Fc region variants with improved protein A binding

Abstract

Herein are reported polypeptides comprising a first polypeptide and a second polypeptide, each of which comprises, in N-terminal to C-terminal direction, at least a portion of an immunoglobulin hinge region comprising one or more cysteine ​​residues, an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, wherein the first polypeptide, the second polypeptide or the first and the second polypeptide comprise the mutation Y436A (numbering according to the EU index).

Description

[0001] This application is a divisional application of Chinese patent application No. 201580004548.6. The filing date of the original application is January 12, 2015. The name of the invention is “Fc region variants with improved protein A binding”.

[0002] Herein we report IgG Fc regions that have been modified with regard to their purification properties. Background of the Invention

[0003] The need for cost-effective production processes has led to the necessity to optimize downstream purification processes, including one or more affinity chromatography steps. Larger volumes to be processed and more stringent cleaning-in-place (CIP) protocols require certain features that need to be addressed (Hober, S., J. Chrom. B. 848 (2007) 40-47).

[0004] Purification of monoclonal antibodies with the aid of selective Fc region affinity ligands is the most promising methodology for large-scale production of therapeutic monoclonal antibodies. In fact, this method does not require any interaction with the antigen-specific portion of the antibody, i.e., the Fab domain, which is therefore intact and can retain its properties (see Salvalaglio, M. et al., J. Chrom. A 1216 (2009) 8678-8686).

[0005] Due to its selectivity, affinity-purification steps are used early in the purification chain and thus can reduce the number of sequential unit operations (see Hober supra; MacLennan, J., Biotechnol. 13 (1995) 1180; Harakas, NK, Bioprocess Technol. 18 (1994) 259).

[0006] The most commonly used ligands for selective binding to IgG are Staphylococcal protein A and protein G, which are able to establish highly selective interactions with the Fc region of most IgGs at a region called the "consensus binding site (CBS)" (DeLano, WL et al., Science 287 (2000) 1279), which is located in the hinge region between the CH2 and CH3 domains of the Fc region.

[0007] Staphylococcal protein A (SPA) is a cell wall-associated protein domain exposed on the surface of the Gram-positive bacterium Staphylococcus aureus. SPA has high affinity for IgG from various species, such as human, rabbit, and guinea pig IgG, but only weakly interacts with bovine and mouse IgG (see table below) (see Hober supra; Duhamel, RC et al., J. Immunol. Methods 31 (1979) 211; L. and Kronvall, G., Immunol. J. 133 (1984) 969; Richman, DD et al., J. Immunol. 128 (1982) 2300; Amersham Pharmacia Biotech, Handbook, Antibody Purification (2000)).

[0008]

[0009] ++: strong binding / +: moderate binding / -: weak or no interaction

[0010] The heavy chain hinge region between the CH2 and CH3 domains of IgG is capable of binding several proteins in addition to protein A, such as the neonatal Fc receptor (FcRn) (see DeLano and Salvalaglio, supra).

[0011] The SPA CBS includes a hydrophobic pocket on the antibody surface. The residues that make up the IgG CBS are Ile253, Ser254, Met252, Met423, Tyr326, His435, Asn434, His433, Arg255, and Glu380 (IgG heavy chain residues are numbered according to the Kabat EU index numbering system). Charged amino acids (Arg255, Glu380) are located around the hydrophobic knot formed by Ile253 and Ser254. This (can) lead to the establishment of polar and hydrophilic interactions (see Salvalaglio above).

[0012] In general, the Protein A-IgG interaction can be described using two major binding sites: the first site is located in the heavy chain CH2 domain and is characterized by hydrophobic interactions between Phe132, Leu136, Ile150 (of Protein A) and the IgG hydrophobic knot composed of Ile253 and Ser254, and an electrostatic interaction between Lys154 (Protein A) and Thr256 (IgG). The second site is located in the heavy chain CH3 domain and is characterized by electrostatic interactions between Gln129 and Tyr133 (Protein A) and His433, Asn434 and His435 (IgG) (see Salvalaglio above).

[0013] Lindhofer, H. et al. (J. Immunol. 155 (1995) 219-225) reported species-restricted preferred heavy / light chain pairing in rat / mouse quadromas.

[0014] Jedenberg, L. et al. (J. Immunol. Meth. 201 (1997) 25-34) reported that SPA binding analysis of two Fc variants (Fc13 and Fc31, each containing an isotype dipeptide substitution from another corresponding isotype) showed that Fc1 and Fc31 interacted with SPA, while Fc3 and Fc13 lacked detectable SPA binding. They concluded that SPA binding by the Fc region variant Fc31 was due to the introduced dipeptide substitutions R435H and F436Y.

[0015] Today, the emphasis with regard to therapeutic monoclonal antibodies is on the generation and use of bispecific antibodies or even multispecific antibodies that specifically bind to two or more targets (antigens).

[0016] The fundamental challenge in producing multispecific heterodimeric IgG antibodies from four antibody chains (two different heavy chains and two different light chains) in one expression cell line is the so-called chain association problem (see Klein, C. et al., mAbs 4 (2012) 653-663). The requirement to use different chains as the left and right arms of a multispecific antibody results in a mixture of antibodies when expressed in one cell: the two heavy chains can (theoretically) associate in four different combinations (two of which are identical), and each of these heavy chains can associate in a random manner with a light chain, producing two 4There are 16 theoretically possible chain combinations. Of the 16 theoretically possible combinations, 10 were found, but only one of these corresponded to the desired functional bispecific antibody (De Lau, WB, et al., J. Immunol. 146 (1991) 906-914). The difficulty in isolating the desired bispecific antibody from a complex mixture and the inherently poor yield of a theoretical maximum of 12.5% ​​make the production of bispecific antibodies in an expression cell line extremely challenging.

[0017] To overcome the chain association problem and force the correct association of two different heavy chains, in the late 1990s, Carter et al. from Genentech invented a method called "knot-in-knot" (KiH) method (see Carter, P., J. Immunol. Meth. 248 (2001) 7-15; Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681; Zhu, Z. et al., Prot. Sci. 6 (1997) 781-788; Ridgway, JB et al., Prot. Eng. 9 (1996) 617-621; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35; and US 7,183,076). Basically, this concept relies on modifying the interface between the two CH3 domains of the two heavy chains of an antibody where most of the interactions occur. A bulky residue is introduced into the CH3 domain of one antibody heavy chain and acts like a key ("knot"). In the other heavy chain, a "clasp" is formed that can accommodate this bulky residue, thereby simulating a lock. The resulting heterodimeric Fc region can be further stabilized by introducing / forming artificial disulfide bonds. Notably, all KiH mutations are buried inside the CH3 domain and are not "visible" to the immune system. In addition, the properties of antibodies with KiH mutations, such as (thermal) stability, FcγR binding and effector functions (e.g., ADCC, FcRn binding) and pharmacokinetic (PK) behavior, are not affected.

[0018] Correct heavy chain association can be achieved with heterodimerization yields greater than 97% by introducing the following six mutations: S354C, T366W in the "knot" heavy chain and Y349C, T366S, L368A, Y407V in the "buckle" heavy chain (see Carter supra; residues are numbered according to the Kabat EU index numbering system). Knot-knot homodimers are generally not observed, although they may be present. Buckle-buckle dimers can be depleted by selective purification procedures or by the procedures outlined below.

[0019] Although the random heavy chain association problem has been solved, it is still necessary to ensure correct light chain association.Similar to the KiHCH3 domain approach, efforts have been made to study asymmetric light chain-heavy chain interactions that may eventually lead to complete bispecific IgGs.

[0020] As a possibility, Roche recently developed the CrossMab solution to force the light chains to pair correctly in bispecific heterodimeric IgG antibodies when combining light chains with KiH technology (see Klein above; Schaefer. W. et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192; Cain, C., SciBX 4 (2011) 1-4). This allows the generation of bispecific antibodies or even multispecific antibodies in a generic manner. In this format, one arm of the desired bispecific antibody remains unchanged. In the second arm, the complete Fab region or VH-VL domain or CH1-CL domain is exchanged between the heavy chain and the light chain by domain crossover. As a result, the newly formed "crossed" light chain no longer associates with the (normal, i.e., non-crossed) heavy chain Fab region of the other arm of the bispecific antibody. Thus, correct "light chain" association can be forced by this minimal change in domain pairing (see Schaefer supra).

[0021] Zhu et al. introduced several sterically complementary mutations and disulfide bonds into the two VL / VH interfaces of a diabody variant. When mutations VL Y87A / F98M and VH V37F / L45W were introduced into the anti-p185HER2 VL / VH interface, heterodimeric diabodies were recovered in >90% yield compared to the parental diabody, while maintaining overall yield and affinity (see Zhu supra).

[0022] Researchers from Chugai have similarly designed bispecific diabodies by introducing mutations into the VH-VL interface that strengthen light chain association, primarily converting Q39 in VH and Q38 in VL into charged residues (WO 2006 / 106905; Igawa, T. et al., Prot. Eng. Des. Sel. 23 (2010) 667-677).

[0023] In WO 2011097603, common light chain mice were reported.

