, the bispecific antibody contains the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (an additional Y349C mutation in one CH3 domain and an additional S354C mutation in the other CH3 domain form an interchain disulfide bond) (according to Kabat's EU index number (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). In a preferred embodiment, the bispecific antibody contains an S354C, T366W mutation in one of the two CH3 domains and a Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (an additional S354C mutation in one CH3 domain and an additional Y349C mutation in the other CH3 domain form an interchain disulfide bond) (according to Kabat's EU index number (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

Other protrusion-in-pore techniques, such as those described in EP 1870459A1, may also be used alternatively or additionally. Thus, another example of a bispecific antibody is the R409D and K370E mutations in the CH3 domain of the "protrusion chain" and the D399K and E357K mutations in the CH3 domain of the "pore chain" (according to Kabat's EU index number (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

In another embodiment, the bispecific antibody contains a T366W mutation in the CH3 domain of the "protrusion chain" and T366S, L368A, and Y407V mutations in the CH3 domain of the "pore chain," and additionally contains R409D and K370E mutations in the CH3 domain of the "protrusion chain" and D399K and E357K mutations in the CH3 domain of the "pore chain."

In one embodiment, the bispecific antibody contains the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, and Y407V mutation in the other of the two CH3 domains; or the bispecific antibody contains the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, and Y407V mutation in the other of the two CH3 domains, and additionally contains the R409D, K370E mutation in the CH3 domain of the "protrusion chain" and the D399K, E357K mutation in the CH3 domain of the "pore chain".

In one embodiment, the bispecific antibody contains S354C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains; or the bispecific antibody contains S354C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains, and additionally contains R409D, K370E mutations in the CH3 domain of the "protrusion chain" and D399K, E357K mutations in the CH3 domain of the "pore chain".

In one implementation, the bispecific antibody, as reported herein, is characterized by having one or more of the following properties:

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (measured in the assay described in Example 6).

- Compared to the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration in the right eye of mice with FcRn deficiency but hemizygous transgenes to human FcRn) (as determined by the assay described in Example 6).

- It shows that it does not bind to human neonatal Fc receptors.

In one embodiment, the bispecific bivalent antibody is characterized by comprising a first Fc region polypeptide and a second Fc region polypeptide, wherein

a) The peptides in the first and second Fc regions contain the mutant Y436A, or

b) The peptides in the first and second Fc regions contain mutants I253A, H310A, and H435A, or

c) The peptides in the first and second Fc regions contain mutants H310A, H433A, and Y436A, or

d) The first and second Fc region peptides contain mutants L251D, L314D, and L432D, or

e) The peptides in the first and second Fc regions contain mutants L251S, L314S, and L432S, or

f) The first Fc region polypeptide contains the mutant Y436A and the second Fc region polypeptide contains

- Mutations in I253A, H310A, and H435A, or

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

g) The first Fc region polypeptide contains mutants I253A, H310A, and H435A, and the second Fc region polypeptide contains...

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

h) The first Fc region polypeptide contains mutants H310A, H433A, and Y436A, and the second Fc region polypeptide contains...

a) Mutate L251D, L314D, and L432D, or

b) Mutations in L251S, L314S, and L432S,

or

i) The first Fc region polypeptide contains mutants L251D, L314D, and L432D, and the second Fc region polypeptide contains...

a) Mutation of L251S, L314S and L432S.

In one embodiment, the bispecific antibody comprises an Fc region containing first and second Fc region polypeptides of human IgG1 or human IgG4 subclass (i.e., human-derived). The first Fc region polypeptide contains one or two mutations selected from group i) I253A, H310A, H435A, or group ii) H310A, H433A, Y436A, or group iii) L251D, L314D, L432D, or group iv) L251S, L314S, L432S (numbered according to the Kabat EU index system), and the second Fc region polypeptide contains a mutation selected from the L2 mutation. One or two mutations of 51D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, Y436A (numbered according to the Kabat EU index numbering system) result in all mutations in the peptides of the first and second Fc regions being included in the Fc region when all mutations are considered together.

In one embodiment, the bispecific antibody comprises an Fc region containing first and second Fc region polypeptides of human IgG1 or human IgG4 subclass (derived from human origin), wherein the polypeptide contains mutant I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or combinations thereof (numbered according to the Kabat EUindex numbering system), thus all All mutations are in the first or second Fc region polypeptide, or one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, such that when considered together, all mutations in the first and second Fc region polypeptides result in mutations i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S being contained in the Fc region.

In one embodiment, the bispecific bivalent antibody is characterized by containing

The first antigen-binding site specifically binds to human VEGF and the second antigen-binding site specifically binds to human ANG-2, characterized in that...

i) The first antigen-binding site contains the amino acid sequence of SEQ ID NO:20 as a heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:21 as a light chain variable domain (VL), and

ii) The second antigen binding site contains the amino acid sequence of SEQ ID NO:28 as a heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:29 as a light chain variable domain (VL), and

iii) The bispecific antibody comprises an Fc region containing a first and second Fc region polypeptide of human IgG1 or human IgG4 subclass (i.e., derived from human sources). The first Fc region polypeptide contains one or two mutations selected from group i) I253A, H310A, H435A, or group ii) H310A, H433A, Y436A, or group iii) L251D, L314D, L432D, or group iv) L251S, L314S, L432S (numbered according to the Kabat EU index system), and the second Fc region polypeptide contains a mutation selected from the mutant L25. A mutation in one or two of the following: I251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, Y436A (numbered according to the Kabat EUindex numbering system), such that, when considered together, all mutations in the peptides of the first and second Fc regions result in mutations i) I253A, H310A, and H435A, or ii) H310A, H433A, and Y436A, or iii) L251D, L314D, and L432D, or iv) L251S, L314S, and L432S being contained in the Fc region.

And it has one or more of the following characteristics

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (measured in the assay described in Example 6).

- Compared to the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (as determined in the assay described in Example 6).

- It shows that it does not bind to human neonatal Fc receptors.

In one embodiment, the bispecific bivalent antibody is characterized by containing

The first antigen-binding site specifically binds to human VEGF and the second antigen-binding site specifically binds to human ANG-2, characterized in that...

i) The first antigen-binding site contains the amino acid sequence of SEQ ID NO:20 as a heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:21 as a light chain variable domain (VL), and

ii) The second antigen binding site contains the amino acid sequence of SEQ ID NO:28 as a heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:29 as a light chain variable domain (VL), and

iii) Bispecific antibodies contain a first Fc region polypeptide and a second Fc region polypeptide, wherein

a) The peptides in the first and second Fc regions contain the mutant Y436A, or

b) The peptides in the first and second Fc regions contain mutants I253A, H310A, and H435A, or

c) The peptides in the first and second Fc regions contain mutants H310A, H433A, and Y436A, or

d) The first and second Fc region peptides contain mutants L251D, L314D, and L432D, or

e) The peptides in the first and second Fc regions contain mutants L251S, L314S, and L432S, or

f) The first Fc region polypeptide contains the mutant Y436A and the second Fc region polypeptide contains

- Mutations in I253A, H310A, and H435A, or

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

g) The first Fc region polypeptide contains mutants I253A, H310A, and H435A, and the second Fc region polypeptide contains...

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

h) The first Fc region polypeptide contains mutants H310A, H433A, and Y436A, and the second Fc region polypeptide contains...

a) Mutate L251D, L314D, and L432D, or

b) Mutate L251S, L314S, and L432S.

or

i) The first Fc region polypeptide contains mutants L251D, L314D, and L432D, and the second Fc region polypeptide contains...

a) Mutate L251S, L314S, and L432S.

And it has one or more of the following characteristics

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (measured in the assay described in Example 6).

- Compared to the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration to the right eye in mice with FcRn deficiency but hemizygous transgenes for human FcRn) (as determined in the assay described in Example 6).

- It shows that it does not bind to human neonatal Fc receptors.

In one embodiment, the bispecific antibody is characterized by comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that...

i) The first antigen-binding site contains the amino acid sequence of SEQ ID NO:20 with 1, 2, or 3 amino acid substitutions as the heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:21 with 1, 2, or 3 amino acid substitutions as the light chain variable domain (VL).

ii) The second antigen binding site contains the amino acid sequence of SEQ ID NO:28 with 1, 2, or 3 amino acid substitutions as the heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:29 with 1, 2, or 3 amino acid substitutions as the light chain variable domain (VL).

iii) The bispecific antibody comprises an Fc region containing a first and second Fc region polypeptide of human IgG1 or human IgG4 subclass (i.e., derived from human sources). The first Fc region polypeptide contains one or two mutations selected from group i) I253A, H310A, H435A, or group ii) H310A, H433A, Y436A, or group iii) L251D, L314D, L432D, or group iv) L251S, L314S, L432S (numbered according to the Kabat EU index system), and the second Fc region polypeptide contains a mutation selected from the mutant L251. Mutations in one or two of the following Fc regions (D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, Y436A, or their corresponding Kabat EUindex numbers) when considered together result in all mutations in the peptides of the first and second Fc regions being included in the Fc region: i) I253A, H310A, and H435A; or ii) H310A, H433A, and Y436A; or iii) L251D, L314D, and L432D; or iv) L251S, L314S, and L432S.

And it has one or more of the following characteristics

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (measured in the assay described in Example 6).

- Compared to the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration to the right eye in mice with FcRn deficiency but hemizygous transgenes for human FcRn) (as determined in the assay described in Example 6).

- It shows that it does not bind to human neonatal Fc receptors.

In one embodiment, the bispecific antibody is characterized by comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2, characterized in that...

i) The first antigen-binding site contains the amino acid sequence of SEQ ID NO:20 with 1, 2, or 3 amino acid substitutions as the heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:21 with 1, 2, or 3 amino acid substitutions as the light chain variable domain (VL).

ii) The second antigen binding site contains the amino acid sequence of SEQ ID NO:28 with 1, 2, or 3 amino acid substitutions as the heavy chain variable domain (VH) and the amino acid sequence of SEQ ID NO:29 with 1, 2, or 3 amino acid substitutions as the light chain variable domain (VL).

iii) Bispecific antibodies contain a first Fc region polypeptide and a second Fc region polypeptide, wherein...

a) The peptides in the first and second Fc regions contain the mutant Y436A, or

b) The peptides in the first and second Fc regions contain mutants I253A, H310A, and H435A, or

c) The peptides in the first and second Fc regions contain mutants H310A, H433A, and Y436A, or

d) The first and second Fc region peptides contain mutants L251D, L314D, and L432D, or

e) The peptides in the first and second Fc regions contain mutants L251S, L314S, and L432S, or

f) The first Fc region polypeptide contains the mutant Y436A and the second Fc region polypeptide contains

- Mutations in I253A, H310A, and H435A, or

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

g) The first Fc region polypeptide contains mutants I253A, H310A, and H435A, and the second Fc region polypeptide contains...

- Mutations in H310A, H433A, and Y436A, or

- Mutations in L251D, L314D, and L432D, or

- Mutations in L251S, L314S, and L432S,

or

h) The first Fc region polypeptide contains mutants H310A, H433A, and Y436A, and the second Fc region polypeptide contains...

a) Mutate L251D, L314D, and L432D, or

b) Mutate L251S, L314S, and L432S.

or

i) The first Fc region polypeptide contains mutants L251D, L314D, and L432D, and the second Fc region polypeptide contains...

a) Mutate L251S, L314S, and L432S.

And it has one or more of the following characteristics

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice with FcRn deficiency but hemizygous transgenes to human FcRn) (measured in the assay described in Example 6).

- Compared to the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration to the right eye in mice with FcRn deficiency but hemizygous transgenes for human FcRn) (as determined in the assay described in Example 6).

- It shows that it does not bind to human neonatal Fc receptors.

The antigen-binding sites of the bispecific antibodies reported in this paper contain six complementarity-determining regions (CDRs) that contribute to the affinity of the binding site for its antigen to varying degrees. Three heavy chain variable domain CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2, and CDRL3) are present. The extent of the CDRs and framework regions (FRs) is determined by comparison with a compiled amino acid sequence database, in which these regions have been defined according to the variability between sequences.

In one embodiment of all aspects, the antibody does not specifically bind to human FcRn. In another embodiment of all aspects, the antibody additionally specifically binds to staphylococcal protein A.

In one embodiment of all aspects, the antibody does not specifically bind to human FcRn. In another embodiment of all aspects, the antibody additionally does not specifically bind to staphylococcal protein A.

In one embodiment of all aspects, the first polypeptide further comprises the mutations Y349C, T366S, L368A, and Y407V (“pores”) and the second polypeptide comprises the mutations S354C and T366W (“protrusions”).

In one embodiment of all aspects, the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V (“pores”) and the second polypeptide comprises the mutations Y349C and T366W (“protrusions”).

In one embodiment of all aspects, the Fc region polypeptide belongs to the human IgG1 subclass. In one embodiment, the first Fc region polypeptide and the second Fc region polypeptide further comprise the mutations L234A and L235A. In one embodiment, the first Fc region polypeptide and the second Fc region polypeptide further comprise the mutation P329G.

In one embodiment across all aspects, the Fc region polypeptide belongs to the human IgG4 subclass. In one embodiment, the first and second Fc region polypeptides further comprise the mutations S228P and L235E. In one embodiment, the first and second Fc region polypeptides further comprise the mutation P329G.

The antibodies reported herein are generated via recombinant methods. Therefore, one aspect, as reported herein, is a nucleic acid encoding the antibody as reported herein, and another aspect is a cell containing the nucleic acid encoding the antibody as reported herein. Methods for recombinant generation are widely known in the art and include protein expression in prokaryotic and eukaryotic cells, along with subsequent antibody isolation and typically purification to pharmaceutically acceptable purity. To express the aforementioned antibody in host cells, nucleic acids encoding the respective (modified) light and heavy chains are inserted into an expression vector using standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibody is recovered from the cells (culture supernatant or lysed cells). The general methods for generating antibodies through recombinant therapy are well known in the art and are described, for example, in the following review articles: Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-160; and Werner, R.G., Drug Res. 48 (1998) 870-880.

Therefore, one aspect as reported herein is a method for preparing a bispecific antibody as reported herein, the method comprising the steps of

a) Transform host cells using a vector containing nucleic acid molecules encoding antibodies.

b) Culture host cells under conditions that allow for antibody synthesis, and

c) Recover antibodies from the culture.

In one embodiment, the recovery step under c) includes the use of a light chain constant domain-specific capture reagent (which is, for example, specific to the κ or λ constant light chain, depending on whether the κ or λ light chain is contained in the bispecific antibody). In one embodiment, this light chain-specific capture reagent is used in a binding-elution mode. Examples of such light chain constant domain-specific capture reagents are, for example, KappaSelect ^™ and LambdaFabSelect ^™ (available from GE Healthcare/BAC), which are based on a highly rigid agarose-based matrix that allows for high flow rates and low back pressure at large scale. These materials contain ligands that bind to the constant regions of the κ or λ light chain, respectively (i.e., constant region fragments lacking the light chain will not bind; Figure 1). Thus, both are capable of binding to other target molecules containing the light chain constant region, such as IgG, IgA, and IgM. The ligands are attached to the matrix by means of hydrophilic long spacer arms to make them readily available for binding to target molecules. They are based on single-chain antibody fragments selected against human Igκ or λ.

In one embodiment, the recovery step under c) includes the use of an Fc region-specific capture reagent. In one embodiment, the Fc region-specific capture reagent is used in a binding-elution mode. Examples of such Fc region-specific capture reagents include affinity chromatography materials based on staphylococcal protein A.

Bispecific antibodies can be appropriately separated from the culture medium by conventional immunoglobulin purification methods such as affinity chromatography (protein A-agarose gel, or KappaSelect ^™ , LambdaFabSelect ^™ ), hydroxyapatite chromatography, gel electrophoresis, or dialysis.

Using conventional methods, the DNA and RNA encoding monoclonal antibodies can be easily isolated and sequenced. B cells or hybridoma cells can serve as sources of this DNA and RNA. Once isolated, this DNA can be inserted into an expression vector, which is then transfected into host cells that do not produce additional immunoglobulins (such as HEK293 cells, CHO cells, or myeloma cells) to achieve the synthesis of recombinant monoclonal antibodies in the host cells.

Some molecules, such as those reported herein, provide convenient separation/purification by including Fc regions that have undergone different modifications, wherein at least one modification results in i) a differential affinity of the molecule for (Staphylococcus) protein A and ii) a differential affinity of the molecule for human FcRn, and the molecule can be separated from ruptured cells, culture media, or molecular mixtures based on its affinity for protein A.

Antibody purification is performed using standard techniques (including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis) and other techniques well known in the art, with the aim of removing cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) (see, for example, Ausubel, F. et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Different methods have been established and are widely used for protein purification, such as affinity chromatography using microbial proteins (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed-mode exchange), thiophilic adsorption (e.g., using β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., using phenyl-agarose gel, aza-arenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., using Ni(II)- and Cu(II)- affinity materials), size exclusion chromatography, and electrophoresis methods (e.g., gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl. Biochem. Biotech. 75 (1998) 93-102).

The bivalent bispecific antibodies against human VEGF and human ANG-2 reported in this article can possess a valuable potency/safety profile and provide benefits to patients requiring anti-VEGF and anti-ANG-2 therapy.

One aspect reported herein is a pharmaceutical formulation comprising an antibody as reported herein. Another aspect reported herein is the use of the antibody as reported herein in the manufacture of a pharmaceutical formulation. Yet another aspect reported herein is a method for manufacturing a pharmaceutical formulation comprising an antibody as reported herein. In another aspect, formulations, such as pharmaceutical formulations, are provided containing an antibody as reported herein formulated together with a pharmaceutical carrier.

The formulations of the present invention can be administered by a variety of methods known in the art. Those skilled in the art will understand that the route and/or mode of administration will vary depending on the desired outcome. To administer compounds as reported herein via certain routes of administration, it may be necessary to coat the compound with a material that prevents its inactivation or to administer it co-with such a material. For example, the compound may be administered to the subject in a suitable carrier such as a liposome or a diluent. Pharmaceutically acceptable diluents include saline and buffered aqueous solutions. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the on-site preparation of sterile injectable solutions or dispersions. The use of such media and substances for pharmaceutically active substances is known in the art.

Many possible delivery modalities can be used, including but not limited to intraocular or local application. In one embodiment, application is intraocular and includes, but is not limited to, subconjunctival injection, intracranial injection, injection into the anterior chamber via the limbus, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, subfascial injection, or a persistent delivery device, and intravitreal injection (e.g., anterior, middle, or posterior vitreous injection). In one embodiment, application is local and includes, but is not limited to, instillation onto the cornea.

In one embodiment, a bispecific antibody or pharmaceutical formulation as reported herein is administered via intravitreal application, such as by intravitreal injection. This can be done according to standard methods known in the art (see, for example, Ritter et al., J. Clin. Invest. 116 (2006) 3266-3276; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185).

In some embodiments, the therapeutic kits reported herein may contain one or more doses of bispecific antibodies present in the pharmaceutical formulation described herein, a suitable device for intravitreal injection of the pharmaceutical formulation, and a description of the intended recipients and the protocol for administering the injection. In these embodiments, the formulation is generally administered to the subject requiring treatment via intravitreal injection. This can be done according to standard methods known in the art (see, for example, Ritter et al., J. Clin. Invest. 116 (2006) 3266-3276; Russelakis-Carneiro et al., Neuropathol. Appl. Neurobiol. 25 (1999) 196-206; and Wray et al., Arch. Neurol. 33 (1976) 183-185).

These formulations may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization methods described above and by incorporating various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to incorporate isotonic agents such as sugars and sodium chloride into the formulation. Furthermore, prolonged absorption of injectable drug forms can be achieved by incorporating substances that delay absorption, such as aluminum monostearate and gelatin.

Regardless of the chosen route of administration, the compounds as reported herein (which may be used in a suitable hydrated form) and/or pharmaceutical formulations as reported herein shall be formulated into pharmaceutically usable dosage forms using conventional methods known to those skilled in the art.

The actual dose level of the active ingredient in a pharmaceutical formulation, as reported herein, can be altered to obtain an amount of the active ingredient that, for a specific patient, formulation, and route of administration, effectively achieves the desired therapeutic response while being non-toxic to the patient. The chosen dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific formulation used, the route of administration, the time of administration, the excretion rate of the specific compound being used, the duration of treatment, other drugs, compounds, and/or materials used in combination with the formulation, and factors well-known in the medical field such as the patient's age, sex, weight, condition, general health, and medical history.

The formulation must be sterile and fluid enough to be delivered via a syringe. Besides water, the carrier is preferably an isotonic buffered saline solution.

Suitable flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable to include isotonic agents in the formulation, such as sugars, polyols such as mannitol or sorbitol, and sodium chloride.

Formulations may include ophthalmic depot formulations containing an active substance for subconjunctival administration. Ophthalmic depot formulations contain microparticles of substantially pure active substance (e.g., bispecific antibodies as reported herein). Microparticles containing bispecific antibodies as reported herein may be embedded in a biocompatible pharmaceutically acceptable polymer or lipid encapsulating agent. The depot formulation may be adapted to release all or substantially all of the active substance over an extended period of time. If present, the polymer or lipid matrix may be adapted to sufficiently degrade to allow translocation from the application site after the release of all or substantially all of the active substance. The depot formulation may be a liquid formulation containing a pharmaceutically acceptable polymer and a dissolved or dispersed active substance. Once injected, the polymer forms a reservoir at the injection site, for example, through a gelation or precipitation process.

Another aspect reported in this article is the use of bispecific antibodies, such as those reported in this article, for the treatment of ocular vascular diseases.

Another aspect reported in this article is the use of pharmaceutical preparations for the treatment of ocular vascular diseases, as described in this article.

One aspect reported in this article is the use of antibodies in the manufacture of drugs for the treatment of ocular vascular diseases.

Another aspect, as reported in this article, is the treatment of patients with ocular vascular diseases by administering antibodies, as reported in this article, to patients who require such treatment.

It is explicitly stated herein that the term "comprising" as used herein includes the term "consisting of". Therefore, all aspects and embodiments containing the term "comprising" are similarly disclosed using the term "consisting of".

Modification

In another aspect, antibodies according to any one or more embodiments may combine, alone or in combination, any of the features described in sections 1-6 below:

1. Antibody affinity

In some embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤100 nM or ≤10 nM (e.g., ^10⁻⁷ M or less, e.g., from ^10⁻⁷ M to ^10⁻¹³ M, e.g., from ^10⁻⁸ M to ^10⁻¹³ M).

According to another embodiment, Kd is measured using a surface plasmon resonance assay. For example, the assay is performed at 25°C using an immobilized antigen CM5 chip with approximately 10 response units (RUs) using a method from GE Healthcare Inc., Piscataway, NJ. In one embodiment, the carboxymethylated dextran biosensor chip (CM5, GE Healthcare, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg/mL (~0.2 μM) with 10 mM sodium acetate at pH 4.8, and then loaded at a flow rate of 5 μL/min to achieve approximately 10 response units (RUs) of the conjugate protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two serial dilutions of Fab (0.78 nM to 500 nM) were injected at 25 °C into PBS containing 0.05% polysorbate 20 (Tween-20 ^™ ) surfactant (PBST). The association rate (k_on) and dissociation rate (k _{_off}) were calculated by simultaneously fitting association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (evaluation software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio k_{_off} /k_{_on} (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). If the association rate is shown to exceed ^10⁶ M⁻¹ _s⁻¹ by the above surface plasmon resonance assay, the association rate can be determined using fluorescence quenching techniques. These fluorescence quenching techniques involve measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass ⁾ of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C in the presence of an increased concentration of antigen, as measured by a spectrometer such as an Aviv Instruments spectrophotometer equipped with a stop-flow method or an 8000-series SLM-AMINCO™ spectrophotometer with a stirred cuvette.

2. Chimeric and humanized antibodies

In some embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a “class-switching” antibody, wherein the class or subclass has been altered relative to the parent antibody. A chimeric antibody comprises its antigen binding to its fragment.

In some embodiments, the chimeric antibody is a humanized antibody. Generally, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody contains one or more variable domains in which the HVR, such as the CDR (or a portion thereof), is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may also optionally contain at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

Humanized antibodies and methods for their manufacture are reviewed, for example, in Almagro, J.Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, and, for example, in Riechmann, I. et al., Nature 332 (1988) 323-329; Queen, C. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; US 5,821,337, US 7,527,791, US 6,982,321 and US 7,087,409; Kashmiri, S.V. et al., Me Methods 36 (2005) 25-34 (description of specific region-determining (SDR) transplantation); Padlan, E.A., Mol. Immunol. 28 (1991) 489-498 (description of “resurfacing”); Dall'Acqua, W.F. et al., Methods 36 (2005) 43-60 (description of “FR reorganization”); Osbourn, J. et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (description of “guided selection” scheme for FR reorganization) further describe this.

Human scaffold regions that can be used for humanization include, but are not limited to: scaffold regions selected using the “best fit” method (e.g., see Sims, M.J. et al., J. Immunol. 151 (1993) 2296-2308); and scaffold regions derived from the common sequence of the light chain variable region or heavy chain variable region of a specific subgroup of human antibodies (e.g., see Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, L.G. et al., J. Immunol. 151 (1992) 2296-2308). 3) 2623-2632); human mature (somatic mutation) framework regions or human germline framework regions (e.g., see Almagro, J.C. and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (e.g., see Baca, M. et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, M.J. et al., J. Biol. Chem. 271 (1996) 22611-22618).

3. Human antibodies

In some embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

Human antibodies can be prepared by administering an immunogen to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. These animals generally contain all or part of human immunoglobulin loci that have replaced endogenous immunoglobulin loci or are located extrachromosomally or randomly integrated into the animal's chromosome. In these transgenic mice, endogenous immunoglobulin loci are usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, for example, US 6,075,181 and US 6,150,584 describing the XENOMOUSE ^™ technology; US 5,770,429 describing the technology; US 7,041,870 describing the KM technology; and US 2007/0061900 describing the Veloci technology. The human variable regions of intact antibodies produced from these animals can be further modified, for example, by combining them with different human constant regions.

Human antibodies can also be generated using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to generate human monoclonal antibodies have been described (e.g., see Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, B.R. et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp. 51-63; and Boerner, P. et al., J. Immunol. 147 (1991) 86-95). Human antibodies generated using human B-cell hybridoma technology have also been described in Li, J. et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Additional methods include, for example, those described in US 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (which describes human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers, H.P. and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, H.P. and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-derived antibodies

Antibodies, as reported herein, can be isolated by screening combinatorial libraries for antibodies possessing one or more desired activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics. These methods are reviewed, for example, in Hoogenboom, H.R. et al., Methods in Molecular Biology 178 (2001) 1-37 and, for example, in McCafferty, J. et al., Nature 348 (1990) 552-554; Clackson, T. et al., Nature 352 (1991) 624-628; Marks, J.D. et al., J. Mol. Biol. 222 (1992) 581-597; Marks, J.D. and Bradbury, A., Methods in Further descriptions can be found in Molecular Biology 248(2003)161-175; Sidhu, S.S. et al., J.Mol.Biol. 338(2004)299-310; Lee, C.V. et al., J.Mol.Biol. 340(2004)1073-1093; Fellouse, F.A., Proc.Natl.Acad.Sci.USA 101(2004)12467-12472 and Lee, C.V. et al., J.Immunol.Methods 284(2004)119-132.

In some phage display methods, VH and VL gene libraries are cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, whereby the phage library can subsequently be screened for phages that bind to the antigen, as described in Winter, G., et al., Ann. Rev. Immunol., 12 (1994) 433-455. Phages generally display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural libraries can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self antigens and also against self antigens without any immunization, as described in Griffiths, A.D., et al., EMBO J. 12 (1993) 725-734. Finally, natural libraries can also be synthesized by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and to achieve in vitro rearrangement, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example: US5,750,373 and US2005/0079574, US2005/0119455, US2005/0266000, US2007/0117126, US2007/0160598, US2007/0237764, US2007/0292936 and US2009/0002360.

The antibodies or antibody fragments isolated from the human antibody library are considered to be the human antibodies or human antibody fragments described in this paper.

5. Multispecific antibodies

In some embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two distinct sites. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing one or more target antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO93/08829 and Traunecker, A. et al., EMBO J.10 (1991) 3655-3659) and “protrusion-in-pore” engineering (e.g., see US5,731,168). Multispecific antibodies can also be generated in the following ways: engineered electrostatic manipulation effects to generate antibody Fc-heterodimer molecules (WO 2009/089004); crosslinking two or more antibodies or fragments (e.g., see US 4,676,980 and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to generate bispecific antibodies (e.g., see Kostelny, S.A. et al., J. Immunol. 148 (1992) 1547-1553); using "bimeric antibodies" The "(diabody)" technique is used to generate bispecific antibody fragments (e.g., see Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and to use single-chain Fv (sFv) dimers (e.g., see Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and to prepare trispecific antibodies as described, for example, in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

This article also includes engineered antibodies with three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006/0025576).

The antibodies or fragments described herein also include “dual-function Fab” or “DAF”, which contain antigen-binding sites that bind to a first antigen and another different antigen (see, for example, US 2008/0069820).

The antibodies or fragments mentioned in this article also include multispecific antibodies described in WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792 and WO 2010/145793.

6. Antibody variants

In some embodiments, amino acid sequence variants of the antibodies provided herein are conceived. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deleting residues from the amino acid sequence of the antibody and/or inserting residues into said amino acid sequence and/or substituting residues in said amino acid sequence. Any combination of deletions, insertions, and substitutions can be produced to obtain a final construct, provided that said final construct possesses the desired characteristics, such as antigen binding.

a) Substitution variants, insertion variants, and deletion variants

In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conserved substitutions are shown in the table below under the heading “Preferred Substitutions.” More obvious variations are shown in the table below under the heading “Exemplary Substitutions” and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody and screen the product for desired activities, such as retained/improved antigen binding or reduced immunogenicity, or improved ADCC or CDC.

surface

Original residues Exemplary permutation Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys；Gln；Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser；Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu, Val; Met; Ala; Phe; Leucine Leu Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg；Gln；Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity: Leucine, Met, Ala, Val, Leu; Ile;

(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn; Gln;

(3) Acidity: Asp, Glu;

(4) Alkaline: His, Lys, Arg;

(5) Residues that affect chain direction: Gly, Pro;

(6) Fang tribe: Trp, Tyr, Phe.

Non-conservative permutations will swap members of one of these categories with members of another category.

A substitution variant type involves replacing one or more hypervariable residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant, selected for further research, is modified (e.g., improved) relative to the parent antibody in terms of certain biological properties (e.g., increased affinity, decreased immunogenicity) and/or retains some biological properties substantially preserved by the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

Alterations (e.g., substitutions) can be made in the HVR, for example, to improve antibody affinity. Such alterations can be made in HVR “hotspots” (i.e., residues encoded by codons that undergo mutations at high frequency during somatic maturation (see, e.g., Chowdhury, P.S., Methods Mol. Biol. 207 (2008) 179-196) and/or residues that contact the antigen, while simultaneously testing binding affinity for the resulting variant VH or VL. Affinity maturation by constructing a secondary library and reselecting from it has been described, for example, in Hoogenboom, H.R. et al., cited in Methods in Molecular Biology 178 (2002) 1-37). In some embodiments of affinity maturation, diversity is introduced into the selected variant gene for maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-guided protocols, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be specifically identified, for example, by mutagenesis or modeling using alanine scanning. CDR-H3 and CDR-L3 are particularly frequently targeted.

In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not materially reduce the antibody's ability to bind to the antigen. For example, conserved alterations that do not materially reduce binding affinity (e.g., conserved substitutions as provided herein) may be made in the HVR. Such alterations, for example, may be external to the antigen-contacting residues of the HVR. In some embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham, B.C. and Wells, J.A., Science, 244 (1989) 1081-1085. In this method, a residue or target group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Additional substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions of peptides ranging in length from one residue to hundreds or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertive variants of antibody molecules include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., an enzyme targeting ADEPT) or a peptide that increases the serum half-life of the antibody.

b) Glycosylation variants

In some implementations, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation. Adding or deleting glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

In the case of antibodies containing an Fc region, the sugars linked to it can be modified. Naturally occurring antibodies produced by mammalian cells generally contain branched biantennary oligosaccharides, which are typically linked to the CH2 domain of the Fc region via an N-linked Asn297. See, for example, Wright, A. and Morrison, S.L., TIBTECH 15 (1997) 26-32. Oligosaccharides can include a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in antibodies as reported herein can be modified to produce antibody variants with improved properties.