[0024] In WO 2010151792, a bispecific antibody format is provided that results in readily isolable bispecific antibodies comprising immunoglobulin heavy chain variable domains that are differentially modified (i.e., heterodimeric) in the CH3 domain, wherein the differential modifications are non-immunogenic or substantially non-immunogenic relative to the CH3 modifications, and at least one modification results in a differential affinity of the bispecific antibodies for an affinity reagent such as protein A, and the bispecific antibodies are isolable from disrupted cells, from culture medium, or from a mixture of antibodies based on their affinity for protein A.

[0025] Neonatal Fc-receptor (FcRn) is important for the metabolic fate of IgG class antibodies in vivo. FcRn plays a role in utilizing IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein composed of two polypeptides: 50kDa major histocompatibility complex-like protein class I (α-FcRn) and 15kDa β2-microglobulin (β2m). FcRn binds to the CH2-CH3 portion of the Fc region of IgG class antibodies with high affinity. The interaction between IgG class antibodies and FcRn is pH-dependent and occurs in a 1:2 stoichiometry, i.e., one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, e.g., Huber, AH et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0026] Therefore, the in vitro FcRn binding properties / characteristics of IgG are indicative of its in vivo pharmacokinetic properties in blood circulation.

[0027] Different amino acid residues in the heavy chain CH2 and CH3 domains are involved in the interaction between FcRn and the Fc region of IgG class antibodies.

[0028] Different mutations that affect FcRn binding and half-life in blood circulation are known. Fc region residues that are critical for mouse Fc region-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF et al., J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (numbered according to the KabatEU index numbering system) 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, JK et al., Eur. J. Immunol. 24 (1994) 542-548). Residues 1253, H310, and H435 were found to be critical for the interaction of the human Fc region with murine FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819-2885).

[0029] Methods to increase Fc region (and likewise IgG) binding to FcRn have been implemented by mutating multiple amino acid residues in the Fc region: Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433, and Asn 434 (see Kuo, TT et al., J. Clin. Immunol. 30 (2010) 777-789; Ropeenian, DC et al., Nat. Rev. Immunol. 7 (2007) 715-725).

[0030] Through protein-protein interaction studies, Dall'Acqua et al. have described that the combination of M252Y, S254T, and T256E mutations improves FcRn binding (Dall'Acqua, WF et al., J. Biol. Chem. 281 (2006) 23514-23524). Studies of the human Fc region-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, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671 ), various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and studied.

[0031] In WO 2014 / 006217, a dimeric protein with triple mutations is reported. The 2.8 angstrom crystal structure of the FcRn / heterodimeric Fc complex with pH-dependent binding mechanism was reported by Martin, W. et al. (Mol. Cell. 7 (2001) 867-877. In US 6,277,375, WO 2013 / 004842 reported immunoglobulin-like domains with increased half-life. Shields, RL et al. reported high-resolution mapping of the binding sites of FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 and the design of IgG1 variants with improved FcγR binding (Biochem. Mol. Biol. 276 (2001) 6591-6604). Medesan, C. et al. (J. Immunol. 158 (1997) 2211-2217) elucidated the mouse IgG1 amino acid residues involved in transcytosis and catabolism. In US 2010 / 0272720 reports an antibody fusion protein with a modified FcRn binding site. WO 2013 / 060867 reports the production of heterodimeric proteins. Qiao, S.-W. et al. report that antibody-mediated antigen presentation is FcRn-dependent (Proc. Natl. Acad. Sci. USA 105 (2008) 9337-9342). SUMMARY OF THE INVENTION

[0032] Reported herein are variant Fc regions that specifically bind to Staphylococcus protein A and do not bind to human FcRn. These variant Fc regions contain specific amino acid mutations in the CH2 and CH3 domains. It has been found that when used in a tether or linker of a heterodimeric Fc region, these mutations allow purification of the heterodimeric Fc region, i.e., separation of the heterodimeric Fc region from the homodimeric Fc region.

[0033] One aspect as reported herein is the use of the mutation Y436A to increase the binding of (dimeric) Fc-region polypeptides to Protein A.

[0034] One aspect as reported herein is a (dimeric) polypeptide comprising

[0035] a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminal to the C-terminal direction, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, and the second polypeptide comprises, from the N-terminal to the C-terminal direction, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain,

[0036] wherein the first polypeptide, the second polypeptide or the first and second polypeptides comprise the mutation Y436A (numbering according to the Kabat EU index numbering system), and

[0037] wherein the first polypeptide and the second polypeptide are linked by one or more disulfide bonds in at least a portion of the immunoglobulin hinge region.

[0038] In one embodiment, the first and second polypeptides comprise the mutation Y436A.

[0039] In one embodiment the (dimeric) polypeptide does not specifically bind to human FcRn and specifically binds to Staphylococcal protein A.

[0040] In one embodiment the (dimeric) polypeptide is a homodimeric polypeptide.

[0041] In one embodiment the (dimeric) polypeptide is a heterodimeric polypeptide.

[0042] In one embodiment, the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V ("knob") and the second polypeptide comprises the mutations S354C and T366W ("knob").

[0043] In one embodiment, the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V ("knob") and the second polypeptide comprises the mutations Y349C and T366W ("knob").

[0044] In one embodiment

[0045] i) the first and second polypeptides each comprise the mutations H310A, H433A and Y436A, or

[0046] ii) the first and second polypeptides each further comprise the mutations L251D, L314D and L432D, or

[0047] iii) the first and second polypeptides each further comprise the mutations L251S, L314S and L432S, or

[0048] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0049] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0050] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0051] In one embodiment, the immunoglobulin hinge region, immunoglobulin CH2 domain, and immunoglobulin CH3 domain of the first and second polypeptides are of human IgG1 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutations L234A and L235A. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutations P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutations L234A, L235A, and P329G.

[0052] In one embodiment, the immunoglobulin hinge region, immunoglobulin CH2 domain and immunoglobulin CH3 domain of the first and second polypeptides are of human IgG2 subclass.In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutations H268Q, V309L, A330S and P331S.

[0053] In one embodiment, the immunoglobulin hinge region, immunoglobulin CH2 domain and immunoglobulin CH3 domain of the first and second polypeptides are of human IgG2 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutations V234A, G237A, P238S, H268A, V309L, A330S and P331S.

[0054] In one embodiment, the immunoglobulin hinge region, immunoglobulin CH2 domain, and immunoglobulin CH3 domain of the first and second polypeptides are of human IgG4 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutations S228P and L235EA. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutation P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutations S228P, L235E, and P329G.

[0055] In one embodiment, the immunoglobulin hinge region, immunoglobulin CH2 domain, and immunoglobulin CH3 domain of the first and second polypeptides are of human IgG4 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutations S228P, L234A, and L235A. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutation P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutations S228P, L234A, L235A, and P329G.

[0056] In one embodiment the (dimeric) polypeptide is an Fc region fusion polypeptide.

[0057] In one embodiment the (dimeric) polypeptide is a (full length) antibody.

[0058] In one embodiment, the (full length) antibody is a monospecific antibody. In one embodiment, the monospecific antibody is a monovalent monospecific antibody. In one embodiment, the monospecific antibody is a bivalent monospecific antibody.

[0059] In one embodiment, the (full length) antibody is a bispecific antibody. In one embodiment, the bispecific antibody is a bivalent bispecific antibody. In one embodiment, the bispecific antibody is a tetravalent bispecific antibody.

[0060] In one embodiment, the (full length) antibody is a trispecific antibody. In one embodiment, the trispecific antibody is a trivalent trispecific antibody. In one embodiment, the trispecific antibody is a tetravalent trispecific antibody.

[0061] One aspect as reported herein is a method of treating a patient suffering from an ocular vascular disease by administering to a patient in need of such treatment a (dimeric) polypeptide or an antibody as reported herein.

[0062] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for intravitreal administration.

[0063] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use as a medicament.

[0064] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in the treatment of an ocular vascular disease.

[0065] One aspect as reported herein is a pharmaceutical formulation comprising a (dimeric) polypeptide or antibody as reported herein and optionally a pharmaceutically acceptable carrier.

[0066] For the use of antibodies that target / bind to antigens present in the eye and the rest of the body, a short systemic half-life from the eye into the blood after crossing the blood-ocular barrier is beneficial in order to avoid systemic side effects.

[0067] Additionally, antibodies that specifically bind to a receptor ligand are only effective in treating eye diseases if the antibody-antigen complex is removed from the eye, ie, the antibody acts as a transporter for the receptor ligand outside the eye and thereby inhibits receptor signaling.

[0068] The present inventors have found that antibodies comprising an Fc region that does not bind to the human neonatal Fc-receptor (i.e., (dimeric) polypeptides as reported herein) are transported across the blood-ocular barrier. This is surprising because antibodies do not bind to human FcRn, although binding to FcRn is thought to be required for transport across the blood-ocular barrier.

[0069] One aspect as reported herein is the use of a (dimeric) polypeptide or antibody as reported herein for transporting a soluble receptor ligand from the eye across the blood-eye barrier into the blood circulation.