In one embodiment, an antibody variant is provided having a sugar structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex structures, hybrid structures, and high-mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008/077546. Asn297 refers to the asparagine residue (EU number of Fc region residues) located approximately at position 297 within the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. These fucosylated variants can possess improved ADCC function. See, for example, US 2003/0157108; US 2004/0093621. Examples of publications related to “defucosylated” or “fucosylated” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109 865; WO2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO2002/031140; Okazaki, A. et al., J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable of producing defucosylation antibodies include Lec13CHO cells defective in protein fucosylation (Ripka, J. et al., Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; and WO 2004/056312, particularly at Example 11) and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (e.g., see Yamane-Ohnuki, N. et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003/085107).

Antibody variants may also be provided with bimeric oligosaccharides, for example, wherein the biantennary oligosaccharide linked to the Fc region of the antibody is bimeric by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878; US6,602,684; and US 2005/0123546. Antibody variants are also provided with at least one galactose residue in the oligosaccharide linked to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087; WO 1998/58964 and WO1999/22764.

c) Fc region variants

In some embodiments, one or more additional amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant. This Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions/mutations) at one or more amino acid positions.

In some embodiments, the present invention conceives of antibody variants possessing some, but not all, effector functions, making these variants attractive candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduced/depleted CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary cells mediating ADCC—NK cells—express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in US 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al., Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); and US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive analytical methods can be used (see, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc., Mountain View, CA; and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in animal models such as those disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, WO 2006/029879 and WO C1q and C3c binding ELISA in 2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro, H. et al., J. Immunol. Methods 202(1996) 163-171; Cragg, MS et al., Blood 101(2003) 1045-1052; and Cragg, MS and MJ Glennie, Blood 103(2004) 2738-2743). FcRn binding and in vivo clearance/half-life can also be determined using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18(2006) 1759-1769).

Antibodies with reduced effector function include those that substitute one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US 6,737,056). These Fc region variants include Fc regions with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc region mutant (US 7,332,581) with residues 265 and 297 substituted with alanine.

Certain antibody variants with improved or weakened FcR binding activity have been described (see, for example, US 6,737,056; WO 2004/056312, and Shields, R.L. et al., J. Biol. Chem. 276(2001)6591-6604).

In some embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and/or 334 of the Fc region) (EU numbers of the residues).

In some implementations, changes are made within the Fc region that result in altered (i.e., improved or weakened) C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as described in US 6,194,551, WO 99/51642, and Idusogie, E.E. et al., J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus, are described in US 2005/0014934 (Guyer, R.L. et al., J. Immunol. 117 (1976) 587-593 and Kim, J.K. et al., J. Immunol. 24 (1994) 2429-2434). These antibodies contain an Fc region having one or more substitutions, wherein the substitutions improve the binding of the Fc region to the FcRn. These Fc region variants include those with a substitution (e.g., a substitution of Fc region residue 434) at one or more Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 (US 7,371,826).

For other examples of Fc region variants, see Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821 and WO 94/29351.

d) Cysteine-engineered antibody variants

In some embodiments, it may be desirable to generate cysteine-engineered antibodies, such as “thioMAb”, wherein one or more residues of the antibody are replaced with cysteine residues. In specific embodiments, the replaced residues are located at accessible sites on the antibody. By replacing these residues with cysteine, a reactive thiol group is thus positioned at an accessible site on the antibody and can be used to conjugate the antibody to other parts, such as pharmaceutical portions or linker-pharmaceutical portions, to generate immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues may be replaced with cysteine: V205 (Kabat number) of the light chain and A118 (EU number) of the heavy chain; and S400 (EU number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described in US7,521,541.

e) Antibody derivatives

In some embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly(1,3-dioxolane), poly(1,3,6-triane), ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can have manufacturing advantages due to its stability in water. This polymer can have any molecular weight and can be branched or unbranched. The number of polymers linked to the antibody can vary, and if more than one polymer is linked, they can be the same or different molecules. Typically, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in a limited therapeutic context, and so on.

In another embodiment, an antibody and a conjugate of a non-protein portion that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein portion is carbon nanotubes (Kam, N.W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature that kills cells adjacent to the antibody-non-protein portion.

f) Heterodimerization

Several schemes exist for modifying CH3 to enhance heterodimerization, which are fully described, for example, in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, and WO 2013096291. Generally, in all such schemes, the first and second CH3 domains are engineered in a complementary manner, so that each CH3 domain (or the heavy chain containing it) cannot homodimerize with itself for a long time, but is forced to heterodimerize with the other complementary engineered CH3 domain (thus the first and second CH3 domains heterodimerize and no homodimer is formed between the two first CH3 domains or between the two second CH3 domains). These different schemes conceived to improve heavy chain heterodimerization are alternatives to heavy chain-light chain modifications in the multispecific antibodies of this invention (VH and VL exchange/replacement in a binding arm and introduction of oppositely charged amino acid substitutions at the CH1/CL interface), which reduces Bence-Jones-type byproducts of light chain mispairing.

In a preferred embodiment of the invention (in which the multispecific antibody contains a CH3 domain in the heavy chain), the CH3 domain of the multispecific antibody of the invention can be altered by a "protrusion-in-pore" technique, detailed in examples such as WO 96/027011; Ridgway, J.B. et al., Protein Eng. 9 (1996) 617-621; and Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-681; WO 98/050431. In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two heavy chains containing the two CH3 domains. One of the two CH3 domains (of the two heavy chains) can be a "protrusion," and the other a "pore." The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, A.M. et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

Therefore, in one embodiment of the invention, the multispecific antibody (containing a CH3 domain in each heavy chain) is further characterized in that...

The first CH3 domain of the first heavy chain of the antibody in a) and the second CH3 domain of the second heavy chain of the antibody in b) each meet at the interface containing the initial interface between the antibody CH3 domains.

The interface is modified to promote the formation of multispecific antibodies, wherein the modification is characterized by:

i) This alters the CH3 domain of a heavy chain.

Thus, within the multispecific antibody, at the initial interface of the CH3 domain of one heavy chain, it meets with the initial interface of the CH3 domain of another heavy chain.

Replacing amino acid residues with amino acid residues having a larger side chain volume creates a protrusion inside the interface of a CH3 domain of a heavy chain, which can be placed in a cavity inside the interface of a CH3 domain of another heavy chain.

and

ii) This alters the CH3 domain of the other heavy chain.

Thus, within the multispecific antibody, at the initial interface of the second CH3 domain, which meets the initial interface of the first CH3 domain,

By replacing amino acid residues with amino acid residues having smaller side chain volumes, a cavity is created inside the interface of the second CH3 domain, in which the protrusion inside the interface of the first CH3 domain can be placed.

Preferably, the amino acid residues with larger side chain volume are selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

Preferably, the amino acid residues with smaller side chain volume are selected from alanine (A), serine (S), threonine (T), and valine (V).

In one aspect of the invention, the two CH3 domains are further modified by introducing cysteine (C) as an amino acid at the corresponding position in each CH3 domain, thereby allowing disulfide bonds to be formed between the two CH3 domains.

In one embodiment, the multispecific antibody contains the amino acid T366W mutation in the first CH3 domain of the "protrusion chain" and the amino acid T366S, L368A, and Y407V mutations in the second CH3 domain of the "pore chain". Additional interchain disulfide bonds between the CH3 domains can also be used (Merchant, A.M. et al., Nature Biotech 16 (1998) 677-681), for example, by introducing the amino acid Y349C mutation into the CH3 domain of the "pore chain" and the amino acid E356C or S354C mutation into the CH3 domain of the "protrusion chain".

In a preferred embodiment, the multispecific antibody (which contains a CH3 domain in each heavy chain) contains the amino acid S354C, T366W mutation in one of the two CH3 domains and the amino acid Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (the additional amino acid S354C mutation in one CH3 domain and the additional amino acid Y349C mutation in the other CH3 domain form an interchain disulfide bond) (according to Kabat numbering).

Other techniques for modifying CH3 to enhance heterodimerization are conceived as alternatives to the present invention and are described, for example, in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, and WO 2013/096291.

In one embodiment, the heterodimerization scheme described in EP 1 870 459A1 may be used alternatively. This method is based on introducing charged amino acid substitutions/mutations with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two heavy chains. A preferred embodiment of the multispecific antibody is a mutation of amino acids R409D and K370E in the first CH3 domain and a mutation of amino acids D399K and E357K (according to Kabat numbering) in the second CH3 domain of the multispecific antibody.

In another embodiment, the multispecific antibody contains the amino acid T366W mutation in the CH3 domain of the "protrusion chain" and the amino acid T366S, L368A, Y407V mutations in the CH3 domain of the "pore chain," and additionally contains the amino acid R409D, K370E mutations in the CH3 domain of the "protrusion chain" and the amino acid D399K, E357K mutations in the CH3 domain of the "pore chain."

In another embodiment, the multispecific antibody contains the amino acid S354C, T366W mutation in one of the two CH3 domains and the amino acid Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains; or the multispecific antibody contains the amino acid Y349C, T366W mutation in one of the two CH3 domains and the amino acid S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains, and additionally contains the amino acid R409D, K370E mutation in the CH3 domain of the "protrusion chain" and the amino acid D399K, E357K mutation in the CH3 domain of the "pore chain".

In one embodiment, the heterodimerization scheme described in WO 2013/157953 may be used alternatively. In one embodiment, the first CH3 domain contains the amino acid T366K mutation and the second CH3 domain polypeptide contains the amino acid L351D mutation. In yet another embodiment, the first CH3 domain contains other amino acid L351K mutations. In still another embodiment, the second CH3 domain contains other amino acid mutations selected from Y349E, Y349D, and L368E (preferably L368E).

In one embodiment, the heterodimerization scheme described in WO 2012/058768 may be used alternatively. In one embodiment, the first CH3 domain contains the amino acid L351Y, Y407A mutation and the second CH3 domain contains the amino acid T366A, K409F mutation. In yet another embodiment, the second CH3 domain contains other amino acid mutations at positions T411, D399, S400, F405, N390, or K392, such amino acid mutations being selected, for example, from a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W; b) D399R, D399W, D399Y, or D399K; c) S400E, S400D, S400R, or S400K; F405I, F405M, F405T, F405S, F405V, or F405W; N390R, N390K, or N390D; K392V, K392M, K392R, K392L, K392F, or K392E. In yet another embodiment, the first CH3 domain contains the amino acid mutations L351Y and Y407A, and the second CH3 domain contains the amino acid mutations T366V and K409F. In yet another embodiment, the first CH3 domain contains the amino acid mutation Y407A, and the second CH3 domain contains the amino acid mutations T366A and K409F. In yet another embodiment, the second CH3 domain contains other amino acid mutations K392E, T411E, D399R, and S400R.

In one embodiment, the heterodimerization scheme described in WO 2011/143545 may be used alternatively, for example, with amino acid modifications at positions selected from 368 and 409.

In one embodiment, the heterodimerization scheme described in WO 2011/090762 may alternatively be used, which also employs the protrusion-in-pore technique described above. In one embodiment, the first CH3 domain contains the amino acid T366W mutation and the second CH3 domain contains the amino acid Y407A mutation. In one embodiment, the first CH3 domain contains the amino acid T366Y mutation and the second CH3 domain contains the amino acid Y407T mutation.

In one implementation, the multispecific antibody is of the IgG2 isotype and may alternatively use the heterodimerization scheme described in WO 2010/129304.

In one embodiment, the heterodimerization scheme described in WO 2009/089004 may be used alternatively. In one embodiment, the first CH3 domain comprises a substitution of K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) and the second CH3 domain comprises a substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In yet another embodiment, the first CH3 domain further comprises a substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In yet another embodiment, the first CH3 domain further or additionally comprises an amino acid substitution of K439 and/or K370 by a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)).

In one embodiment, the heterodimerization scheme described in WO 2007/147901 may be used alternatively. In one embodiment, the first CH3 domain contains amino acid mutations of K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations of D239K, E240K, and K292D.

In one implementation, the heterodimerization scheme described in WO 2007/110205 may be used alternatively.

Recombinant methods and formulations

Antibodies can be generated using recombinant methods and formulations, such as those described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody as described herein is provided. This nucleic acid may encode an amino acid sequence comprising antibody VL and/or an amino acid sequence comprising antibody VH (e.g., the light chain and/or heavy chain of the antibody). In yet another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In yet another embodiment, a host cell comprising such nucleic acid is provided. In such an embodiment, the host cell comprises (e.g., has been transformed therewith): (1) a vector comprising nucleic acid encoding an amino acid sequence comprising antibody VL and an amino acid sequence comprising antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VH. In one embodiment, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for generating an antibody as reported herein is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

To recombinantly generate a variant Fc region, the nucleic acid encoding the variant Fc region (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and/or expression in host cells. This nucleic acid can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the variant Fc region polypeptide or antibody heavy and light chains).

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, particularly when glycosylation and Fc effector function are not required. For examples of antibody fragment and peptide expression in bacteria, see US 5,648,237, US 5,789,199, and US 5,840,523 (see also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in *E. coli*). After expression, antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of antibodies with partial or complete human glycosylation patterns (see Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains that can be used in conjunction with insect cells have been identified, particularly for transfecting fall armyworm (Spodoptera frugiperda) cells.

Plant cell cultures can also be used as hosts (see, for example, US5,959,177, US6,040,498, US6,420,548, US7,125,978 and US6,417,429 (description of PLATNIBODIES ^™ technology for generating antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension growth can be used. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell lines (described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, HEK293 or 293 cells); young hamster kidney cells (BHK); mouse supporting cells (described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252, TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); described, for example in Mather, JP et al., Annals TRI cells, MRC 5 cells, and FS4 cells are mentioned in NYAcad.Sci. 383 (1982) 44-68. Other useful mammalian cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2/0. A review of certain mammalian host cell lines suitable for antibody production can be found, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

Determination method

The physical/chemical properties and/or biological activity of the antibodies provided herein can be identified, screened, or characterized using a variety of assays known in the art.

In one aspect, for example, the antigen-binding activity of antibodies such as those reported in this article can be tested using known methods such as ELISA, Western blotting, etc.

Immunoconjugates

The present invention also provides immunoconjugates comprising antibodies as reported herein conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal protein toxins, enzyme-active toxins) or radioisotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see US5,208,020, US5,416,064 and EP0425235B1); auristatins such as monomethyl auristatin drug portions DE and DF (MMAE and MMAF) (see US5,635,483 and US5,780,588 and US7,498,298); and dolastatin. Ciprofloxacin or its derivatives (see US 5,712,374, US 5,714,586, US 5,739,116, US 5,767,285, US 5,770,701, US 5,770,710, US 5,773,001, and US 5,877,296; Hinman, L.M. et al., Cancer Res. 53 (1993) 3336-3342; and Lode, H.N. et al., Cancer Res. 58 (1998) 292) 5-2928); anthracyclines such as daunorubicin or doxorubicin (Kratz, F. et al., Curr. Med. Chem. 13 (2006) 477-523; Jeffrey, S.C. et al., Bioorg. Med. Chem. Lett. 16 (2006) 358-362; Torgov, M.Y. et al., Bioconjug. Chem. 16 (2005) 717-721; Nagy, A. et al., Proc. Natl. Acad. Sci. USA 97 (2 000)829-834; Dubowchik, G.M. et al., Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, H.D. et al., J. Med. Chem. 45 (2002) 4336-4343; and US 6,630,579); methotrexate; vinca disine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel and ortataxel; trichothecene compounds; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzyme-active toxin or a fragment thereof, said enzyme-active toxin or fragment thereof including, but not limited to, diphtheria toxin A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, acacia toxin A chain, senna root toxin A chain, α-broomrin, tung oil (Aleurites fordii) protein, carnation toxin, phytolacca americana protein (PAPI, PAPII, and PAP-5), momordica charantia inhibitory protein, jatropha toxin, croton toxin, sapaonaria officinalis inhibitory protein, white jatropha toxin, mitogellin, localized aspergillin, phenolmycin, enoxamin, and trichothecene compounds.

In another embodiment, the immunoconjugate comprises an antibody, as described herein, conjugated with a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to generate the radioconjugate. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu. When the radioconjugate is used for detection, it may contain radioactive atoms used for scintillation studies, such as TC ^99m or I ¹²³ , or spin-labeled materials for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

A variety of bifunctional protein conjugates can be used to generate conjugates of antibodies and cytotoxic agents, such as N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), iminothiones (IT), bifunctional derivatives of iminoesters (such as dimethyl hexamethylenediimide hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-diazonyl benzoyl)hexamethylenediamine), bis-diazonium salt derivatives (such as bis(p-diazonyl benzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bi-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta, E.S. et al., Science 238 (1987) 1098-1104. Carbon-14 labeled 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionuclides to antibodies. See WO94/11026. The adapter can be a “cleavable adapter” that facilitates the release of cytotoxic agents into cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or adapters containing disulfide bonds can be used (Chari, R.V. et al., Cancer Res. 52 (1992) 127-131; US 5,208,020).

Immunoconjugates or ADCs are explicitly conceived herein, but are not limited to such conjugates prepared using cross-linking agents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, and sulfon-SMPB, and commercially available SVSB (succinimide-(4-vinyl sulfone)benzoate) (e.g., obtained from Ppierce Biotechnology, Inc., Rockford, Illinois, USA).

Methods and preparations for diagnosis and detection

In some embodiments, any of the antibodies provided herein can be used to detect the presence of one or more of their associated antigens in a biological sample. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some embodiments, the biological sample includes cells or tissues.

In one implementation, an antibody reported herein is provided for use in diagnostic or detection methods.

In some embodiments, labeled antibodies as reported herein are provided. Labels include, but are not limited to, directly detectable labels or portions (such as fluorescent, chromogenic, electronically dense, chemiluminescent, and radioactive labels), and indirectly detectable portions (e.g., by means of enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary markers include, but are not limited to, radioisotopes ^32P , ^14C , ^125I , ^3H , and ^131I ; fluorophores such as rare earth element chelates or luciferin and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (US4,737,456); luciferin; 2,3-dihydrodiazanaphthalenedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases such as uricase and xanthine oxidase, coupled with enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase; biotin/avidin; spin markers; phage markers; stable free radicals; etc.

pharmaceutical preparations

Pharmaceutical formulations of antibodies as described herein are prepared in lyophilized or aqueous solutions by mixing the antibody, having the desired purity, with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers such as phosphates, citric acid, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecylbenzyldimethylammonium chloride; hexamethyl chloride; benzalkonium chloride, benzalkonium bromide; phenol, butanol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); and low molecular weight (less than about 10 residues) peptides. Proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannose, trehalose, or sorbitol; anti-charge ions that form salts, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers described herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rhuPH20 (Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US2005/0260186 and US2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006/044908, the latter containing a histidine-acetate buffer.

The formulations described herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other, depending on the specific indication being treated. Such active ingredients are appropriately present in an amount effective for the intended purpose.

The active ingredient can be encapsulated in, for example, microcapsules (e.g., hydroxymethyl cellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or emulsions prepared by coagulation techniques or interfacial polymerization, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

Sustained-release articles can be prepared. Suitable examples of sustained-release articles include solid hydrophobic polymer semi-permeable matrices containing antibodies, said matrices being in the form of molded articles (e.g., films or microcapsules).

Preparations intended for internal administration are typically sterile. Sterility can be easily achieved, for example, by filtration using a sterile filter membrane.

Treatment methods and preparations

Any antibodies described in this article can be used in treatments.

In one respect, antibodies, as reported in this article, are provided for use as medicines.

In some embodiments, an antibody is provided for use in the treatment method. In one such embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent, such as the therapeutic agent described below, to the individual. In a preferred embodiment, the "individual" according to any of the more than one embodiment is a human being.

In another aspect, the present invention provides the use of antibodies in the manufacture or preparation of pharmaceuticals. The "individual" according to any of the above embodiments can be a human being.

In another aspect, the present invention provides pharmaceutical formulations comprising any antibody provided herein, for example, for use in any of the foregoing treatment methods. In one embodiment, the pharmaceutical formulation comprises any antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any antibody provided herein and at least one additional therapeutic agent, for example, as described below.

The antibodies reported in this article can be used alone or in combination with other active agents in therapy. For example, the antibodies reported in this article can be co-administered with at least one additional therapeutic agent.

The antibodies (and any additional therapeutic agents) reported herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if local treatment requires). Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out via any suitable route, such as by injection, like intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing regimens are conceived herein, including but not limited to single or multiple administrations at various time points, rapid concentrated administration, and pulsatile infusion.

The antibodies reported herein will be formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery, the method of administration, the administration schedule, and other factors known to the medical practitioner. The antibody does not need to be formulated with one or more drugs currently used to prevent or treat the disease in question, but may optionally be formulated with them. The effective amount of such other drugs depends on the amount of antibody present in the formulation, the type of disease or therapy, and other factors discussed above. These drugs are typically used at the same dose and via the same route of administration as described herein, or at approximately 1% to 99% of the dose described herein, or at any dose and via any route determined empirically/clinically to be appropriate.

For the prevention or treatment of disease, the appropriate dose of the antibody, as reported herein (when used alone or in combination with one or more other additional therapeutic agents), will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the attending physician's decision. The antibody is appropriately administered to the patient as a single dose or in a series of treatments. Depending on the type and severity of the disease, an antibody dose of approximately 1 μg/kg to 15 mg/kg (e.g., 0.5 mg/kg–10 mg/kg) may be a starting candidate dose for administration to the patient, whether administered, for example, by one or more single doses or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from approximately 1 μg/kg to 100 mg/kg or more. For repeated administration over a period of several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms is achieved. An exemplary dose of the antibody would be in the range of approximately 0.05 mg/kg to approximately 10 mg/kg. Therefore, one or more doses (or any combination thereof) of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg can be administered to the patient. These doses can be administered intermittently, for example weekly or every 3 weeks (e.g., thus the patient receives about 2 to about 20 doses of the antibody, or for example about 6 doses). A higher initial loading dose can be administered, followed by one or more lower doses. The progress of this therapy can be easily monitored using routine techniques and assays.

It is understood that any of the aforementioned formulations or treatments may be implemented using an immunoconjugate as reported herein instead of an antibody as reported herein, or may be implemented using an immunoconjugate as reported herein in addition to an antibody as reported herein.

Manufactured products

In another aspect reported herein, an article is provided containing a substance described above that can be used to treat, prevent, and/or diagnose a condition. The article includes a container and a label or packaging insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains the preparation that is effective in treating, preventing, and/or diagnosing a condition on its own or in combination with another preparation and may have a sterile access point (e.g., the container may be an intravenous infusion bag or a vial with a stopper permeable by a hypodermic needle). At least one active substance in the preparation is an antibody as reported herein. The label or packaging insert indicates that the preparation is used to treat a selected condition. Furthermore, the article may include (a) a first container containing the preparation, wherein the preparation contains an antibody as reported herein; and (b) a second container containing the preparation, wherein the preparation contains other cytotoxic agents or therapeutic agents. In this embodiment reported herein, the article may also include a packaging insert indicating that the preparation can be used to treat a specific condition. Alternatively or additionally, the manufacture may also include a second (or third) container containing pharmaceutically acceptable buffers such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. It may also include other materials desirable from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

It is understood that any of the foregoing manufactures may include an immunoconjugate as reported herein in place of an antibody as reported herein, or may include an immunoconjugate as reported herein in addition to an antibody as reported herein.

III. Specific Implementation Plan

1. An IgG-like Fc region comprising a first variant Fc region polypeptide and a second variant Fc region polypeptide,

in

a) The first variant Fc region polypeptide is derived from the first parental IgG class Fc region polypeptide, and the second variant Fc region polypeptide is derived from the second parental IgG class Fc region polypeptide. Therefore, the first parental IgG class Fc region polypeptide may be the same as or different from the second parental IgG class Fc region polypeptide.

b) The first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues, excluding those amino acid residues in which the first parental IgG class Fc region polypeptide differs from the second parental IgG class Fc region polypeptide.

c) An IgG-type Fc region containing a first variant Fc region polypeptide and a second variant Fc region polypeptide has a different affinity for the human Fc receptor than an IgG-type Fc region containing a first parental IgG-type Fc region polypeptide and a) a second parental IgG-type Fc region polypeptide.

2. The IgG class Fc region according to implementation scheme 1, wherein the human Fc receptor is the human neonatal Fc receptor (FcRn) or the human FcγIII receptor (FcγRIII).

3. The IgG class Fc region according to any one of the implementation schemes 1 to 2, wherein the human Fc-receptor is the human neonatal Fc-receptor.

4. An IgG-type Fc region according to any one of embodiments 1 to 3, wherein, compared with an IgG-type Fc region containing a) a first parental IgG-type Fc region polypeptide and a) a second parental IgG-type Fc region polypeptide, the IgG-type Fc region containing the first variant Fc region polypeptide and the second variant Fc region polypeptide has an increased or decreased affinity for human Fc-receptor by 10% or more as determined by surface plasmon resonance (SPR).

5. According to any one of the embodiments 1 to 4, in the IgG Fc region, at least some of the amino acid residues that are different between the first parent IgG Fc region polypeptide and the second parent IgG Fc region polypeptide promote the formation of the heterodimeric IgG Fc region.

6. The IgG class Fc region according to any one of the implementation schemes 1 to 5, wherein...

i) The Fc region polypeptide of the first parental IgG was selected from

-Human IgG1Fc region polypeptide,

-Human IgG2Fc region polypeptide,

-Human IgG3Fc region polypeptide,

-Human IgG4Fc region polypeptide,

-A human IgG1Fc region polypeptide containing mutant L234A and L235A,

-A human IgG1 Fc region polypeptide containing mutants Y349C, T366S, L368A, and Y407V.

-A human IgG1 Fc region polypeptide containing mutants S354C, T366S, L368A, and Y407V.

- A human IgG1 Fc region polypeptide containing mutations L234A, L235A, Y349C, T366S, L368A, and Y407V.

- A human IgG1 Fc region polypeptide containing mutations L234A, L235A, S354C, T366S, L368A, and Y407V.

-A peptide containing the mutant P329G in the Fc region of human IgG1.

-A human IgG1Fc region polypeptide containing mutants L234A, L235A, and P329G.

- A human IgG1 Fc region polypeptide containing mutant P329G, Y349C, T366S, L368A, and Y407V.

-A human IgG1 Fc region polypeptide containing mutant P329G, S354C, T366S, L368A, and Y407V.

- A human IgG1 Fc region polypeptide containing mutations in L234A, L235A, P329G, Y349C, T366S, L368A, and Y407V.

- A human IgG1 Fc region polypeptide with mutations in L234A, L235A, P329G, S354C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutant S228P and L235E.

-A human IgG4Fc region polypeptide with mutant S228P, L235E, and P329G.

-A human IgG4Fc region polypeptide containing mutant Y349C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutants S354C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutants S228P, L235E, Y349C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutants S228P, L235E, S354C, T366S, L368A, and Y407V.

-A polypeptide containing the mutant P329G human IgG4Fc region,

- A human IgG4Fc region polypeptide containing mutant P329G, Y349C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutant P329G, S354C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutants S228P, L235E, P329G, Y349C, T366S, L368A, and Y407V.

-A human IgG4Fc region polypeptide containing mutants S228P, L235E, P329G, S354C, T366S, L368A, and Y407V.

-People with the K392D mutation have IgG1, IgG2, or IgG4, and

-Human IgG3 with mutation N392D,

and

ii) The second parental IgG Fc region polypeptide was selected from

-Human IgG1Fc region polypeptide,

-Human IgG2Fc region polypeptide,

-Human IgG3Fc region polypeptide,

-Human IgG4Fc region polypeptide,

-A human IgG1Fc region polypeptide containing mutant L234A and L235A,

-A human IgG1Fc region polypeptide with mutant S354C and T366W

-A human IgG1Fc region polypeptide with mutant Y349C and T366W

- A human IgG1 Fc region polypeptide containing mutant L234A, L235A, S354C, and T366W.

- A human IgG1 Fc region polypeptide containing mutants L234A, L235A, Y349C, and T366W.

-A peptide containing the mutant P329G in the Fc region of human IgG1.

-A human IgG1Fc region polypeptide containing mutants L234A, L235A, and P329G.

-A human IgG1 Fc region polypeptide with mutant P329G, S354C, and T366W

-A human IgG1Fc region polypeptide with mutant P329G, Y349C, and T366W.

- A human IgG1 Fc region polypeptide containing mutants L234A, L235A, P329G, S354C, and T366W.

- A human IgG1 Fc region polypeptide containing mutants L234A, L235A, P329G, Y349C, and T366W.

-A human IgG4Fc region polypeptide containing mutant S228P and L235E.

-A human IgG4Fc region polypeptide with mutant S228P, L235E, and P329G.

-A human IgG4Fc region polypeptide with mutant S354C and T366W

-A human IgG4Fc region polypeptide with mutant Y349C and T366W.

-A human IgG4Fc region polypeptide with mutated S228P, L235E, S354C, and T366W.

-A human IgG4Fc region polypeptide with mutant S228P, L235E, Y349C, and T366W.

-A polypeptide containing the mutant P329G human IgG4Fc region,

-A human IgG4Fc region polypeptide containing mutant P329G, S354C, and T366W.

-A human IgG4Fc region polypeptide with mutant P329G, Y349C, and T366W.

-A human IgG4Fc region polypeptide with mutant S228P, L235E, P329G, S354C, and T366W.

-A human IgG4Fc region polypeptide with mutant S228P, L235E, P329G, Y349C, and T366W.

-Human IgG1 with mutations in D399K, D356K, and/or E357K, and

-People with IgG2, IgG3, or IgG4 who have mutations in D399K, E356K, and/or E357K.

7. The IgG class Fc region according to any one of the implementation schemes 1 to 6, wherein

i) The Fc region polypeptide of the first parental IgG class has an amino acid sequence selected from SEQ ID NO: 60, 61, 62, 63, 64, 65, 67, 69, 70, 71, 73, 75, 76, 78, 80, 81, 82 and 84.

and

ii) The second parent IgG Fc region polypeptide has an amino acid sequence selected from SEQ ID NO: 60, 61, 62, 63, 64, 66, 68, 69, 70, 72, 74, 75, 76, 77, 79, 81, 83 and 85.

8. The IgG class Fc region according to any one of the implementation schemes 1 to 7, wherein

i) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide, or

ii) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A and L235A, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A and L235A, or

iii) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, and P329G, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, and P329G, or

iv) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, S354C, or T366W, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, Y349C, T366S, L368A, or Y407V, or

v) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, P329G, S354C, or T366W; the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations L234A, L235A, P329G, Y349C, T366S, L368A, or Y407V.

vi) The first parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide and the second parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide, or

vii) The first parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutant S228P and L235E, and the second parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutant S228P and L235E, or

viii) The first parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, and P329G, and the second parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, and P329G, or

ix) The first parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, S354C, and T366W, and the second parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, Y349C, T366S, L368A, and Y407V, or

x) The first parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, P329G, S354C, and T366W, and the second parental IgG Fc region polypeptide is a human IgG4 Fc region polypeptide with mutations S228P, L235E, P329G, Y349C, T366S, L368A, and Y407V.

9. The IgG class Fc region according to any one of the implementation schemes 1 to 5 and 7, wherein

i) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:60 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:60, or

ii) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:64 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:64, or

iii) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:70 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:70, or

iv) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:68 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:67, or

v) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:74 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:73, or

vi) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:63 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:63, or

vii) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:75 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:75, or

viii) The first parental IgG Fc region polypeptide has the amino acid sequence SEQ ID NO:76 and the second parental IgG Fc region polypeptide has the amino acid sequence SEQ ID NO:76, or

ix) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:79 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:80, or

x) The first parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:85 and the second parental IgG Fc region polypeptide has the amino acid sequence of SEQ ID NO:84.

10. An IgG-type Fc region according to any one of embodiments 1 to 9, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in 1 to 8 amino acid residues, excluding those amino acid residues in which the first parental IgG-type Fc region polypeptide differs from the second parental IgG-type Fc region polypeptide.

11. An IgG-type Fc region according to any one of embodiments 1 to 10, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in 1 to 6 amino acid residues, excluding those amino acid residues in which the first parental IgG-type Fc region polypeptide differs from the second parental IgG-type Fc region polypeptide.