[0070] One aspect as reported herein is the use of a (dimeric) polypeptide or antibody as reported herein for removing one or more soluble receptor ligands from the eye.

[0071] One aspect as reported herein is the use of a (dimeric) polypeptide or antibody as reported herein for the treatment of eye diseases, in particular ocular vascular diseases.

[0072] One aspect as reported herein is the use of a (dimeric) polypeptide or antibody as reported herein for transporting one or more soluble receptor ligands from the intravitreal cavity to the blood circulation.

[0073] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in the treatment of an eye disease.

[0074] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in transporting a soluble receptor ligand from the eye across the blood-eye barrier into the blood circulation.

[0075] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in removing one or more soluble receptor ligands from the eye.

[0076] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in the treatment of an eye disease, in particular an ocular vascular disease.

[0077] One aspect as reported herein is a (dimeric) polypeptide or antibody as reported herein for use in transporting one or more soluble receptor ligands from the intravitreal cavity into the blood circulation.

[0078] One aspect as reported herein is a method of treating an individual suffering from an ocular vascular disease, said method comprising administering to the individual an effective amount of a (dimeric) polypeptide or antibody as reported herein.

[0079] One aspect as reported herein is a method for transporting a soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation in an individual, comprising administering to the individual an effective amount of a (dimeric) polypeptide or antibody as reported herein to transport the soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation.

[0080] One aspect as reported herein is a method of removing one or more soluble receptor ligands from the eye in an individual, comprising administering to the individual an effective amount of a (dimeric) polypeptide or antibody as reported herein to remove the one or more soluble receptor ligands from the eye.

[0081] One aspect as reported herein is a method for transporting one or more soluble receptor ligands from the intravitreal cavity into the blood circulation in an individual, comprising administering to the individual an effective amount of a (dimeric) polypeptide or antibody as reported herein to transport one or more soluble receptor ligands from the intravitreal cavity into the blood circulation.

[0082] One aspect as reported herein is a method for transporting a soluble receptor ligand from the vitreous cavity or the eye across the blood-ocular barrier into the blood circulation in an individual, comprising administering to the individual an effective amount of a (dimeric) polypeptide or antibody as reported herein to transport the soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation.

[0083] In one embodiment, the (dimeric) polypeptide is a bispecific antibody. In one embodiment, the bispecific antibody is a bivalent bispecific antibody. In one embodiment, the bispecific antibody is a tetravalent bispecific antibody.

[0084] In one embodiment, the (dimeric) polypeptide is a trispecific antibody. In one embodiment, the trispecific antibody is a trivalent trispecific antibody. In one embodiment, the trispecific antibody is a tetravalent trispecific antibody.

[0085] In one embodiment, the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide further comprises the mutations S354C and T366W.

[0086] In one embodiment, the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide further comprises the mutations Y349C and T366W.

[0087] In one embodiment

[0088] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0089] ii) the first and second polypeptides further comprise the mutations L251D, L314D and L432D, or

[0090] iii) the first and second polypeptides further comprise the mutations L251S, L314S and L432S, or

[0091] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0092] v) the first polypeptide further comprises the mutations I253A, H310A and H435A and the second polypeptide further comprises the mutations L251D, L314D and L432D, or

[0093] vi) the first polypeptide further comprises the mutations I253A, H310A and H435A and the second polypeptide further comprises the mutations L251S, L314S and L432S.

[0094] In one embodiment the (dimeric) polypeptide is a CrossMab.

[0095] In one embodiment the (dimeric) polypeptide is an Fc region fusion polypeptide.

[0096] In one embodiment, the antibody or Fc region fusion polypeptide is of subclass IgG1. In one embodiment, the antibody or Fc region fusion polypeptide further comprises the mutations L234A and L235A. In one embodiment, the antibody or Fc region fusion polypeptide further comprises the mutation P329G.

[0097] In one embodiment, the antibody or Fc region fusion polypeptide is of subclass IgG2. In one embodiment, the antibody or Fc region fusion polypeptide further comprises the mutations V234A, G237A, P238S, H268A, V309L, A330S and P331S.

[0098] In one embodiment, the antibody or Fc region fusion polypeptide is of subclass IgG4. In one embodiment, the antibody or Fc region fusion polypeptide further comprises the mutations S228P and L235E. In one embodiment, the antibody or Fc region fusion polypeptide further comprises the mutation P329G. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 : Schematic diagram of the concept and advantages of an anti-VEGF / ANG2 antibody of the IgG1 or IgG4 subclass having the IHH-AAA mutation (a combination of mutations I253A, H310A and H435A (numbering according to the Kabat EU index numbering system)).

[0100] Figure 2: Small-scale DLS-based viscosity measurements: Extrapolated viscosity at 150 mg / mL in 200 mM arginine / succinate buffer, pH 5.5 (anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutations) compared to reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutations)).

[0101] Figure 3 : Temperature-dependent aggregation of DLS (including DLS aggregation onset temperature) in 20 mM histidine buffer, 140 mM NaCl, pH 6.0 (comparing the anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutations) as reported herein with the reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutations)).

[0102] Figure 4 : Storage at 100 mg / mL for 7 days at 40°C (decrease in the main peak and increase in high molecular weight (HMW)) (comparison of the anti-VEGF / ANG2 antibody VEGF / ANG2-0016 as reported herein (with IHH-AAA mutations) which shows less aggregation with the reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutations)).

[0103] Figure 5A and Figure 5B : FcRn steady-state affinity A: VEGF / ANG2-0015 (without IHH-AAA mutation) and B: VEGF / ANG2-0016 (with IHH-AAA mutation).

[0104] Figure 6 : FcγRIIIa interaction measurement of VEGF / ANG2-0015 without IHH-AAA mutation and VEGF / ANG2-0016 with IHH-AAA mutation (both IgG1 subclass with P329G LALA mutation; as controls, anti-digoxigenin antibody (anti-Dig antibody) of IgG1 subclass and IgG4-based antibody were used).

[0105] Figure 7A : Schematic pharmacokinetic (PK) ELISA assay principle for determining anti-VEGF / ANG2 antibody concentrations in serum and whole-eye lysates.

[0106] Figure 7B : Serum concentrations after intravenous (iv) administration: comparison of VEGF / ANG2-0015 without IHH-AAA mutation and VEGF / ANG2-0016 with IHH-AAA mutation.

[0107] Figure 7C : Serum concentrations after intravitreal administration: comparison of VEGF / ANG2-0015 without IHH-AAA mutation and VEGF / ANG2-0016 with IHH-AAA mutation.

[0108] Figure 7D : Eye lysate concentrations of VEGF / ANG2-0016 (with IHH-AAA mutation) in right and left eyes (after intravitreal administration into the right eye only compared to intravenous administration): Significant concentrations could be detected in the right eye only after intravitreal administration; no concentrations were detectable in eye lysates after intravenous administration due to the low serum half-life of VEGF / ANG2-0016 (with IHH-AAA mutation).

[0109] Figure 7E : Eye lysate concentrations of VEGF / ANG2-0015 (without IHH-AAA mutation) were detectable in both the right and left eyes (only after intravitreal administration into the right eye, compared to intravenous administration): Concentrations of VEGF / ANG2-0015 were detectable in the right eye (and to some extent in the left eye) after intravitreal administration; this indicates 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 VEGF / ANG2-0015 (without IHH-AAA mutation); significant concentrations were also detectable in eye lysates of both eyes after intravenous administration, due to diffusion of serum-stable VEGF / ANG2-0015 (without IHH-AAA mutation) into the eye.

[0110] Figure 8: Antibodies engineered in their ability to bind to FcRn show prolonged (YTE mutation) or shortened (IHH-AAA mutation) in vivo half-life in SPR analysis, enhanced (YTE mutation) or diminished binding (IHH-AAA mutation), and enhanced or reduced retention time in FcRn column chromatography compared to a reference wild-type (wt) antibody; a) PK data following a single intravenous bolus administration of 10 mg / kg to huFcRn transgenic male C57BL / 6J mice + / - 276: AUC data for wild-type IgG and IgG with YTE and IHH-AAA Fc regions modified; b) BIAcore sensorgram; c) FcRn affinity column elution; wild-type anti-IGF-1R antibody (reference), YTE-mutant of anti-IGF-1R antibody, IHH-AAA-mutant of anti-IGF-1R antibody.

[0111] Figure 9 : Changes in retention time in FcRn affinity chromatography depending on the number of mutations introduced into the Fc region.

[0112] Figure 10 : Changes in FcRn binding depending on the asymmetric distribution of mutations introduced into the Fc region.

[0113] Figure 11 : Elution chromatogram of a bispecific anti-VEGF / ANG2 antibody (VEGF / ANG2-0121) having a combination of mutations H310A, H433A and Y436A in both heavy chains from two consecutive Protein A affinity chromatography columns.

[0114] Figure 12 : Elution chromatogram of an anti-IGF-1R antibody (IGF-1R-0045) having mutations H310A, H433A and Y436A in both heavy chains from a protein A affinity chromatography column.