12. An IgG-type Fc region according to any one of embodiments 1 to 11, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in 1 to 3 amino acid residues, excluding those amino acid residues in which the first parental IgG-type Fc region polypeptide differs from the second parental IgG-type Fc region polypeptide.

13. The IgG class Fc region according to any one of embodiments 1 to 12, wherein the first variant Fc region peptide is located at positions 228, 234, 235, 236, 237, 238, 239, 248, 250, 251, 252, 253, 254, 255, 256, 257, 265, 266, 267, 268, 269, 270, 272, 285, 288, 2 One or more amino acid residues at positions 90, 291, 297, 298, 299, 307, 308, 309, 310, 311, 314, 327, 328, 329, 330, 331, 332, 385, 387, 428, 433, 434, 435, and 436 (numbered according to the KabatEU index system) differ from the second variant Fc region polypeptide.

14. An IgG-type Fc region according to any one of embodiments 1 to 2 and 4 to 13, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues at positions 228, 234, 235, 236, 237, 238, 239, 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 327, 328, 329, 330, 331, 332, 434, and 435 (numbered according to the Kabat EU index numbering system).

15. An IgG-type Fc region according to any one of embodiments 1 to 2 and 4 to 14, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues at positions 233, 236, 265, 297, 329 and 331.

16. The IgG class Fc region according to embodiment 15, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide due to one or more amino acid changes E233P, ΔG236, D265A, N297A, N297D, P329A, and P331S.

17. An IgG-type Fc region according to any one of embodiments 1 to 3 and 5 to 13, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues at positions 248, 250, 251, 252, 253, 254, 255, 256, 257, 272, 285, 288, 290, 291, 308, 309, 310, 311, 314, 385, 386, 387, 428, 432, 433, 434, 435, and 436.

18. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 17, wherein the first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues at positions 251, 253, 310, 314, 432, 433, 435 and 436.

19. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 18, wherein the first Fc region polypeptide differs from the second Fc region polypeptide due to one or two mutations selected from group i) I253A, H310A, and H435A, or group ii) H310A, H433A, and Y436A, or group iii) L251D, L314D, and L432D, or group iv) L251S, L314S, and L432S (numbered according to the Kabat EU index numbering system), and the second Fc region polypeptide is selected from mutations L251D, L251S, I253A, H... One or two mutations of 310A, L314D, L314S, L432D, L432S, H433A, H435A, and Y436A (numbered according to the Kabat EU index system) are different from the peptide in the first Fc region, so that when considered together, all mutations in the peptides in the first and second Fc regions result in mutations i) I253A, H310A, and H435A, or ii) H310A, H433A, and Y436A, or iii) L251D, L314D, and L432D, or iv) L251S, L314S, and L432S being included in the Fc region.

20. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 18, wherein the first Fc region polypeptide is different from the second Fc region polypeptide by one or two mutations selected from i) group I253A, H310A and H435A, or ii) group H310A, H433A and Y436A (numbered according to the Kabat EU index numbering system), and the second Fc region polypeptide is different from the first Fc region polypeptide by one or two mutations selected from mutations I253A, H310A, H433A, H435A and Y436A (numbered according to the Kabat EU index numbering system), such that when taken together, all mutations in the first and second Fc region polypeptides result in mutations i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A being included in the Fc region.

21. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 18, which contains the mutant I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or combinations thereof (numbered according to the Kabat EU index numbering system), and therefore i) all mutations are in the peptide in the first or second Fc region. ii) One or two mutations in the first Fc region polypeptide and one or two mutations in the second Fc region polypeptide, such that when considered together, all mutations in the first and second Fc region polypeptides result in mutations i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S contained in the Fc region.

22. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 18, which contains the mutations I253A/H310A/H435A or H310A/H433A/Y436A or combinations thereof (numbered according to the Kabat EU index numbering system), such that i) all mutations are in the first or second Fc region polypeptide, or ii) one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, such that when taken together, all mutations in the first and second Fc region polypeptides result in the mutations i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A being contained in the Fc region.

23. An IgG-type Fc region according to any one of embodiments 1 to 3 and 5 to 22, wherein the IgG-type Fc region has reduced binding to Staphylococcus protein A compared to an IgG-type Fc region comprising a) a first parental IgG-type Fc region polypeptide and a) a second parental IgG-type Fc region polypeptide.

24. An IgG class Fc region according to any one of embodiments 1 to 3 and 5 to 17, which contains the mutation M252Y/S254T/T256E (numbered according to the Kabat EU index numbering system), such that i) all mutations are in the first or second Fc region polypeptide, or ii) one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, such that when taken together, all mutations in the first and second Fc region polypeptides result in the mutation M252Y/S254T/T256E being contained in the IgG class Fc region.

25. An antibody comprising an IgG class Fc region according to any one of embodiments 1 to 24.

26. The antibody according to implementation scheme 25, wherein the antibody is a monoclonal antibody.

27. An antibody according to any one of the implementation schemes 25 to 26, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody.

28. An antibody according to any one of the implementation schemes 25 to 27, wherein the antibody is a bispecific antibody.

29. An antibody according to any one of the implementation schemes 25 to 28, wherein the antibody is a bivalent antibody.

30. An antibody according to any one of embodiments 25 to 29, wherein the antibody is a bispecific bivalent antibody eliminated by FcRn binding, comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2.

31. A bispecific bivalent antibody with FcRn binding elimination, comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2.

in

α) The first antigen-binding site that specifically binds to VEGF includes the CDR3H region of SEQ ID NO:14, the CDR2H region of SEQ ID NO:15, and the CDR1H region of SEQ ID NO:16 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:17, the CDR2L region of SEQ ID NO:18, and the CDR1L region of SEQ ID NO:19 in the light chain variable domain.

The second antigen-binding site that specifically binds to ANG-2 includes the CDR3H region of SEQ ID NO:22, the CDR2H region of SEQ ID NO:23, and the CDR1H region of SEQ ID NO:24 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:25, the CDR2L region of SEQ ID NO:26, and the CDR1L region of SEQ ID NO:27 in the light chain variable domain.

The γ) bispecific antibody contains an IgG class Fc region according to any one of embodiments 1 to 24.

32. The bispecific antibody according to implementation plan 31, wherein...

α) The first antigen-binding site that specifically binds to VEGF contains the amino acid sequence of SEQ ID NO:20 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:21 as the light chain variable domain VL.

The second antigen-binding site that specifically binds to ANG-2 contains the amino acid sequence of SEQ ID NO:28 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:29 as the light chain variable domain VL.

The γ) bispecific antibody contains an IgG class Fc region according to any one of embodiments 1 to 24.

33. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy chain and light chain of a first full-length antibody that specifically binds to VEGF, and a modified heavy chain and modified light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO:38 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:40 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:39 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:41 is used as the modified light chain of the second full-length antibody.

34. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy and light chain of a first full-length antibody that specifically binds to VEGF and a modified heavy and light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO:34 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:36 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:35 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:37 is used as the modified light chain of the second full-length antibody.

35. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy and light chain of a first full-length antibody that specifically binds to VEGF and a modified heavy and light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO:42 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:44 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:43 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:45 is used as the modified light chain of the second full-length antibody.

36. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy chain and light chain of a first full-length antibody that specifically binds to VEGF, and a modified heavy chain and modified light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO: 90 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO: 40 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:91 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:41 is used as the modified light chain of the second full-length antibody.

37. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy chain and light chain of a first full-length antibody that specifically binds to VEGF, and a modified heavy chain and modified light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO:88 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:36 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:89 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:37 is used as the modified light chain of the second full-length antibody.

38. A bispecific bivalent antibody with FcRn binding elimination, comprising a heavy and light chain of a first full-length antibody that specifically binds to VEGF and a modified heavy and light chain of a second full-length antibody that specifically binds to ANG-2, wherein constant domains CL and CH1 are interchanged, the bispecific bivalent antibody comprising

α) The amino acid sequence of SEQ ID NO:92 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:44 serves as the light chain of the first full-length antibody.

β) The amino acid sequence of SEQ ID NO:93 is used as the modified heavy chain of the second full-length antibody and the amino acid sequence of SEQ ID NO:45 is used as the modified light chain of the second full-length antibody.

39. A bispecific antibody with FcRn binding elimination, comprising the heavy and light chains of a first full-length antibody that specifically binds to VEGF and the heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker, the bispecific antibody comprising

α) The amino acid sequence of SEQ ID NO:46 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:48 serves as the light chain of the first full-length antibody.

The amino acid sequence of SEQ ID NO:47 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

40. A bispecific antibody with FcRn binding elimination, comprising the heavy and light chains of a first full-length antibody that specifically binds to VEGF and the heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker, the bispecific antibody comprising

α) The amino acid sequence of SEQ ID NO:49 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:51 serves as the light chain of the first full-length antibody.

The amino acid sequence of SEQ ID NO:50 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

41. A bispecific antibody with FcRn binding elimination, comprising the heavy and light chains of a first full-length antibody that specifically binds to VEGF and the heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker, the bispecific antibody comprising

α) The amino acid sequence of SEQ ID NO:94 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:48 serves as the light chain of the first full-length antibody.

The amino acid sequence of β)SEQ ID NO:95 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

42. A bispecific antibody with FcRn binding elimination, comprising the heavy and light chains of a first full-length antibody that specifically binds to VEGF and the heavy and light chains of a second full-length antibody that specifically binds to ANG-2, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker, the bispecific antibody comprising

α) The amino acid sequence of SEQ ID NO:96 serves as the heavy chain of the first full-length antibody, and the amino acid sequence of SEQ ID NO:51 serves as the light chain of the first full-length antibody.

The amino acid sequence of SEQ ID NO:97 serves as the heavy chain of the second full-length antibody linked to the light chain of the second full-length antibody via a peptide linker.

43. A bispecific antibody according to any one of embodiments 39 to 42, wherein the antibody heavy chain variable domain (VH) and antibody light chain variable domain (VL) of the heavy chain and light chain of the second full-length antibody are stabilized by introducing a disulfide bond between the heavy chain variable domain position 44 and the light chain variable domain position 100 (according to Kabat number).

44. A bispecific antibody according to any one of embodiments 33 to 43, wherein the bispecific antibody contains mutations S354C, T366W in one of the two CH3 domains and mutations Y349C, T366S, L368A, Y407V in the other domain of the two CH3 domains, or wherein the bispecific antibody contains mutations Y349C, T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, Y407V in the other domain of the two CH3 domains.

45. A bispecific antibody according to any one of embodiments 33 to 43, wherein the bispecific antibody contains mutants S354C and T366W in one of the two CH3 domains and mutants Y349C, T366S, L368A, and Y407V in the other of the two CH3 domains, and in addition to mutants Y349C, T366S, L368A, and Y407V, also contains mutants R409D and K370E in the CH3 domain, and in addition to mutants S354C and T366W, also contains... The bispecific antibody contains mutations D399K and E357K, or contains mutations Y349C and T366W in one of the two CH3 domains and mutations S354C, T366S, L368A, and Y407V in the other of the two CH3 domains, and contains mutations R409D and K370E in addition to mutations S354C, T366S, L368A, and Y407V in the CH3 domain, and contains mutations D399K and E357K in addition to mutations Y349C and T366W in the CH3 domain.

46. A bispecific antibody according to any one of embodiments 25 to 45, wherein the antibody has one or more of the following properties:

- Compared with the corresponding bispecific antibody without the mutation described in (iii), it showed lower serum concentrations (96 hours after intravitreal administration in mice that were FcRn-deficient but hemizygous transgenic for human FcRn), and/or,

- Compared with the corresponding bispecific antibody without the mutation described in (iii), similar (fold 0.8 to 1.2) concentrations were observed in the whole right eye lysate (96 hours after intravitreal administration in the right eye of mice that were FcRn-deficient but hemizygous transgenic for human FcRn), and/or

- Shows no binding to human neonatal Fc receptors, and/or

- Indicates no binding to Staphylococcus protein A, and/or

- Shows binding to Staphylococcus protein A.

47. An Fc region fusion polypeptide comprising an IgG-type Fc region according to any one of embodiments 1 to 24.

48. A pharmaceutical preparation comprising an antibody according to any one of embodiments 25 to 46 or an Fc region fusion polypeptide according to embodiment 47.

49. A pharmaceutical preparation according to implementation scheme 48, wherein the pharmaceutical preparation is used to treat ocular vascular diseases.

50. An antibody according to any one of embodiments 25 to 46 or an Fc region fusion polypeptide according to embodiment 47, which is used as a drug.

51. The use according to implementation scheme 50, wherein the use is for the treatment of ocular vascular diseases.

52. Use of an antibody according to any one of embodiments 25 to 46 or an Fc region fusion polypeptide according to embodiment 47 in the manufacture of a pharmaceutical.

53. The use according to embodiment 52, wherein the use is for manufacturing a drug for treating ocular vascular diseases.

54. An antibody according to any one of claims 25 to 46 or an Fc region fusion polypeptide according to claim 47, for the treatment of ocular vascular diseases.

55. A method of treating a patient with ocular vascular disease by administering to the patient in need of such treatment an antibody according to any one of embodiments 25 to 46 or an Fc region fusion peptide according to embodiment 47.

IV. Examples

The following are examples of the methods and formulations reported herein. It should be understood that, given the general description provided above, many other implementation schemes may be implemented.

While the invention has been described in detail to some extent by way of illustration and example for the purpose of clarity, these descriptions and examples should not be construed as limiting the scope reported herein. All disclosures of patents and scientific literature cited herein are expressly and fully incorporated by way of reference.

method

Electrospray ionization mass spectrometry (ESI-MS)

The protein aliquot (50 μg) was deglycosylated by adding 0.5 μL of N-glycanase plus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) to obtain a final sample volume of 115 μL. The mixture was incubated at 37 °C for 18 h. Subsequently, for reduction and denaturation, 60 μL of 0.5 M TCEP (Pierce) in 4 M guanidine*HCl (Pierce) and 50 μL of 8 M guanidine*HCl were added. The mixture was incubated at 37 °C for 30 min. The sample was desalted by size exclusion chromatography (Sepharose G-25, isostatic, 40% acetonitrile, containing 2% formic acid). ESI mass spectra (+ve) were recorded on a Q-TOF instrument (maXis, Bruker) equipped with a nano ESI source (TriVersa NanoMate, Advion). MS parameters were set as follows: Transfer: funnel RF, 400 Vpp; ISCID energy, 0 eV; multi-electrode RF, 400 Vpp; quadrupole: ion energy, 4.0 eV; low mass, 600 m/z; source: dry gas, 8 L/min; dry gas temperature, 160 °C; collision chamber: collision energy, 10 eV; collision RF: 2000 Vpp; ion cooler: ion cooler RF, 300 Vpp; transfer time: 120 μs; pre-pulse storage, 10 μs; scan range m/z 600 to 2000. For data evaluation, proprietary software (MassAnalyzer) was used.

FcRn Surface Plasmon Resonance (SPR) Analysis

The binding properties of wild-type antibodies and mutants to FcRn were analyzed using surface plasmon resonance (SPR) technology on a BIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This system was well-established for studying molecular interactions. It allows for continuous, real-time monitoring of ligand/analyte binding and thus the determination of kinetic parameters in a variety of analytical settings. SPR technology is based on measuring the refractive index near the surface of a gold-plated biosensor chip. Changes in refractive index represent the mass change resulting from the interaction of the immobilized ligand on that surface with the analyte injected in solution. If a molecule binds to the immobilized ligand on the surface, the mass increases; in the case of dissociation, the mass decreases. In the current assay, the FcRn receptor was immobilized on a BIAcore CM5 biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) to a level of 400 response units (RU) using an amine coupling method. The assay was performed at room temperature with PBS, 0.05% Tween-20 ^™ pH 6.0 (GE Healthcare Bioscience) as the run and dilution buffers. A 200 nM antibody sample was injected at room temperature at a flow rate of 50 μL/min. The binding time was 180 seconds, and the dissociation phase took 360 seconds. Regeneration of the chip surface was achieved by brief injection of HBS-P at pH 8.0. SPR data were evaluated by comparing the biological response signal height at 180 seconds and 300 seconds post-injection. The corresponding parameters were the highest RU level (180 seconds post-injection) and late stability (300 seconds post-injection).

Protein A surface plasmon resonance (SPR) analysis

This assay is based on surface plasmon resonance spectroscopy (SPR). Protein A is immobilized on the surface of the SPR biosensor. Once the sample is injected into the flow cell of the SPR spectrometer, it forms a complex with the immobilized protein A, resulting in an increase in mass on the sensor chip surface and thus a higher response (e.g., 1 RU is defined as 1 pg/ ^mm² ). Subsequently, the sensor chip is regenerated by dissolving the sample-protein A complex. The obtained response is then evaluated in response units (RU) to assess signal intensity and dissociation behavior.

Approximately 3,500 response units (RU) of protein A (20 μg/ml) were coupled to a CM5 chip (GE Healthcare) at pH 4.0 using the GE Healthcare Amine Conjugation Kit.

The sample and system buffers were HBS-P+ (0.01M HEPES, 0.15M NaCl, sterile filtered 0.005% surfactant P20, pH 7.4). The flow chamber temperature was set to 25°C and the sample compartment temperature to 12°C. The system was initiated with the run buffer. Subsequently, a 5 nM sample construct solution was injected at a flow rate of 30 μL/min for 120 seconds, followed by a 300-second dissociation phase. The sensor chip surface was then regenerated by two long injections of glycine-HCl pH 1.5 at a flow rate of 30 μL/min for 30 seconds each. Each sample was measured in triplicate.

Bispecific antibodies and their respective sequences

As used herein, the term "having the mutation IHH-AAA" refers to a combination of mutations in I253A (Ile253Ala), H310A (His310Ala), and H435A (His435Ala) in the constant heavy chain region (according to the Kabat EU index number) of the IgG1 or IgG4 subclass. Similarly, the term "having the mutation HHY-AAA" refers to mutations in H310A (His310Ala), H433A (His433Ala), and Y436A (According to the Kabat EU index number) in the constant heavy chain region (according to the Kabat EU index number) of the IgG1 or IgG4 subclass. The combination of Tyr436Ala, as used herein, “having the mutation P329GLALA” refers to the combination of mutations L234A (Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly) in the constant heavy chain region (according to Kabat’s EU index number), and the combination of mutations SPLE, as used herein, refers to the combination of mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region (according to Kabat’s EU index number).

Overview

General information on the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acid residues of the antibody chain are numbered according to EU designations and mentioned (Edelman, G.M. et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

Recombinant DNA technology

The standard method described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), is used to manipulate DNA. Molecular biology reagents are used according to the manufacturer's instructions.

Gene synthesis

Order the desired gene segment from Geneart (Regensburg, Germany) according to the provided instructions.

DNA sequencing

DNA sequences were determined by double-strand sequencing performed in MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NTI Advance suite version 8.0 are used for sequence generation, plotting, analysis, annotation, and illustration.

expression carrier

To express the described antibody, transient expression was performed using cDNA organization based on or without the CMV-intron A promoter, or genome organization based on the CMV promoter (e.g., in HEK293-F cells).

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

- A single restriction site at the 5' end,

-Immediate early enhancer and promoter from human cytomegalovirus,

-Intron A sequence in the case of cDNA organization

- The 5' untranslated region of the human immunoglobulin gene

- Nucleic acid encoding the immunoglobulin heavy chain signal sequence,

- A nucleic acid encoding a human antibody chain (wild-type or with domain exchange), said nucleic acid being either cDNA or, in genome organization, immunoglobulin exon-intron organized.

-The 3' untranslated region containing a polyadenylation signal sequence, and

- A single restriction site at the 3' end.

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

- The origin of replication that allows the plasmid to replicate in E. coli.

- The β-lactamase gene that confers ampicillin resistance in Escherichia coli, and

- The dihydrofolate reductase gene from the mouse (Mus musculus) serves as a selection marker in eukaryotic cells.

The nucleic acids encoding the antibody chain are generated via PCR and/or gene synthesis and assembled using known recombination methods and techniques by ligating the corresponding nucleic acid segments, for example, using a single restriction enzyme site in each vector. The nucleic acid sequence of the subclones is verified by DNA sequencing. For transient transfection, plasmids are prepared from transformed *E. coli* cultures (Nucleobond AX, Macherey-Nagel) to obtain a relatively large quantity of plasmids.

Cell culture technology

Use standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J., and Yamada, K.M. (eds.), John Wiley & Sons, Inc.

As described below, bispecific antibodies were expressed by transiently co-transfecting their respective expression plasmids into HEK293-F cells grown in suspension.

Example 1

Expression and purification

Transient transfection in the HEK293-F system

Using the HEK293-F system (Invitrogen), monospecific and bispecific antibodies are generated by transient transfection with their respective plasmids (e.g., plasmids encoding the heavy chain and modified heavy chain, and corresponding light chain and modified light chain) according to the manufacturer's instructions. In short, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle ^™ 293 expression medium (Invitrogen) in shake flasks or stirred fermenters are transfected with their respective expression plasmids and a mixture of 293fectin ^™ or fectin (Invitrogen). For 2L shake flasks (Corning), HEK293-F cells are seeded at a density of 1 × ^10⁶ cells/mL in 600 mL and incubated at 120 rpm in 8% _CO₂ . Following this day, cells at a density of approximately 1.5 × ^10⁶ cells/mL were transfected with approximately 42 mL of the following mixture: A) 20 mL of Opti-MEM (Invitrogen) mixed with 600 μg of total plasmid DNA (1 μg/mL) encoding the heavy chain or modified heavy chain and the corresponding light chain in an equimolar ratio; and B) 20 mL of Opti-MEM mixed with 1.2 mL of 293 fectin or fectin (2 μL/mL). During fermentation, glucose solution was added according to glucose consumption. After 5–10 days, the supernatant containing secretory antibodies was harvested, and the antibodies were either purified directly from the supernatant or the supernatant was frozen and stored.

purification

Bispecific antibodies were purified from cell culture supernatant using affinity chromatography with MabSelectSure-Sepharose ^™ (for the non-IHH-AAA mutant) (GE Healthcare, Sweden) or KappaSelect-Agarose (for the IHH-AAA mutant) (GE Healthcare, Sweden), hydrophobic interaction chromatography with butyl-Sepharose (GE Healthcare, Sweden), and size exclusion chromatography with Superde x 200 (GE Healthcare, Sweden).

In summary, sterile filtered cell _culture supernatant was captured on MabSelect SuRe resin (free of _-IHH -AAA mutant and wild-type antibody) equilibrated with PBS buffer (10 mM _Na₂HPO₄ , 1 mM _KH₂PO₄ , 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 IHH-AAA mutant was captured on KappaSelect resin equilibrated with 25 mM Tris, 50 mM NaCl, pH 7.2, washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 2.9. The eluted antibody fractions were combined and neutralized with 2 M Tris at pH 9.0. The antibody pool was prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution to a final concentration of 0.8 M ammonium sulfate and adjusting the pH to 5.0 with acetic acid. After equilibrating butyl-Sepharose resin with 35 mM sodium acetate, 0.8 M ammonium sulfate, and pH 5.0, the antibody was applied to the resin, washed with equilibration buffer, and eluted with a linear gradient to 35 mM sodium acetate at pH 5.0. Fractions containing (monospecific or bispecific) antibodies were pooled and further purified by size exclusion chromatography using a Superde x 20026/60GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, and pH 6.0. Fractions containing (monospecific or bispecific) antibodies were pooled, concentrated to the required concentration using a Vivaspin ultrafiltration system (Sartorius Stedim Biotech S.A., France), and stored at -80°C.

Table: Yield of bispecific <VEGF-ANG-2> antibody

After each purification step, purity and antibody integrity were analyzed by CE-SDS using Labchip microfluidic technology (Caliper Life Science, USA). Following the manufacturer's instructions, 5 μL of protein solution for CE-SDS analysis was prepared using the HT Protein Expression Reagent Kit and analyzed on a LabChip GXII system using the HT Protein Expression Chip. Data were analyzed using LabChip GX software.

Table: Removal of typical byproducts by different sequential purification steps as determined by CE-SDS

Aggregate content of antibody samples was analyzed by high-performance SEC using a Superde x 200 analytical size exclusion column (GE Healthcare, Sweden) at 25°C in 2x PBS ( ₂₀ mM _Na₂HPO₄ , ₂ mM _KH₂PO₄ , 274 mM NaCl and 5.4 mM KCl, pH 7.4) run buffer. 25 μg of protein was injected onto the column at a flow rate of 0.75 mL/min and eluted at isostatic concentrations for 50 min.

Similarly, the bispecific antibodies VEGFang2-0012 and VEGFang2-0201 were prepared and purified in the following yields:

In addition, the following bispecific antibodies against VEGF-ANG-2 with IHH-AAA and SPLE mutations can be prepared and purified similarly: VEGF-ANG-2 CrossMAb IgG4 (SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45), VEGF-ANG-2 OAscFab IgG1 with IHH-AAA mutation (SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48), VEGF-ANG-2 OAscFab IgG4 with IHH-AAA and SPLE mutations (SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51), and VEGF-ANG-2 with HHY-AAA and P329GLALA mutations. 2> CrossMab IgG1 (SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:40, SEQ ID NO:41), <VEGF-ANG-2>CrossMab IgG4 with HHY-AAA and SPLE mutations (SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:44, SEQ ID NO:45), <VEGF-ANG-2>OAscFab IgG1 with HHY-AAA mutation (SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:48), and <VEGF-ANG-2>OAscFab IgG4 with HHY-AAA and SPLE mutations (SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:51).

Example 2

Analysis and Extensibility

Viscosity measurement based on small-scale DLS:

Viscosity measurements were performed essentially as described in (He, F. et al., Analytical Biochemistry 399 (2009) 141-143). Briefly, the sample was concentrated to various protein concentrations in 200 mM arginine succinate, pH 5.5, followed by the addition of polystyrene latex beads (300 nm diameter) and polysorbate 20 (0.02% v/v). The sample was transferred to an optical 384-well plate via centrifugation through a 0.4 μm filter and covered with paraffin oil. The apparent diameter of the latex beads was determined by dynamic light scattering at 25 °C. The viscosity of the solution was calculated as η = η0(rh/rh,0) (η: viscosity; η0: viscosity of water; rh: apparent hydrodynamic radius of the latex beads; rh,0: hydrodynamic radius of the latex beads in water).

To allow for comparison of various samples at the same concentration, viscosity-concentration data were fitted with the Mooney equation (Equation 1) (Mooney, M., Colloid. Sci., 6 (1951) 162-170; Monkos, K., Biochem. Biophys. Acta 304 (1997) 1339) and interpolated accordingly.

Equation 1

(S: Hydrodynamic interaction parameter of protein; K: Self-aggregation factor; Φ: Volume fraction of dissolved protein)

Figure 2 shows the results: Compared with VEGFang2-0015, which has no IHH-AAA mutation in the Fc region, VEGFang2-0016, which has an IHH-AAA mutation in the Fc region, showed lower viscosity at all measurement temperatures.

DLS aggregation initiation temperature

Samples were prepared at a concentration of 1 mg/mL in 20 mM histidine/histidine hydrochloride, 140 mM NaCl, pH 6.0, centrifuged, filtered through a 0.4 μm filter, and transferred into an optical 384-well plate covered with paraffin oil. The hydrodynamic radius was repeatedly measured by dynamic light scattering while the sample was heated from 25 °C to 80 °C at a rate of 0.05 °C/min. The aggregation onset temperature was defined as the temperature at which the hydrodynamic radius began to increase. The results are shown in Figure 3. Figure 3 shows the aggregation of VEGFang2-0015 without the IHH-AAA mutation, relative to VEGFang2-0016 with the IHH-AAA mutation in the Fc region. VEGFang2-0016 showed an aggregation onset temperature of 61 °C, while VEGFang2-0015 without the IHH-AAA mutation showed an onset temperature of 60 °C.

DLS Timeline

Samples were prepared at a concentration of 1 mg/mL in 20 mM histidine/histidine hydrochloride, 140 mM NaCl, pH 6.0, centrifuged, filtered through a 0.4 μm filter, and transferred to an optical 384-well plate covered with paraffin oil. The samples were maintained at a constant temperature of 50 °C for 145 hours, while the hydrodynamic radius was repeatedly measured using dynamic light scattering (DLS). In this experiment, the tendency of naturally unfolded proteins to aggregate at elevated temperatures leads to an increase in average particle size over time. This DLS-based method is highly sensitive to aggregates, as these aggregates promote the intensity of scattered light in a superproportional manner. Even after 145 hours at 50 °C (close to the aggregation initiation temperature, see above), only VEGFang2-0015 and VEGFang2-0016 showed an increase in average particle size of less than 0.5 nm.

Store at 40℃ for 7 days at 100 mg/mL.

The sample was concentrated to a final concentration of 100 mg/mL in 200 mM arginine succinate at pH 5.5, aseptically filtered, and stored statically at 40°C for 7 days. Before and after storage, the contents of high molecular weight and low molecular weight species (HMW and LMW, respectively) were determined by size exclusion chromatography. Differences in HMW and LMW contents between stored samples and samples measured immediately after preparation were reported as “HMW increase” and “LMW increase,” respectively. The results are shown in the table below and Figure 4. These results show that VEGFang2-0015 (without the IHH-AAA mutation) exhibited a larger peak drop and a higher HMW increase compared to VEGFang2-0016 (with the IHH-AAA mutation). Surprisingly, VEGFang2-0016 (with the IHH-AAA mutation) showed a lower aggregation tendency compared to VEGFang2-0015 (without the IHH-AAA mutation).

Table: Δ main peak, HMW peak, and LMW peak after 7 days at 40℃

Functional analysis of the <VEGF-ANG-2> bispecific antibody was performed using a T200 instrument (GE Healthcare) at 25°C via surface plasmon resonance (SPR). The system was well-established for studying molecular interactions. SPR technology is based on measuring the refractive index near the surface of a gold-plated biosensor chip. Changes in refractive index represent the mass change caused by the interaction of the immobilized ligand on that surface with the analyte injected in solution. If a molecule binds to the immobilized ligand on the surface, the mass increases, and vice versa; if the analyte dissociates from the immobilized ligand (reflecting complex dissociation), the mass decreases. SPR allows for continuous, real-time monitoring of ligand/analyte binding and thus determination of the binding rate constant (ka), dissociation rate constant (kd), and equilibrium constant (KD).

Example 3

Binding to VEGF, ANG-2, FcγR and FcRn

VEGF isoform kinetic affinity, including assessment of species cross-reactivity.

A capture system of approximately 12,000 resonance units (RU) was conjugated on a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine conjugation kit supplied by GE Healthcare (10 μg/mL goat anti-human F(Ab)'₂; order code: 28958325; GE Healthcare Bio-Sciences AB, Sweden). Sample and system buffers were PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween 20 ^™ ) pH 7.4. The flow chamber was set to 25°C – and the sample block was set to 12°C – and initiated twice with run buffer. Bispecific antibodies were captured by injecting 50 nM solution at a flow rate of 5 μL/min for 30 seconds. Binding was measured by injecting multiple concentrations (starting from 300 nM diluted 1:3) of human hVEGF121, mouse mVEGF120, or rat rVEGF164 into solution at a flow rate of 30 μL/min for 300 seconds. Interphase monitoring was conducted for up to 1200 seconds and initiated by switching from the sample solution to the run buffer. Surface regeneration was achieved by washing with glycine solution at pH 2.1 at a flow rate of 30 μL/min for 60 seconds. Bulk refractive index differences were corrected by subtracting the response values obtained from goat anti-human F(Ab') ₂ surfaces. Blank injections (= dual references) were also subtracted. A Langmuir 1:1 model was used to calculate apparent K_{_D} and other kinetic parameters. Results are shown below.

ANG-2 solution affinity, including assessment of species cross-reactivity.