[0115] Figure 13 : Binding of IgG Fc region-modified anti-VEGF / ANG-2 antibody to immobilized protein A on a CM5 chip.

[0116] Figure 14 : Elution chromatograms of different anti-VEGF / ANG2 antibodies on FcRn affinity column.

[0117] Figure 15 : Binding effect of different fusion peptides on Staphylococcal protein A (SPR).

[0118] Figure 16 : Binding of different anti-VEGF / ANG2 antibodies and anti-IGF-1R antibody mutants to immobilized protein A (SPR).

[0119] Figure 17 : Comparison of serum concentrations after intravenous administration of antibodies IGF-1R 0033, 0035 and 0045.

[0120] Figure 18 : Comparison of ocular lysate concentrations following intravitreal and intravenous administration of the antibody IGF-1R 0033.

[0121] Figure 19 : Comparison of ocular lysate concentrations following intravitreal and intravenous administration of the antibody IGF-1R 0035.

[0122] Figure 20 : Comparison of ocular lysate concentrations following intravitreal and intravenous administration of the antibody IGF-1R 0045.

[0123] Detailed description of embodiments of the present invention

[0124] I. Definition

[0125] The term "about" refers to a range of + / - 20% of the following numerical value. In one embodiment, the term "about" refers to a range of + / - 10% of the following numerical value. In one embodiment, the term "about" refers to a range of + / - 5% of the following numerical value.

[0126] For the purposes herein, an "acceptor human framework" is a framework that comprises the 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 consensus framework as defined below. An acceptor human framework that is "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, 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 acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0127] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) where such alterations result in improved affinity of the antibody for antigen, compared to a parent antibody which does not possess those alterations.

[0128] The term "alteration" refers to the mutation (replacement), insertion (addition) or deletion of one or more amino acid residues in a parent antibody or fusion polypeptide (e.g., a fusion polypeptide comprising at least the FcRn binding portion of an Fc region) to obtain a modified antibody or fusion polypeptide. The term "mutation" refers to the replacement of a specific amino acid residue with a different amino acid residue. For example, the mutation L234A refers to the replacement of the amino acid residue lysine at position 234 in the Fc region (polypeptide) of an antibody with the amino acid residue alanine (lysine replaced with alanine) (numbered according to the Kabat EU index numbering system).

[0129] As used herein, the amino acid positions of all constant regions and domains of 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 are referred to herein as "numbered according to Kabat." 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 light chain constant domains CL of the kappa and lambda isotypes and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains of (CH1, hinge, CH2, and CH3).

[0130] "Naturally occurring amino acid residue" refers to an amino acid residue from the group consisting of alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0131] The term "amino acid mutation" refers to the replacement of at least one existing amino acid residue by another, different amino acid residue (= substituting amino acid residue). The replacement amino acid residue can be a "naturally occurring amino acid residue" and is selected from 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 replacement amino acid residue can be a "non-naturally occurring amino acid residue". See, for example, US 6,586,207, WO 98 / 48032, WO 03 / 073238, US 2004 / 0214988, WO 2005 / 35727, WO 2005 / 74524, Chin, JW et al., J. Am. Chem. Soc. 124 (2002) 9026-9027; Chin, JW and Schultz, P. G., ChemBioChem 11 (2002) 1135-1137; Chin, JW et al., PICAS United States of America 99 (2002) 11020-11024; and Wang, L. and Schultz, P. G., Chem. (2002) 1-10 (all of which are incorporated herein by reference in their entirety).

[0132] The term "amino acid insertion" refers to the (additional) incorporation of at least one amino acid residue at a predetermined position in an amino acid sequence. In one embodiment, the insertion will be the insertion of one or two amino acid residues. The inserted amino acid residue may be any naturally occurring or non-naturally occurring amino acid residue.

[0133] 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.

[0134] 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, PC et al., Science 277 (1997) 55-60 and Cheung, AH et al., Genomics 48 (1998) 389-91. Angiopoietin-1 (SEQ ID NO: 32) and -2 are found as ligands for the Tie tyrosine kinase family, which is selectively expressed within the vascular endothelium (Yancopoulos, GD et al., Nature 407 (2000) 242-248). There are currently four identified members of the angiopoietin family. Angiopoietin-3 and -4 (ANG-3 and ANG-4) may broadly represent diverse counterparts of 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 originally 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, PC et al., Science 277 (1997) 55-60). All known angiopoietins bind primarily to Tie2 (SEQ ID NO: 33), and both ANG-1 and -2 bind to Tie2 with an affinity of 3 nM (Kd) (Maisonpierre, PC et al., Science 277 (1997) 55-60).

[0135] The term "antibody" is used herein in the broadest sense and encompasses 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, so long as they exhibit the desired antigen- and / or protein A and / or FcRn-binding activity.

[0136] The term "asymmetric Fc region" refers to a pair of Fc region polypeptides that have different amino acid residues at corresponding positions according to the Kabat EU index numbering system.

[0137] The term "asymmetric Fc region for FcRn binding" refers to an Fc region composed of two polypeptide chains having different amino acid residues at corresponding positions, wherein the positions are determined according to the Kabat EU index numbering system, and thus the different positions affect the binding of the Fc region to the human neonatal Fc-receptor (FcRn). For the purposes of this article, the differences between the two polypeptide chains of the Fc region in an "asymmetric Fc region for FcRn binding" do not include differences that have been introduced to promote the formation of heterodimeric Fc regions (e.g., for the production of bispecific antibodies). These differences can also be asymmetric, that is, the two chains have differences at non-corresponding amino acid residues according to the Kabat EU index numbering system. These differences promote heterodimerization and reduce homodimerization. Examples of such differences are so-called "knob-in-knob" substitutions (see, for example, US Pat. No. 7,695,936 and US 2003 / 0078385). The following knot and hook substitutions in a single polypeptide chain of the Fc region of an IgG antibody of the IgG1 subclass have been found to increase heterodimer formation: 1) Y407T in one chain and T366Y in the other chain; 2) Y407A in one chain and T366W in the other chain; 3) F405A in one chain and T394W in the other chain; 4) F405W in one chain and T394S in the other chain; 5) Y407T in one chain and T394Y in the other chain. 66Y; 6) T366Y and F405A in one chain and T394W and Y407T in the other chain; 7) T366W and F405W in one chain and T394S and Y407A in the other chain; 8) F405W and Y407A in one chain and T366W and T394S in the other chain; and 9) T366W in one chain and T366S, L368A and Y407V in the other chain, with the last listed being particularly suitable. In addition, changes that create new disulfide bonds between the two Fc region polypeptide chains promote heterodimer formation (see, e.g., US 2003 / 0078385). The following substitutions that create appropriately spaced cysteine ​​residues for forming new intrachain disulfide bonds in a single polypeptide chain of the Fc region of an IgG antibody of the IgG1 subclass have been found to increase heterodimer formation: Y349C in one chain and S354C in the other chain; Y349C in one chain and E356C in the other chain; Y349C in one chain and E357C in the other chain; L351C in one chain and S354C in the other chain; T394C in one chain and E397C in the other chain; or D399C in one chain and K392C in the other chain. Other examples of amino acid changes that promote heterodimerization are so-called "charge pair substitutions" (see, e.g., WO 2009 / 089004).The following charge pair substitutions in a single polypeptide chain of the Fc region of an IgG antibody of 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 E356R in the other chain. 7K; 7) K409D and K392D plus D399K, E356K in one chain and E357K in the other chain; 8) K409D and K392D in one chain and D399K in the other chain; 9) K409D and K392D in one chain and D399K and E356K in the other chain; 10) K409D and K392D in one chain and D399K and D357K in the other chain; 11) K409D and K370D in one chain and D399K and D357K in the other chain; 12) D399K in one chain and K409D and K360D in the other chain; and 13) K409D and K439D in one chain and D399K and E356K on the other chain.

[0138] The term "binding (to an antigen)" refers to the binding of an antibody to its antigen in an in vitro assay, in one embodiment a binding assay in which the antibody is bound to a surface and antigen-antibody binding is measured by surface plasmon resonance (SPR). -8 M or less, in some embodiments 10 -13 to 10 -8 M. In some embodiments 10 -13 to 10 -9 The affinity of M (K D ).

[0139] Binding can be studied by BIAcore assay (GE Healthcare Biosensor AB, Uppsala, Sweden). The affinity of binding is expressed in terms of k a (association rate constant of the antibody from the antibody / antigen complex), k D (dissociation constant) and K D (k D / k a )definition.

[0140] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0141] The term "CH2 domain" refers to the portion of an antibody heavy chain polypeptide extending from EU position 231 to EU position 340 (EU numbering system according to Kabat). 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.

[0142] The term "CH3 domain" refers to the portion of an 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.

[0143] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0144] The term "comparable length" means that the two polypeptides comprise the same number of amino acid residues or may differ in length by one or more and up to a maximum of 10 amino acid residues. In one embodiment, the (Fc region) polypeptides comprise the same number of amino acid residues or may differ by 1 to 10 amino acid residues. In one embodiment, the (Fc region) polypeptides comprise the same number of amino acid residues or may differ by 1 to 5 amino acid residues. In one embodiment, the (Fc region) polypeptides comprise the same number of amino acid residues or may differ by 1 to 3 amino acid residues.