Solution affinity assays determine the affinity of interactions by determining the concentration of free interacting couples in the equilibrium mixture. The solution affinity assay involves mixing a constant concentration of the <VEGF-ANG-2> bispecific antibody with varying concentrations of the ligand (=ANG-2). The maximum possible resonance units (e.g., 17,000 resonance units (RU)) of the antibody immobilized on the surface of a CM5 chip (GE Healthcare BR-1005-30) are determined at pH 5.0 using an amine conjugation kit supplied by GE Healthcare. Sample and system buffers are HBS-P pH 7.4. The flow chamber is set to 25°C and the sample block is set to 12°C and induced twice with run buffer. To generate a calibration curve, incremental concentrations of ANG-2 are injected into BIAcore flow chambers containing the immobilized <VEGF-ANG-2> bispecific antibody. The amount of ANG-2 bound is determined as resonance units (RU) and plotted against concentration. Solutions of each ligand (11 concentrations, from 0 to 200 nM for the <VEGF-ANG-2> bispecific antibody) were incubated with 10 nM ANG-2 and allowed to equilibrate at room temperature. The free ANG-2 concentration was determined from calibration curves generated before and after measuring the response values of solutions with known amounts of ANG-2. A 4-parameter fit was established using Model 201, with the free ANG-2 concentration as the y-axis and the antibody concentration used for inhibition as the x-axis, using XLfit4 (IDBS Software). Affinity was calculated by determining the inflection point of this curve. The surface was regenerated by washing once for 30 seconds with 0.85% _{H₃PO₄ solution} at a flow rate of 30 μL/min. The bulk refractive index difference was corrected by subtracting the response values obtained from the blank-coupled surface. The results are shown below.

FcRn steady-state affinity

For FcRn measurements, steady-state affinity was used to compare bispecific antibodies. Human FcRn was diluted in conjugation buffer (10 μg/mL, sodium acetate, pH 5.0) and immobilized on a C1 chip (GE Healthcare BR-1005-35) using the BIAcore wizard with a directional immobilization method to a final response value of 200 RU. The flow chamber was set to 25°C and the sample block was set to 12°C and induced twice with run buffer. The sample and system buffers were PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween 20 ^™ ) pH 6.0. To evaluate different IgG concentrations for each antibody, concentrations of 62.5 nM, 125 nM, 250 nM, and 500 nM were prepared. Different samples were serially injected onto the chip surface at a flow rate set to 30 μL/min and with a selected binding time of 180 seconds. Surface regeneration was achieved by injecting PBS-T at a flow rate of 30 μL/min for 860 seconds. Bulk refractive index differences were corrected by subtracting the response values obtained from the blank surface. Buffer injection (= dual reference) was also subtracted. Steady-state affinity was calculated using a method from the BIA-evaluation software. In short, RU values were plotted against the analyzed concentrations to generate dose-response curves. Based on a 2-parameter fit, upper asymptotes were calculated, allowing the determination of the half-maximum RU value and thus affinity. The results are shown in Figure 5 and the table below. Similarly, affinities for cynomolgus monkey, mouse, and rabbit FcRn were determined.

FcγRIIIa measurement

For FcγRIIIa measurement, a direct binding assay was used. A capture system (1 μg/ml Penta-His; Qiagen) of approximately 3,000 resonance units (RU) was coupled to a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine conjugation kit supplied by GE Healthcare. Sample and system buffers were HBS-P+ pH 7.4. The flow chamber was set to 25°C – and the sample block was set to 12°C – and initiated twice with run buffer. FcγRIIIa-His receptor was captured by injecting 100 nM solution at a flow rate of 5 μL/min for 60 seconds. Binding was measured by injecting 100 nM bispecific antibody or monospecific control antibody (anti-digitoxin antibody (anti-Dig) for 180 seconds at a flow rate of 30 μL/min). The surface was regenerated by washing with a glycine solution at pH 2.5 at a flow rate of 30 μL/min for 120 seconds. Because FcγRIIIa binding differs from the Langmuir 1:1 model, this assay was used alone to determine binding/non-binding. FcγRIa and FcγRIIa binding were determined in a similar manner. The results are shown in Figure 6, where subsequent introduction of the mutant P329G LALA failed to detect further binding to FcγRIII.

Evaluation of the independent VEGF binding and ANG-2 binding of the <VEGF-ANG-2> bispecific antibody.

A capture system of approximately 3,500 resonance units (RU) (10 μg/mL goat anti-human IgG; GE Healthcare Bio-Sciences AB, Sweden) was coupled to a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. Sample and system buffers were PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween20 ^™ ) at pH 7.4. The flow chamber temperature was set to 25°C and the sample block temperature was set to 12°C. The flow chamber was induced twice with run buffer prior to capture.

Bispecific antibodies were captured by injecting 10 nM solution at a flow rate of 5 μL/min for 60 seconds. The independent binding activity of each ligand to the bispecific antibody was analyzed by determining the effective binding capacity of each ligand added sequentially or simultaneously (at a flow rate of 30 μL/min).

1. Inject human VEGF at a concentration of 200 nM for 180 seconds (to identify single binding of the antigen).

2. Inject human ANG-2 at a concentration of 100 nM for 180 seconds (to identify the single binding effect of the antigen).

3. Human VEGF was injected at a concentration of 200 nM for 180 seconds, followed by an additional injection of human ANG-2 at a concentration of 100 nM for 180 seconds (to identify the binding effect of ANG-2 in the presence of VEGF).

4. Human ANG-2 was injected at a concentration of 100 nM for 180 seconds, followed by an additional injection of human VEGF at a concentration of 200 nM (to identify VEGF binding in the presence of ANG-2).

5. Human VEGF at a concentration of 200 nM and human ANG-2 at a concentration of 100 nM were co-injected for 180 seconds (to simultaneously identify the binding effects of VEGF and ANG-2).

The surface was regenerated by washing with 3M MgCl₂ _solution at a flow rate of 30 μL/min for 60 seconds. The difference in bulk refractive index was corrected by subtracting the response value obtained from the goat anti-human IgG surface.

If the final signals obtained from schemes 3, 4, and 5 are equal to or similar to the sum of the independent final signals from schemes 1 and 2, then the bispecific antibodies can bind to both antigens independently. The results are shown in the table below, where both antibodies, VEGFang2-0016 and VEGFang2-0012, show that they can bind to VEGF and ANG-2 independently.

Evaluation of simultaneous VEGF binding and ANG-2 binding with the <VEGF-ANG-2> bispecific antibody.

First, approximately 1,600 resonance units (RU) of VEGF (20 μg/mL) were conjugated on a CM4 chip (GE Healthcare BR-1005-34) at pH 5.0 using an amine conjugation kit supplied by GE Healthcare. Sample and system buffers were PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween 20 ^™ ) at pH 7.4. The flow chamber was set to 25°C and the sample block was set to 12°C and initiated twice with run buffer. Second, a 50 nM bispecific antibody solution was injected at a flow rate of 30 μL/min for 180 seconds. Third, hANG-2 was injected at a flow rate of 30 μL/min for 180 seconds. The binding response of hANG-2 depended on the amount of bispecific antibody binding to VEGF and showed simultaneous binding. Surface regeneration was achieved by washing with 0.85% _{H₃PO₄ solution} at a flow rate of 60 μL/min for 60 seconds. Simultaneous binding was indicated by additional specific binding signals from hANG-2 to the previously VEGF-bound <VEGF-ANG-2> bispecific antibody. For the two bispecific antibodies VEGFang2-0015 and VEGFang2-0016, simultaneous binding to both VEGF and ANG-2 with the <VEGF-ANG-2> bispecific antibody was detected (data not shown).

Table: Results: Kinetic affinity for VEGF isoforms from different species

Table: Results: Solution Affinity to ANG-2

Table: Results: Affinity of FcRn to <VEGF-ANG-2> bispecific antibody

Table: Binding results with FcγRI–IIIa

Table: Results: Independent binding of VEGF and ANG-2 to the <VEGF-ANG-2> bispecific antibody

Example 4

Mass spectrometry

This section describes the characterization of the <VEGF-ANG-2> bispecific antibody, focusing on its proper assembly. The expected primary structure of the <VEGF-ANG-2> bispecific antibody was confirmed by electrospray ionization mass spectrometry (ESI-MS) using deglycosylation and either intact or IdeS-digested (using an IgG-degrading enzyme from *Streptococcus pyogenes*). IdeS-digestion was performed with 100 μg of purified antibody and 2 μg of IdeS protease (Fabricator) incubated at 37°C for 5 h in 100 mmol/L _NaH₂PO₄ / _{Na₂HPO₄ ,} pH 7.1 _. Subsequently, the antibody was deglycosylated at a protein concentration of 1 mg/mL in ₁₀₀ mmol/L _NaH₂PO₄ / _Na₂HPO₄ , pH _7.1 using N-glycosidase F, neuraminidase, and O-glycosidase (Roche) at 37 °C for up to 16 hours, and then desalted by HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

The mass obtained from IdeS-digested deglycosylated (see table below) or fully deglycosylated (see table below) molecules corresponds to the predicted mass, which is inferred from the amino acid sequence of the <VEGF-ANG-2> bispecific antibody composed of two different light chains LC _ANG-2 and LC _Lucentis and two different heavy chains HC _ANG-2 and HC _Lucentis .

Table: Quality of deglycosylated and IdeS-digested bispecific <VEGF/ANG2> antibodies VEGFang2-0201 (without IHH-AAA mutation) and VEGFang2-0012 (with IHH-AAA mutation)

Table: Quality of deglycosylated VEGF/ANG2 antibodies VEGFang2-0016 (with IHH-AAA mutation) and VEGFang2-0015 (without IHH-AAA mutation)

Example 5

FcRn chromatography

Coupled with streptavidin sepharose:

Add 1 gram of streptavidin sepharose (GE Healthcare) to the biotinylated and dialyzed receptor and incubate with shaking for two hours. Pack the receptor-derived sepharose into a 1 mL XK column (GE Healthcare).

Chromatography using FcRn affinity columns:

condition:

Column dimensions: 50mm x 5mm

Bed height: 5cm

Loading capacity: 50 μg sample

Equilibration buffer: 20 mM MES containing 150 mM NaCl, adjusted to pH 5.5.

Elution buffer: 20 mM Tris/HCl, containing 150 mM NaCl, adjusted to pH 8.8.

Elution: 7.5 CV equilibration buffer, 30 CV to 100% elution buffer, 10 CV elution buffer

Human FcRn affinity column chromatography

The table below shows the retention times of the <VEGF-ANG-2> bispecific antibody on an affinity column containing human FcRn. Data were obtained using the conditions described above.

Table: Results: Retention time of <VEGF-ANG-2> bispecific antibody

Example 6

Pharmacokinetic (PK) characteristics of antibodies with IHH-AAA mutation

PK data of human FcRn transgenic FcRn mice

During life stages:

This study included female C57BL/6J mice (background); and mice that were FcRn deficient but hemizygous for human FcRn (huFcRn, strain 276-/tg).

Part 1:

All mice were injected once into the right eye via intravitreal injection with 2 μL of appropriate solution/animal (i.e., 21 μg compound/animal (VEGFAng2-0015 (without IHH-AAA mutation)) or 23.6 μg compound/animal (VEGFAng2-0016 (with IHH-AAA mutation)).

Mice were divided into two groups of six. Blood samples were collected from group 1 at 2, 24, and 96 hours post-administration and from group 2 at 7, 48, and 168 hours post-administration.

The compound was injected into the vitreous humor of the right eye of a mouse using the NanoFil microinjector system for nanoliter injection from World Precision Instruments, Inc., Berlin, Germany. The mice were anesthetized with 2.5% isoflurane, and a Leica MZFL3 microscope with 40x magnification and a ring lamp featuring a Leica KL 2500LCD flash was used for observation of the mouse eye. Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

Blood was collected from each animal via the posterior venous plexus of the contralateral eye to determine the levels of compounds in the serum.

After 1 hour at room temperature, at least 50 μL of serum sample was obtained from the blood by centrifugation (9300 x g) for 3 minutes at 4 °C. The serum sample was immediately frozen after centrifugation and stored at -80 °C until analysis. The treated eyes of animals in group 1 were isolated 96 hours after treatment, and the treated eyes of animals in group 2 were isolated 168 hours after treatment. The samples were stored at -80 °C until analysis.

Part 2:

All mice were injected once via the tail vein intravenously with 200 μL of suitable solution per animal (i.e., 21 μg compound per animal (VEGFAng2-0015 without IHH-AAA mutation)) or 23.6 μg compound per animal (VEGFAng2-0016 with IHH-AAA mutation)).

Mice were divided into two groups of five animals each. Blood samples were collected from group 1 at 1, 24, and 96 hours post-administration and from group 2 at 7, 48, and 168 hours post-administration. Blood was collected from each animal via the retroocular venous plexus to determine the levels of compounds in the serum.

After 1 hour at room temperature, at least 50 μL of serum sample was obtained from the blood by centrifugation at 4 °C (9300 x g) for 3 minutes. The serum sample was frozen immediately after centrifugation and stored at -80 °C until analysis.

Preparation of whole-eye lysate (mouse)

Ocular lysates were obtained from whole eyes of laboratory animals via physicochemical disintegration. For mechanical lysis, each eye was transferred to a 1.5 mL conical-bottom microvial. After freezing and thawing, the eye was washed once with 1 mL of cell washing buffer (Bio-Rad, Bio-Plex Cell Lysis Kit, catalog number 171-304011). In the following steps, 500 μL of freshly prepared cell lysis buffer was added, and the eye was ground using a 1.5 mL tissue mortar and pestle (Kimble Chase, 1.5 mL pestle, product number 749521-1500). The mixture was then frozen and thawed five times and ground again. To separate the lysates from the remaining tissue, the sample was centrifuged at 4,500 g for 4 minutes. After centrifugation, the supernatant was collected and stored at -20°C until further analysis in a quantitative ELISA.

analyze

The concentration of <VEGF-ANG-2> antibody in mouse serum and ocular lysates was determined by enzyme-linked immunosorbent assay (ELISA).

To quantify VEGF-ANG-2 antibodies in mouse serum samples and ocular lysates, a standard solid-phase sandwich immunoassay was performed using biotinylated and digitoxin-modified monoclonal antibodies as capture and detection antibodies, respectively. To verify the integrity of the analyte's bispecificity, the biotinylated capture antibody recognized the VEGF-binding site, while the digitoxin-modified detection antibody bound to the ANG-2 binding site of the analyte. The immune complex of the capture antibody, analyte, and detection antibody bound on the solid phase of a streptavidin-coated microtiter plate (SA-MTP) was then detected using horseradish peroxidase conjugated with an anti-digitoxin antibody. After washing away unbound material from the SA-MTP and adding ABTS-substrate, the obtained signal was proportional to the amount of analyte bound on the SA-MTP solid phase. The measured signal of the sample was then converted to concentration using a calibrator referenced in parallel analyses for quantification.

In the first step, SA-MTP was coated on an MTP shaker at 500 rpm for 1 hour with 100 μL/well of biotinylated capture antibody solution (anti-idiotype antibody mAb<Id<VEGF>>M-2.45.51-IgG-Bi(DDS)) at a concentration of 1 μg/mL for 1 hour. Simultaneously, calibrators, QC samples, and samples were prepared. The calibrators and QC samples were diluted to 2% serum matrix; the samples were diluted until the signal was within the linear range of the calibrators.

After coating the SA-MTP with the capture antibody, the plates were washed three times with washing buffer and 300 μL/well. Then, 100 μL/well of calibrator, QC sample, and sample were pipetted onto the SA-MTP and incubated again at 500 rpm for 1 hour. The analytes now bind to the SA-MTP via their anti-VEGF binding sites through the capture antibody. After incubation and removal of unbound analytes by washing the plates, 100 μL/well of the first detection antibody (anti-idiotype antibody mAb<Id-<ANG-2>>M-2.6.81-IgG-Dig(XOSu)) at a concentration of 250 ng/mL was added to the SA-MTP. Again, the plates were incubated on a shaker at 500 rpm for 1 hour. After washing, 100 μL/well of the second detection antibody (pAb<Digoxigenin> S-Fab-POD(poly)) at a concentration of 50 mU/mL was added to the wells of the SA-MTP plate, and the plate was incubated again at 500 rpm for 1 hour. After a final washing step to remove excess detection antibody, 100 μL/well of substrate (ABTS) was added. The antibody-enzyme conjugate catalyzed the color reaction of the substrate. The signal was then measured at 405 nm (reference wavelength: 490 nm [405/490] nm) using an ELISA reader.

Pharmacokinetic evaluation

Pharmacokinetic parameters were calculated using non-compartmental analysis using WinNonlin™ (Pharsight) version 5.2.1, a pharmacokinetic evaluation program.

result:

A) Serum concentration

The results of serum concentrations are shown in the table below and in Figures 7B to 7C.

Table: Comparison of serum concentrations of VEGFAng2-0015 (without IHH-AAA mutation) after intravitreal and intravenous administration .