[0145] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the 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 receptor); and B-cell activation.

[0146] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective to achieve the desired therapeutic or prophylactic result, at dosages and for periods of time necessary to achieve that result.

[0147] The term "Fc-fusion polypeptide" refers to a fusion of a binding domain (e.g., an antigen binding domain such as a single-chain antibody, or a polypeptide such as a receptor ligand) and an antibody Fc region that exhibits the desired target binding activity, protein A binding activity, and FcRn-binding activity.

[0148] The term "human Fc region" refers to the C-terminal region heavy chain of a human immunoglobulin, which contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl 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.

[0149] The term "FcRn" refers to the human neonatal Fc receptor. FcRn plays a role in salvaging IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. FcRn is a heterodimeric protein composed of two polypeptides: the 50kDa major histocompatibility complex-like protein class I (α-FcRn) and the 15kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a 1:2 stoichiometry, i.e., one IgG binds to two FcRn molecules via its two heavy chains (Huber, AH et al., J. Mol. Biol. 230 (1993) 1077-1083). FcRn binding occurs at acidic pH (pH < 6.5) in endosomes, and IgG is released on the surface of neutral cells (pH approximately 7.4). The pH-sensitive nature of this interaction promotes FcRn-mediated protection of IgG pinocytosed into cells from intracellular degradation by binding to the receptor within the acidic environment of endosomes. FcRn then promotes the recycling of IgG to the cell surface and subsequent release into the bloodstream when the FcRn-IgG complex is exposed to the neutral pH environment outside the cell.

[0150] The term "FcRn binding portion of an Fc region" refers to the portion of an antibody heavy chain polypeptide that extends from about EU position 243 to EU position 261, and from about EU position 275 to EU position 293, and from about EU position 302 to EU position 319, and from about EU position 336 to EU position 348, and from about EU position 367 to EU position 393 and EU position 408, and from about EU position 424 to EU position 440. In one embodiment, one or more of the following amino acid residues, F243, P244, P245 according to Kabat EU numbering, are altered. P,K246,P247,K248,D249,T250,L251,M252,I253,S254,R255,T256,P257,E258 ,V259,T260,C261,F275,N276,W277,Y278,V279,D280,V282,E283,V284,H285,N 286,A287,K288,T289,K290,P291,R292,E293,V302,V303,S304,V305,L306,T3 07,V308,L309,H310,Q311,D312,W313,L314,N315,G316,K317,E318,Y319,I336 ,S337,K338,A339,K340,G341,Q342,P343,R344,E345,P346,Q347,V348,C367, V369,F372,Y373,P374,S375,D376,I377,A378,V379,E380,W381,E382,S383,N3 84, 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 numbering).

[0151] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0152] The term "full-length antibody" refers to an antibody having a structure substantially similar to a natural antibody structure comprising four polypeptides or having heavy chains containing an Fc region as defined herein. A full-length antibody may comprise additional domains, such as scFv or scFab, conjugated to one or more chains of a full-length antibody. These conjugates are also encompassed by the term full-length antibody.

[0153] The term "dimeric polypeptide" refers to a complex comprising at least two covalently bound polypeptides. The complex may comprise other polypeptides, which are also covalently or non-covalently bound to the remaining polypeptides. In one embodiment, the dimeric polypeptide comprises two or four polypeptides.

[0154] The term "heterodimer" or "heterodimeric" refers to a molecule comprising two polypeptides (e.g., of comparable length), wherein the two polypeptides have amino acid sequences that differ by at least one amino acid residue at corresponding positions, wherein corresponding positions are determined according to the Kabat EU index numbering system.

[0155] The terms "homodimer" and "homodimeric" refer to a molecule comprising two polypeptides of comparable length, wherein the two polypeptides have identical amino acid sequences at corresponding positions, as determined according to the Kabat EU index numbering system.

[0156] As reported herein, the dimeric polypeptides may be homodimeric or heterodimeric, as determined with respect to the mutation or property of interest. For example, with respect to FcRn and / or protein A binding (i.e., the property of interest), the dimeric polypeptide is homodimeric with respect to mutations H310A, H433A, and Y436A (i.e., both polypeptides of the dimeric polypeptide comprise these mutations) (which are of interest with respect to the FcRn and / or protein A binding properties of the dimeric polypeptide), but is heterodimeric with respect to mutations Y349C, T366S, L368A, and Y407V, respectively (which are not of interest because they involve dimeric polypeptide heterodimerization and not FcRn / protein A binding properties), and with respect to mutations S354C and T366W (the first group being contained only in the first polypeptide, while the second group being contained only in the second polypeptide). As a further example, a dimeric polypeptide as reported herein may be heterodimeric with respect to the mutations I253A, H310A, H433A, H435A and Y436A (i.e. these mutations are all involved in the FcRn and / or Protein A binding properties of the dimeric polypeptide), i.e. one polypeptide comprises the mutations I253A, H310A and H435A and the other polypeptide comprises the mutations H310A, H433A and Y436A.

[0157] 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 "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny are included herein as having the same function or biological activity as the progeny screened or selected for in the originally transformed cell.

[0158] A "human antibody" is an antibody that possesses an amino acid sequence corresponding to an antibody produced by a human or human cell, or is derived from a non-human source utilizing human antibody libraries or other sequences encoding human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0159] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH framework sequences is from a subgroup of variable domain sequences. Typically, the sequence subgroup is a subgroup as described in Kabat, EA 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, the subgroup is subgroup κI as described above in Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described above in Kabat et al.

[0160] The term "derived from" refers to an amino acid sequence that is introduced into a derived from parent amino acid sequence by changing at least one position. Therefore, the derived amino acid sequence is different from the corresponding parent amino acid sequence at at least one corresponding position (according to the Kabat EU index in the antibody Fc region). In one embodiment, the amino acid sequence derived from the parent amino acid sequence is different by 1 to 15 amino acid residues at the corresponding position. In one embodiment, the amino acid sequence derived from the parent amino acid sequence is different by 1 to 10 amino acid residues at the corresponding position. In one embodiment, the amino acid sequence derived from the parent amino acid sequence is different by 1 to 6 amino acid residues at the corresponding position. 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.

[0161] The term "human Fc region polypeptide" refers to an amino acid sequence identical to a "native" or "wild-type" human Fc region polypeptide. The term "variant (human) Fc region polypeptide" refers to an amino acid sequence derived from a "native" or "wild-type" Fc region amino acid sequence by 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, both of which may be variant (human) Fc region polypeptides, or one may be a human Fc region polypeptide and the other a variant (human) Fc region polypeptide.

[0162] In one embodiment, the human Fc-region polypeptide has the amino acid sequence of a human IgG1 Fc-region polypeptide of SEQ ID NO: 60, or the amino acid sequence of a human IgG2 Fc-region polypeptide of SEQ ID NO: 61, or the amino acid sequence of a polypeptide of a human IgG4 Fc-region of SEQ ID NO: 63, said amino acid sequence having a mutation as reported herein. In one embodiment, the variant (human) Fc-region polypeptide is derived from the Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63 and has at least one amino acid mutation compared to the Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63. In one embodiment, the variant (human) Fc-region polypeptide comprises / has from about 1 to about 10 amino acid mutations, and in one embodiment has about 1 to about 5 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 62, or 63. In one embodiment, the variant (human) Fc-region polypeptide is at least about 90% homologous to the human Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63. In one embodiment, the variant (human) Fc-region polypeptide is at least about 95% homologous to the human Fc-region polypeptide of SEQ ID NO: 60, or 62, or 63.

[0163] Variant (human) Fc-region polypeptides derived from the human Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63 are defined by the amino acid changes they contain. 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 the human Fc-region polypeptide of SEQ ID NO: 60, or 61, or 63.

[0164] All positions discussed in the present invention are numbered according to the Kabat EU index numbering system.

[0165] The human IgG1 Fc region polypeptide has the following amino acid sequence:

[0166] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:60).

[0167] The human IgG1 Fc region-derived Fc region polypeptide having mutations L234A, L235A has the following amino acid sequence:

[0168] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:64).

[0169] A human IgG1 Fc region-derived Fc region polypeptide having Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

[0170] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:65).

[0171] The human IgG1 Fc region-derived Fc region polypeptide having S354C, T366W mutations has the following amino acid sequence:

[0172] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:66).

[0173] A human IgG1 Fc region-derived Fc region polypeptide having L234A, L235A mutations and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0174] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:67).

[0175] The human IgG1 Fc region-derived Fc region polypeptide having L234A, L235A and S354C, T366W mutations has the following amino acid sequence:

[0176] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:68).

[0177] The human IgG1 Fc region-derived Fc region polypeptide having the P329G mutation has the following amino acid sequence:

[0178] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:69).

[0179] The human IgG1 Fc region-derived Fc region polypeptide having L234A, L235A mutations and P329G mutations has the following amino acid sequence:

[0180] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:70).

[0181] A human IgG1 Fc region-derived Fc region polypeptide having a P239G mutation and Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

[0182] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:71).

[0183] The human IgG1 Fc region-derived Fc region polypeptide having a P329G mutation and S354C and T366W mutations has the following amino acid sequence:

[0184] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:72).

[0185] A human IgG1 Fc region-derived Fc region polypeptide having L234A, L235A, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0186] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:73).

[0187] The human IgG1 Fc region-derived Fc region polypeptide having L234A, L235A, P329G mutations and S354C, T366W mutations has the following amino acid sequence:

[0188] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:74).

[0189] The human IgG4 Fc region polypeptide has the following amino acid sequence:

[0190] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:63).

[0191] A human IgG4 Fc region-derived Fc region polypeptide having S228P and L235E mutations has the following amino acid sequence:

[0192] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:75).

[0193] A human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E, and P329G mutations has the following amino acid sequence:

[0194] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:76).

[0195] The human IgG4 Fc region-derived Fc region polypeptide having S354C and T366W mutations has the following amino acid sequence:

[0196] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:77).

[0197] A human IgG4 Fc region-derived Fc region polypeptide having Y349C, T366S, L368A, and Y407V mutations has the following amino acid sequence:

[0198] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:78).

[0199] The human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E and S354C, T366W mutations has the following amino acid sequence:

[0200] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:79).

[0201] A human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0202] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:80).

[0203] The human IgG4 Fc region-derived Fc region polypeptide having a P329G mutation has the following amino acid sequence:

[0204] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:81).

[0205] A human IgG4 Fc region-derived Fc region polypeptide having P239G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0206] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:82).

[0207] The human IgG4 Fc region-derived Fc region polypeptide having P329G, S354C, and T366W mutations has the following amino acid sequence:

[0208] ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:83).

[0209] A human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E, P329G and Y349C, T366S, L368A, Y407V mutations has the following amino acid sequence:

[0210] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:84).

[0211] A human IgG4 Fc region-derived Fc region polypeptide having S228P, L235E, P329G and S354C, T366W mutations has the following amino acid sequence:

[0212] ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEK TISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:85).

[0213] The following shows the alignment of different human Fc regions (Kabat EU index numbering system):

[0214]

[0215]

[0216] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one and generally two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FR regions correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0217] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and form structurally defined loops ("hypervariable loops"), and / or contain antigen contact residues ("antigen contacts"). Generally, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs, as referred to herein, include

[0218] (a) Hypervariable loops occurring 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, AM, J. Mol. Biol. 196 (1987) 901-917);

[0219] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242);

[0220] (c) antigenic contacts occurring 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

[0221] (d) A combination of (a), (b) and / or (c), comprising 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).

[0222] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to the Kabat EU index numbering system (Kabat et al., supra).

[0223] As used herein, the term "IGF-1R" refers to any native IGF-1R from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) unless otherwise indicated. The term encompasses "full-length," unprocessed IGF-1R as well as any form of IGF-1R resulting from processing in cells. The term also encompasses naturally occurring IGF-1R variants, e.g., splice variants or allelic variants. The amino acid sequence of human IGF-1R is shown in SEQ ID NO: 11.

[0224] An "individual" or "subject" 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 certain embodiments, the individual or subject is a human.

[0225] An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., size exclusion chromatography, ion exchange, or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0226] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0227] "Isolated nucleic acid encoding an anti-IGF-1R antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors and such nucleic acid molecules present at one or more locations in a host cell.

[0228] As used herein, term " monoclonal antibody " refers to the antibody obtained from substantially homogeneous antibody colony, that is, each antibody constituting the colony is identical and / or in conjunction with identical epi-position, except possible variant antibody (for example, containing naturally occurring mutation or occurring during the production of monoclonal antibody preparations), this type of variant is usually present in minute amount. Contrary to the polyclonal antibody preparations generally including the different antibodies for different determinants (epi-positions), each monoclonal antibody of monoclonal antibody preparations is for the single determinant on the antigen. Therefore, the modifier " monoclonal " indicates that the feature of this antibody is obtained from substantially homogeneous antibody colony, and should not be interpreted as requiring to produce this antibody by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be produced by multiple techniques, including but not limited to hybridoma method, recombinant DNA method, phage display method and the method for utilizing the transgenic animal containing all or part of human immunoglobulin locus, and this type of method and other exemplary methods for producing monoclonal antibodies are described herein.

[0229] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with an indefinite structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N-terminal to the C-terminal, each heavy chain has a variable region (VH), also known as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminal to the C-terminal, each light chain has a variable region (VL), also known as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be divided into one of two types based on the amino acid sequence of its constant domain, referred to as kappa (κ) and lambda (λ).

[0230] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0231] "Percent amino acid sequence identity (%)" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, relative to a reference polypeptide sequence, after aligning the sequences and introducing gaps as needed to achieve maximum sequence homology and not considering any conservative substitutions as part of sequence identity. Alignment for determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full-length sequences being compared. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate % amino acid sequence identity values. 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, Washington, D.C. 20559, where it is registered under U.S. Copyright Registration 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 do not vary.

[0232] In case ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (this can alternatively be described as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0233] Multiply 100 by the fraction X / Y

[0234] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that where 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 to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0235] The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0236] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0237] As used herein, the term "peptide linker" refers to a peptide with an amino acid sequence that is of synthetic origin in one embodiment. A peptide linker is a peptide with an amino acid sequence of at least 30 amino acids in length, and in one embodiment, a peptide with an amino acid sequence of 32 to 50 amino acids in length. In one embodiment, a peptide linker is a peptide with an amino acid sequence of 32 to 40 amino acids in length. In one embodiment, a peptide linker is (GxS)n, wherein G=glycine, S=serine, (x=3, n=8, 9 or 10) or (x=4 and n=6, 7 or 8), in one embodiment, wherein x=4, n=6 or 7, in one embodiment, x=4, n=7. In one embodiment, a peptide linker is (G4S)6G2.

[0238] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric, humanized and human antibodies) prepared, expressed, produced or isolated by recombinant means. This includes antibodies isolated from host cells such as NS0 or CHO cells or from animals (e.g., mice) that are transgenic for human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant antibodies have variable and constant regions in rearranged form. Recombinant antibodies can undergo in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but may be sequences that do not naturally occur in the human antibody germline library in vivo.

[0239] As used herein, the noun "treatment" (and its grammatical variants such as the verb "treat" or the participle "treating") refers to a clinical intervention intended to alter the natural course of the individual being treated, and may be intended to prevent or be performed during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies or Fc region fusion polypeptides as reported herein are used to delay the development of the disease or to slow the progression of the disease.

[0240] As used in this application, the term "valency" refers to the presence of a specified number of binding sites in an (antibody) molecule. In this context, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of 2, 4, and 6 binding sites, respectively, in an (antibody) molecule. In a preferred embodiment, the bispecific antibodies as reported herein are "bivalent."

[0241] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. The variable domains of the antibody heavy and light chains (VH and VL, respectively) generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91). A single VH domain or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL domains from antibodies that bind to that antigen to screen libraries of complementary VL or VH domains, respectively (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0242] 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 neovascularization, and retinal degeneration (see, e.g., Garner, A., Vascular diseases, in: Pathobiology of ocular disease, A dynamic approach, Garner, A. and Klintworth, GK, (Eds.), 2nd ed., Marcel Dekker, New York (1994), pp. 1625-1710).

[0243] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0244] As used herein, the term "VEGF" refers to human vascular endothelial growth factor (VEGF / VEGF-A), the 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 the related 121, 189 and 206 vascular endothelial cell growth factor isoforms as described in Leung, DW et al., Science 246 (1989) 1306-1309; Houck et al., Mol. Endocrin. 5 (1991) 1806-1814; Keck, PJ et al., Science 246 (1989) 1309-1312 and Connolly, DT et al., J. Biol. Chem. 264 (1989) 20017-20024; as well as 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 disorders (Ferrara, N. et al., Endocrin. Rev. 18 (1997) 4-25; Berkman, RA et al., J. Clin. Invest. 91 (1993) 153-159; Brown, LF et al., Human Pathol. 26 (1995) 86-91; Brown, LF et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J. et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, HF et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein that has been isolated from several sources and includes several isoforms. VEGF displays highly specific mitogenic activity on endothelial cells.

[0245] The term "having the mutation IHH-AAA" as used herein refers to the combination of the mutations I253A (Ile253Ala), H310A (His310Ala) and H435A (His435Ala) in the constant heavy chain region of the IgG1 or IgG4 subclass, and the term "having the mutation HHY-AAA" as used herein refers to the combination of the mutations H310A (His310Ala), H433A (His433Ala) and Y436A (Tyr436Ala) in the constant heavy chain region of the IgG1 or IgG4 subclass, and the term "having the mutation YTE" as used herein refers to the combination of the mutations M252Y (Met252Tyr), S254T (Ser254Thr) and T256E (Thr256Glu) in the constant heavy chain region of the IgG1 or IgG4 subclass, wherein the numbering is according to the Kabat EU index numbering system.