Table: Comparison of serum concentrations of VEGFAng2-0016 (with IHH-AAA mutation) after intravitreal and intravenous administration .

Table: Comparison of serum concentrations after intravitreal administration of VEGFang2-0015 (without IHH-AAA mutation) and VEGFang2-0016 (with IHH-AAA mutation).

Table: Comparison of serum concentrations after intravenous administration of VEGFang2-0015 (without IHH-AAA mutation) and VEGFang2-0016 (with IHH-AAA mutation).

result:

B) Concentrations of ocular lysates in the left and right eyes

The results for the concentration of ocular lysates are shown in the table below and Figures 7D to 7E.

Table: Concentration of VEGFang2-0015 (without IHH-AAA mutation) in ocular lysates after intravitreal administration to the right eye.

Table: Concentration of VEGFang2-0015 (without IHH-AAA mutation) in ocular lysates after intravenous administration

Table: Concentration of VEGFang2-0016 (with IHH-AAA mutation) in ocular lysates after intravitreal administration to the right eye.

Table: Concentration of VEGFang2-0016 (with IHH-AAA mutation) in ocular lysates after intravenous administration

Summary of results:

After intravitreal administration, the bispecific VEGF-ANG-2 antibody VEGFang2-0016 (with the IHH-AAA mutation), as reported in this paper, showed similar concentrations in ocular lysates (after 96 hours and 168 hours) compared to the bispecific VEGF-ANG-2 antibody VEGFang2-0015 without the IHH-AAA mutation.

In addition, when applied intravitreal, the bispecific VEGF-ANG-2 antibody VEGFang2-0016 (with the IHH-AAA mutation), as reported in this paper, showed additional faster clearance in serum and a shorter half-life compared to the bispecific VEGF-ANG-2 antibody VEGFang2-0015 without the IHH-AAA mutation.

Example 7

Mouse corneal microcapsule angiogenesis assay

To test the in vivo anti-angiogenic effect of bispecific <VEGF-ANG-2> antibodies with VEGF-binding VH and VL of SEQ ID NO:20 and 21, and ANG-2-binding VH and VL of SEQ ID NO:28 and 29, on VEGF-induced angiogenesis, a mouse corneal microcapsule angiogenesis assay was performed. In this assay, VEGF-impregnated Nylaflo discs were implanted at a fixed distance relative to the limbal vessels into avascular corneal capsules. Vessels immediately grew into the cornea pointing towards the forming VEGF gradient. Eight- to ten-week-old female BALB/c mice were purchased from Charles River, Sulzfeld, Germany. The study protocol was adapted from the method described in Rogers, M.S. et al., Nat. Protoc. 2 (2007) 2545-2550. In summary, using a scalpel and pointed forceps in anesthetized mice, microcapsules approximately 500 μm wide were prepared under a microscope, extending about 1 mm from the limbus to the apex of the cornea. 0.6 mm diameter discs (Pall Corporation, Michigan) were implanted, and the implantation area was smoothed. The discs were incubated in the appropriate growth factor or in a carrier for at least 30 minutes. After 3, 5, and 7 days (or alternatively only 3, 5, or 7 days), the eyes were photographed and vascular response was measured. This assay was quantified by calculating the percentage of new vascular area to total corneal area.

Discs were loaded with 300 ng VEGF or PBS as controls and implanted for 7 days. Vascular growth from the limbus to the disc was monitored over time on days 3, 5, and/or 7. One day prior to disc implantation, the antibody was administered intravenously at a dose of 10 mg/kg (attributable to intravenous administration, using the same serum-stable VEGFang2-0015 (without the IHH-AAA mutation) with the same anti-VEGF and anti-ANG-2VH and VL efficacy as the mediator, differing only from VEGFang2-0016 due to the IHH-AAA mutation) to test its anti-angiogenic effect against VEGF-induced angiogenesis in vivo. Animals in the control group received the vector. The administration volume was 10 mL/kg.

Example 8

Pharmacokinetic (PK) characteristics of antibodies with HHY-AAA mutation

PK data of human FcRn transgenic FcRn mice

During life stages:

This study included female C57BL/6J mice (background); and mice that were FcRn deficient but hemizygous for human FcRn (huFcRn, strain 276-/tg).

Part 1:

All mice were injected once into the right eye via intravitreal injection with an appropriate solution of VEGF/ANG2-0016, VEGF/ANG2-0096, VEGF/ANG2-0098, and VEGF/ANG2-0121, at a dose of 2 μL/animal.

Mice were divided into two groups of six. Blood samples were collected from group 1 at 2, 24, and 96 hours post-administration and from group 2 at 7, 48, and 168 hours post-administration.

The compound was injected into the vitreous humor of the right eye of a mouse using the NanoFil microinjector system for nanoliter injection from World Precision Instruments, Inc., Berlin, Germany. The mice were anesthetized with 2.5% isoflurane, and a Leica MZFL3 microscope with 40x magnification and a ring lamp featuring a Leica KL 2500LCD flash was used for observation of the mouse eye. Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

Blood was collected from each animal via the posterior venous plexus of the contralateral eye to determine the levels of compounds in the serum.

After 1 hour at room temperature, at least 50 μL of serum sample was obtained from the blood by centrifugation (9,300 x g) for 3 minutes at 4 °C. The serum sample was immediately frozen after centrifugation and stored at -80 °C until analysis. The treated eyes of animals in group 1 were isolated 96 hours after treatment, and the treated eyes of animals in group 2 were isolated 168 hours after treatment. The samples were stored at -80 °C until analysis.

Part 2:

All mice were injected once via the tail vein intravenously with 200 μL/animal of the appropriate VEGF/ANG2-0096, VEGF/ANG2-0098 or VEGF/ANG2-0121.

Mice were divided into two groups of five animals each. Blood samples were collected from group 1 at 1, 24, and 96 hours post-administration and from group 2 at 7, 48, and 168 hours post-administration. Blood was collected from each animal via the retroocular venous plexus to determine the levels of compounds in the serum.

After 1 hour at room temperature, at least 50 μL of serum sample was obtained from the blood by centrifugation at 4 °C (9,300 x g) for 3 minutes. The serum sample was immediately frozen after centrifugation and stored at -80 °C until analysis.

Preparation of whole-eye lysate (mouse)

Ocular lysates were obtained by physicochemical disintegration of the whole eye. For mechanical lysis, each eye was transferred to a 1.5 mL conical-bottom microvial. After freezing and thawing, the eye was washed once with 1 mL of cell washing buffer (Bio-Rad, Bio-Plex Cell Lysis Kit, catalog number 171-304011). In the following steps, 500 μL of freshly prepared cell lysis buffer was added and the eye was ground using a 1.5 mL tissue mortar and pestle (Kimble Chase, 1.5 mL pestle, product number 749521-1500). The mixture was then frozen and thawed five times and ground again. To separate the lysates from the remaining tissue, the sample was centrifuged at 4,500 g for 4 minutes. After centrifugation, the supernatant was collected and stored at -20°C until further analysis in a quantitative ELISA.

analyze

The concentration of antibodies in mouse serum and ocular lysates was determined using enzyme-linked immunosorbent assay (ELISA).

To quantify antibodies in mouse serum samples and ocular lysates, a standard solid-phase sandwich immunoassay was performed using biotinylated and digitoxin-modified monoclonal antibodies as capture and detection antibodies, respectively. Specifically, to verify the integrity of the analyte's bispecificity, the biotinylated capture antibody recognized the VEGF-binding site, while the digitoxin-modified detection antibody bound to the ANG-2 binding site of the analyte. The bound immune complexes of the capture antibody, analyte, and detection antibody on the solid phase of a streptavidin-coated microtiter plate (SA-MTP) were then detected using horseradish peroxidase conjugated with an anti-digitoxin antibody. After washing away unbound material from the SA-MTP and adding ABTS-substrate, the obtained signal was proportional to the amount of analyte bound on the SA-MTP solid phase. The measured signal of the sample was then converted to concentration using a calibrator referenced in parallel analyses for quantification.

In the first step, SA-MTP was coated on an MTP shaker at 500 rpm for 1 hour with 100 μL/well of biotinylated capture antibody solution (anti-idiotype antibody, e.g., mAb<Id<VEGF>>M-2.45.51-IgG-Bi(DDS)) at a concentration of 1 μg/mL for 1 hour. Simultaneously, calibrators, QC samples, and samples were prepared. The calibrators and QC samples were diluted to 2% serum matrix; the samples were diluted until the signal was within the linear range of the calibrators.

After coating the SA-MTP with capture antibody, the plates were washed three times with washing buffer and 300 μL/well. Subsequently, 100 μL/well of calibrator, QC sample, and sample were pipetted onto the SA-MTP and incubated again at 500 rpm for 1 hour. The analytes now bind to the SA-MTP via one of their anti-VEGF binding sites through the capture antibody. After incubation and removal of unbound analytes by washing the plates, 100 μL/well of the first detection antibody (anti-idiotype antibody, e.g., mAb<Id-<ANG-2>>M-2.6.81-IgG-Dig(XOSu)) at a concentration of 250 ng/mL was added to the SA-MTP. Again, the plates were incubated on a shaker at 500 rpm for 1 hour. After washing, 100 μL/well of a second detection antibody (e.g., pAb<Digoxigenin>S-Fab-POD(poly)) at a concentration of 50 mU/mL was added to the wells of the SA-MTP plate, and the plate was incubated again at 500 rpm for 1 hour. After a final washing step to remove excess detection antibody, 100 μL/well of a substrate antibody-enzyme conjugate was added to catalyze the color reaction of the substrate. The signal was measured at 405 nm (reference wavelength: 490 nm [405/490] nm) using an ELISA reader.

Pharmacokinetic evaluation

Pharmacokinetic parameters were calculated using non-compartmental analysis using WinNonlin™ (Pharsight) version 5.2.1, a pharmacokinetic evaluation program.

While the foregoing invention has been described in detail to some extent by way of illustration and example for the purpose of clarity, such description and example should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly and entirely incorporated herein by reference.

Claims (17)

1. An IgG-like Fc region comprising a first variant Fc region polypeptide and a second variant Fc region polypeptide, in a) The first variant Fc region polypeptide is derived from the first parental IgG-type Fc region polypeptide, and the second variant Fc region polypeptide is derived from the second parental IgG-type Fc region polypeptide, wherein the first parental IgG-type Fc region polypeptide and the second parental IgG-type Fc region polypeptide may be the same as or different from each other. b) The first variant Fc region polypeptide differs from the second variant Fc region polypeptide in one or more amino acid residues, excluding those amino acid residues in which the first parental IgG class Fc region polypeptide differs from the second parental IgG class Fc region polypeptide. c) IgG-type Fc regions containing first-variant Fc region polypeptides and second-variant Fc region polypeptides have different affinities for human Fc receptors than IgG-type Fc regions containing the first parental IgG-type Fc region polypeptide of a) and the second parental IgG-type Fc region polypeptide of a). The human Fc receptor is the human neonatal Fc receptor. The IgG class Fc region is asymmetrically modified in its interaction with human neonatal Fc receptors, and in i) The first parental IgG class Fc region polypeptide is a human IgG1 Fc region polypeptide and the second parental IgG class Fc region polypeptide is a human IgG1 Fc region polypeptide, or ii) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A and L235A, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A and L235A, or iii) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, and P329G, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, and P329G, or iv) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, S354C, and T366W, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, Y349C, T366S, L368A, and Y407V, or v) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, P329G, S354C, and T366W, and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with mutations only L234A, L235A, P329G, Y349C, T366S, L368A, and Y407V, or vi) The first parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with only the K392D mutation and the second parental IgG Fc region polypeptide is a human IgG1 Fc region polypeptide with only the D399K, D356K and/or E357K mutations. The first variant Fc region polypeptide is different from the second variant Fc region polypeptide because it is selected from one or two mutations of group H310A, H433A and Y436A, and the second variant Fc region polypeptide is different from the first variant Fc region polypeptide because it is selected from one or two mutations of H310A, H433A and Y436A, thus all mutations H310A, H433A and Y436A are included in the IgG class Fc region; The mutations mentioned therein are numbered according to the Kabat EU index numbering system. 2. The IgG class Fc region according to claim 1, wherein i) The Fc region polypeptide of the first parental IgG class consists of an amino acid sequence selected from SEQ ID NO: 60, 64, 68, 70, and 74. and ii) The second parent IgG Fc region polypeptide consists of an amino acid sequence selected from SEQ ID NO: 60, 64, 67, 70 and 73. 3. The IgG-type Fc region according to claim 2, wherein i) The first parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:60 and the second parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:60, or ii) The first parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:64 and the second parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:64, or iii) The first parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:70 and the second parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:70, or iv) The first parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:68 and the second parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:67, or v) The first parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:74 and the second parental IgG Fc region polypeptide consists of the amino acid sequence of SEQ ID NO:73. 4. The IgG-type Fc region according to any one of claims 1 to 3, wherein the IgG-type Fc region has a reduced binding effect on staphylococcal protein A compared to the IgG-type Fc region comprising the first parental IgG-type Fc region polypeptide of a) and the second parental IgG-type Fc region polypeptide of a). 5. The IgG class Fc region according to any one of claims 1 to 3, characterized in that the Fc region contains the mutation M252Y/S254T/T256E, such that i) all mutations are in the first or second Fc region polypeptide, or ii) one or two mutations are in the first Fc region polypeptide and one or two mutations are in the second Fc region polypeptide, thereby all mutations M252Y/S254T/T256E are contained in the IgG class Fc region; wherein the mutations are numbered according to the Kabat EU index numbering system. 6. An antibody comprising an IgG-type Fc region according to any one of claims 1 to 5. 7. The antibody of claim 6, wherein the antibody is a monoclonal antibody. 8. The antibody according to any one of claims 6 to 7, wherein the antibody is a human antibody, a humanized antibody, or a chimeric antibody. 9. The antibody according to any one of claims 6 to 7, wherein the antibody is a bispecific antibody. 10. The antibody according to any one of claims 6 to 7, wherein the antibody is a bivalent antibody. 11. The antibody according to any one of claims 6 to 7, wherein the antibody is a bispecific bivalent antibody that eliminates FcRn binding, comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2. 12. A bispecific bivalent antibody with FcRn binding elimination, comprising a first antigen-binding site that specifically binds to human VEGF and a second antigen-binding site that specifically binds to human ANG-2. in α) The first antigen-binding site that specifically binds to VEGF includes the CDR3H region of SEQ ID NO:14, the CDR2H region of SEQ ID NO:15, and the CDR1H region of SEQ ID NO:16 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:17, the CDR2L region of SEQ ID NO:18, and the CDR1L region of SEQ ID NO:19 in the light chain variable domain. The second antigen-binding site that specifically binds to ANG-2 includes the CDR3H region of SEQ ID NO:22, the CDR2H region of SEQ ID NO:23, and the CDR1H region of SEQ ID NO:24 in the heavy chain variable domain, and the CDR3L region of SEQ ID NO:25, the CDR2L region of SEQ ID NO:26, and the CDR1L region of SEQ ID NO:27 in the light chain variable domain. The γ) bispecific antibody comprises an IgG class Fc region according to any one of claims 1 to 5. 13. The bispecific antibody according to claim 12, wherein... α) The first antigen-binding site that specifically binds to VEGF contains the amino acid sequence of SEQ ID NO:20 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:21 as the light chain variable domain VL. The second antigen-binding site that specifically binds to ANG-2 contains the amino acid sequence of SEQ ID NO:28 as the heavy chain variable domain VH and the amino acid sequence of SEQ ID NO:29 as the light chain variable domain VL. The γ) bispecific antibody comprises an IgG class Fc region according to any one of claims 1 to 5. 14. An Fc region fusion polypeptide comprising an IgG-type Fc region according to any one of claims 1 to 5. 15. A pharmaceutical formulation comprising an antibody according to any one of claims 6 to 13 or an Fc region fusion polypeptide according to claim 14. 16. Use of the antibody of any one of claims 6 to 13 or the Fc region fusion polypeptide of claim 14 in the preparation of a pharmaceutical formulation, wherein the pharmaceutical formulation is used to treat ocular vascular diseases. 17. Use of the antibody of any one of claims 6 to 13 or the Fc region fusion polypeptide of claim 14 in the manufacture of a medicament, wherein the medicament is used to treat ocular vascular diseases.