[0246] As used herein, the term "having the mutation P329G LALA" refers to the combination of the mutations L234A (Leu235Ala), L235A (Leu234Ala) and P329G (Pro329Gly) in the constant heavy chain region of the IgG1 subclass, wherein the numbering is according to the Kabat EU index numbering system. As used herein, the term "having the mutation SPLE" refers to the combination of the mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region of the IgG4 subclass, wherein the numbering is according to the Kabat EU index numbering system. As used herein, the term "having the mutation SPLE and P329G" refers to the combination of the mutations S228P (Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly) in the constant heavy chain region of the IgG4 subclass, wherein the numbering is according to the Kabat EU index numbering system.

[0247] II. Compositions and Methods

[0248] In one aspect, the invention is based, in part, on the discovery that introduction of the mutation Y436A into one or both Fc region polypeptides can increase binding of the Fc region to Staphylococcal protein A.

[0249] In one aspect, the present invention is based in part on the discovery that specific mutations or combinations of mutations that affect the binding of the immunoglobulin Fc region to the neonatal Fc-receptor (FcRn) (i.e., reduce or even eliminate the binding of the Fc region to FcRn) do not simultaneously eliminate the binding of the Fc region to Staphylococcal protein A. This has profound consequences for the purification processes that can be used, since, for example, non-specific and species-restricted affinity chromatography materials are required, such as KappaSelect, which only binds to antibodies comprising kappa light chains. Thus, using the combinations of mutations as reported herein, it is possible to simultaneously reduce or even eliminate binding to FcRn while maintaining binding to Staphylococcal protein A.

[0250] In one aspect, the present invention is based in part on the discovery that by using different mutations in the Fc region of each heavy chain of a heterodimeric molecule (e.g., a bispecific antibody), it is possible to provide, on the one hand, reduced or even eliminated binding to FcRn, while on the other hand maintaining the ability to bind to Staphylococcal protein A. This binding to Staphylococcal protein A can be used to separate heterodimeric molecules from homodimeric byproducts. For example, by combining the mutations I253A, H310A, and H435A in one heavy chain Fc region with the mutations H310A, H433A, and Y436A in another heavy chain Fc region using a knob-in-knob approach, one can obtain a heterodimeric Fc region that, on the one hand, does not bind to FcRn (both sets of mutations are silent with respect to human FcRn), but maintains binding to Staphylococcal protein A (the heavy chain Fc region with mutations I253A, H310A, and H435A does not bind to FcRn and does not bind to Staphylococcal protein A, while the heavy chain Fc region with mutations H310A, H433A, and Y436A does not bind to FcRn, but still binds to Staphylococcal protein A). Thus, standard protein A affinity chromatography can be used to remove the homodimeric knob-knob byproduct, since this byproduct no longer binds to Staphylococcal protein A. Therefore, combining the mutations I253A, H310A and H435A in the buckle chain and the mutations H310A, H433A and Y436A in the knot chain by the knot-in-knob method can facilitate the purification / separation of heterodimeric knot-in-knob products from homodimeric knot-knob side products.

[0251] In one aspect, the present invention is based in part on the discovery that antibodies for intravitreal administration that do not bind to FcRn are beneficial because these antibodies can cross the blood-retinal barrier, do not have a significantly prolonged or shortened half-life in the eye, and are rapidly cleared from the blood circulation, resulting in no or very limited systemic side effects outside the eye. The antibodies of the invention can be used, for example, to diagnose or treat ocular vascular diseases.

[0252] The present invention is based, at least in part, on the discovery that antibodies with tailored FcRn binding and, therefore, systemic half-life can be provided by using different mutations in each Fc region polypeptide of a heterodimeric molecule (e.g., a bispecific antibody).

[0253] The combination of mutations I253A, H310A, H435A, or L251D, L314D, L432D, or L251S, L314S, L432S results in loss of protein A binding, while the combination of mutations I253A, H310A, H435A, or H310A, H433A, Y436A, or L251D, L314D, L432D results in loss of binding to the human neonatal Fc receptor.

[0254] The following table presents an exemplary overview of the amino acid residues present in the Fc region that are involved in the interaction or that have been altered to modulate the interaction.

[0255]

[0256]

[0257]

[0258]

[0259] The modifications reported herein, which can be used in combination with the mutation Y436A, alter the binding specificity to one or more Fc receptors, such as human FcRn. At the same time, some mutations that alter binding to human FcRn also alter binding to Staphylococcal protein A. This reduction in binding to Staphylococcal protein A can be attenuated or even overcome by using the additional mutation Y436A.

[0260] In one embodiment, the combination of mutations as reported herein alters or substantially alters the serum half-life of the dimeric polypeptide compared to a corresponding dimeric polypeptide lacking this combination of mutations. In one embodiment, this combination of mutations does not alter or does not substantially alter the binding of the dimeric polypeptide to Staphylococcal protein A compared to a corresponding dimeric polypeptide lacking this combination of mutations.

[0261] A. Neonatal Fc-receptor (FcRn)

[0262] Neonatal Fc-receptor (FcRn) is important for the metabolic fate of IgG class antibodies in vivo. FcRn plays a role in rescuing wild-type IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein composed of two polypeptides: 50kDa major histocompatibility complex-like protein class I (α-FcRn) and 15kDa β2-microglobulin (β2m). FcRn binds to the CH2-CH3 portion of the Fc region of IgG class antibodies with high affinity. The interaction between IgG class antibodies and FcRn depends on pH and occurs in a 1:2 stoichiometry, that is, one IgG antibody molecule can interact with two FcRn molecules through its two heavy chain Fc region polypeptides (see, for example, Huber, AH et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0263] Therefore, the in vitro FcRn binding properties / characteristics of IgG are indicative of its in vivo pharmacokinetic properties in blood circulation.

[0264] Different amino acid residues in the heavy chain CH2 domain and CH3 domain are involved in the interaction between FcRn and the Fc region of IgG class antibodies. The amino acid residues that interact with FcRn are located between approximately EU position 243 and EU position 261, between approximately EU position 275 and EU position 293, between approximately EU position 302 and EU position 319, between approximately EU position 336 and EU position 348, between approximately EU position 367 and EU position 393, at EU position 408, and between approximately EU position 424 and EU position 440. More specifically, the following amino acid residues according to Kabat EU numbering are involved in the interaction between the Fc region and FcRn: F243, P244, P245 P,K246,P247,K248,D249,T250,L251,M252,I253,S254,R255,T256,P257,E25 8,V259,T260,C261,F275,N276,W277,Y278,V279,D280,V282,E283,V284,H285 ,N286,A287,K288,T289,K290,P291,R292,E293,V302,V303,S304,V305,L306, T307,V308,L309,H310,Q311,D312,W313,L314,N315,G316,K317,E318,Y319,I 336,S337,K338,A339,K340,G341,Q342,P343,R344,E345,P346,Q347,V348,C3 67,V369,F372,Y373,P374,S375,D376,I377,A378,V379,E380,W381,E382,S38 3, 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.

[0265] 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, e.g., Kim, JK 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).

[0266] Methods to increase IgG binding to FcRn have been implemented by mutating IgG at various amino acid residues: threonine 250, methionine 252, serine 254, threonine 256, threonine 307, glutamate 380, methionine 428, histidine 433, and asparagine 434 (see Kuo, TT et al., J. Clin. Immunol. 30 (2010) 777-789).

[0267] In some cases, antibodies with reduced half-life in the blood circulation are desirable. For example, a drug administered intravitreally should have a long half-life in the eye and a short half-life in the patient's blood circulation. Such antibodies also have the advantage of increased exposure to disease sites, such as the eye.

[0268] Different mutations that affect FcRn binding and half-life in blood circulation are known. Fc region residues that are critical for mouse Fc region-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF et al., J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering according to Kabat) 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, JK et al., Eur. J. Immunol. 24 (1994) 542-548). Residues I253, H310, and H435 were found to be crucial for the interaction of human Fc with mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819-2855). Through protein-protein interaction studies, Dall'Acqua et al. have described that residues M252Y, S254T, and T256E improve FcRn binding (Dall'Acqua, WF 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, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671), various mutants of residues 248 to 259, 301 to 317, 376 to 382, ​​and 424 to 437 have been reported and studied. The following table lists exemplary mutations and their effects on FcRn binding.

[0269] surface

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276] It has been found that a single mutation on one side of an Fc region polypeptide is sufficient to significantly impair binding. The more mutations introduced into the Fc region, the weaker the binding to FcRn becomes. However, a single asymmetric mutation is insufficient to completely inhibit FcRn binding. Mutations on both sides are required to completely inhibit FcRn binding.

[0277] The table below shows the results of symmetric engineering of the IgG1 Fc region to influence FcRn binding (comparison of mutations and retention time on an FcRn-affinity chromatography column).

[0278] surface

[0279]

[0280] Retention times below 3 minutes correspond to no binding since the material is in the flow-through (void peak).

[0281] The single mutation H310A is the most silent symmetric mutation that abolishes any FcRn binding.

[0282] Symmetrical single mutations I253A and H435A resulted in a relative shift in retention time of 0.3-0.4 minutes, which can generally be considered as undetectable binding.

[0283] The single mutation Y436A resulted in a detectable interaction strength with the FcRn affinity column. Without being bound by this theory, this mutation may affect FcRn-mediated half-life in vitro, which may be different from the zero interaction combination of I253A, H310A and H435A mutations (IHH-AAA mutation).

[0284] The results obtained with symmetrically modified anti-HER2 antibodies are shown in the table below (for reference see WO 2006 / 031370).

[0285] surface

[0286] mutation Retention time [minutes] I253H Not combined M252D Not combined S254D Not combined R255D 41.4 M252H 43.6 K288E 45.2 L309H 45.5 E258H 45.6 T256H 46.0 K290H 46.2 D98E 46.2 wild type 46.3 K317H 46.3 Q311H 46.3 E430H 46.4 T307H 47.0 N434H 52.0

[0287] The effect of introducing asymmetric mutations in the Fc region that affect FcRn binding has been exemplified with bispecific antibodies assembled using knob-in-knob technology (see, e.g., US 7,695,936, US 2003 / 0078385; "knob" mutations: S354C / T366W, "knob" mutations: Y349C / T366S / L368A / Y407V). The effect of asymmetrically introduced mutations on FcRn binding can be readily determined using FcRn affinity chromatography (see, e.g., US 7,695,936, US 2003 / 0078385; "knob" mutations: S354C / T366W, "knob" mutations: Y349C / T366S / L368A / Y407V). Figure 9and the table below). Antibodies that elute later from the FcRn affinity column, i.e., have a longer retention time on the FcRn affinity column, have a longer in vivo half-life, and vice versa.

[0288] surface

[0289]

[0290] The effect of introducing asymmetric mutations that affect FcRn binding in the Fc region has been further exemplified using monospecific anti-IGF-1R antibodies assembled using a knob-in-knob technique to allow for the introduction of asymmetric mutations (see, e.g., US Pat. No. 7,695,936, US 2003 / 0078385; "knob" mutations: S354C / T366W, "knob" mutations: Y349C / T366S / L368A / Y407V). The effect of asymmetrically introduced mutations on FcRn binding can be readily determined using FcRn affinity chromatography (see Table below). Antibodies that elute later from the FcRn affinity column, i.e., with a longer retention time on the FcRn affinity column, have a longer in vivo half-life, and vice versa.

[0291] surface

[0292]

[0293]

[0294] Asymmetric IHH-AAA- and LLL-DDD mutations (LLL-DDD mutation = combination of mutations L251D, L314D and L432D) showed weaker binding than the corresponding parental or wild-type antibody.

[0295] The symmetric HHY-AAA mutation (= combination of mutations H310A, H433A and Y436A) results in an Fc region that no longer binds to human FcRn, while protein A binding is maintained (see Figure 11 、 12 , 13 and 14).

[0296] The effect of introducing asymmetric mutations in the Fc region that affect FcRn binding has been exemplified using monospecific anti-IGF-1R antibodies (IGF-1R), bispecific anti-VEGF / ANG2 antibodies (VEGF / ANG2), and full-length antibodies fused to the C-termini of both heavy chains (fusions), wherein the antibodies were assembled using a knob-in-knob technique to allow for the introduction of asymmetric mutations (see, e.g., US 7,695,936, US 2003 / 0078385; "knob" mutations: S354C / T366W, "knob" mutations: Y349C / T366S / L368A / Y407V). The effects of the 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 Table below).

[0297]

[0298]

[0299]

[0300]

[0301] One aspect as reported herein is an antibody or Fc-region fusion polypeptide comprising a variant human IgG class Fc-region as reported herein.

[0302] When included in an Fc region fusion polypeptide or a full length antibody, the Fc region (dimeric polypeptide) as reported herein confers the above characteristics to the molecule. The fusion partner may 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 clearly defined or tailored for the intended application of the molecule.

[0303] An Fc-region fusion polypeptide may, for example, comprise a variant (human) IgG class Fc-region as reported herein and a receptor protein that binds to a target (including a ligand), such as, for example, a TNFR-Fc-region fusion polypeptide (TNFR = human tumor necrosis factor receptor), or an IL-1R-Fc-region fusion polypeptide (IL-1R = human interleukin-1 receptor), or a VEGFR-Fc-region fusion polypeptide (VEGFR = human vascular endothelial growth factor receptor), or an ANG2R-Fc-region fusion polypeptide (ANG2R = human angiopoietin 2 receptor).

[0304] The Fc-region fusion polypeptide may comprise, for example, a variant (human) IgG class Fc region as reported herein and a target-binding antibody fragment, including, for example, an antibody Fab fragment, a scFv (see, for example, Nat. Biotechnol. 23 (2005) 1126-1136), or a domain antibody (dAb) (see, for example, WO 2004 / 058821, WO 2003 / 002609).

[0305] An Fc-region fusion polypeptide may, for example, comprise a variant (human) human IgG class Fc region as reported herein and a (naturally occurring or artificial) receptor ligand.

[0306] Antibodies, such as full length antibodies or CrossMabs, may comprise a variant (human) human IgG class Fc region as reported herein.

[0307] B. Ocular vascular disease

[0308] Ocular vascular disease is any pathological condition characterized by the altered or unregulated proliferation and invasion of new blood vessels into ocular tissue structures, such as the retina or cornea.

[0309] In one embodiment, the ocular vascular disease is selected from wet age-related macular degeneration (wet AMD), dry age-related macular degeneration (dry AMD), diabetic macular edema (DME), cystoid macular edema (CME), non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoid macular edema, vasculitis (e.g., central retinal vein occlusion), optic disc edema, retinitis, conjunctivitis, uveitis, choroiditis, multifocal choroiditis, ocular histoplasmosis, blepharitis, dry eye (Sjogren's disease), and other ophthalmic diseases in which the ocular disease or disorder is associated with ocular neovascularization, vascular leakage, and / or retinal edema.

[0310] Antibodies comprising the dimeric polypeptides as reported herein are useful for the prevention and treatment of wet AMD, dry AMD, CME, DME, NPDR, PDR, blepharitis, dry eyes and uveitis, and in a preferred embodiment are useful for the prevention and treatment of wet AMD, dry AMD, blepharitis and dry eyes, and in a preferred embodiment are useful for the prevention and treatment of CME, DME, NPDR and PDR, and in a preferred embodiment are useful for the prevention and treatment of blepharitis and dry eyes, especially wet AMD and dry AMD, and also particularly useful for the prevention and treatment of wet AMD.

[0311] In some embodiments, the ocular vascular disease is selected from wet age-related macular degeneration (wet AMD), macular edema, retinal vein occlusion, retinopathy of prematurity, and diabetic retinopathy.

[0312] Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens wear, atopic keratitis, superior limbal keratitis, pterygium keratitis sicca, Sjogren's disease, acne rosacea, phylectenulosis, syphilis, mycobacteria infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoal infection, Kaposi's sarcoma, Mooren ulcer, marginal corneal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener sarcoidosis, scleritis, Steven's Johnson disease, radial keratotomy, and corneal graft rejection.

[0313] Diseases associated with retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusions, arterial occlusions, occlusive disease of the carotid artery, chronic uveitis / vitritis, Mycobacterium tuberculosis infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including macular edema), Eale's disease, Bechet's disease, infections causing retinitis or choroiditis, ocular pseudohistoplasmosis, Best's disease, myopia, optic pits, Stargart's disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

[0314] Other diseases include, but are not limited to, diseases associated with rubeosis iridis (neovascularization of the angle) and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0315] Retinopathy of prematurity (ROP) is an eye disease that strikes premature infants. It is believed to be caused by the chaotic growth of retinal blood vessels, which can lead to scarring and retinal detachment. ROP can be mild and resolve spontaneously, but in severe cases can lead to blindness. Thus, all premature infants are at risk for ROP, and very low birth weight is an additional risk factor. Oxygen toxicity and relative hypoxia can both contribute to the formation of ROP.

[0316] Macular degeneration is a medical disease mainly present in elderly adults, in which the center of the inner lining of the eye (called the macula lutea) appears thinning, atrophy and in some cases bleeding. This can cause central vision loss, which causes not being able to see fine details, read or recognize faces. According to the American Academy of Ophthalmology, it is the leading cause of central vision loss (blindness) in people over 50 years of age in the U.S. today. Although some macular dystrophy that attacks younger individuals is sometimes referred to as macular degeneration, this term is generally referred to as age-related macular degeneration (AMD or ARMD).

[0317] Age-related macular degeneration begins with characteristic yellow deposits in the macula (the central area of ​​the retina that provides fine central vision, called the fovea) between the retinal pigment epithelium and the lower choroid, called drusen. Most people who experience these early changes (called age-related macular degeneration) have good vision. People with drusen can continue to develop into late stage AMD. When drusen are large and numerous and are associated with disorders in the pigment cell layer under the macula, the risk is significantly higher. Large and soft drusen are relevant to increased cholesterol deposits and can respond to cholesterol-lowering drugs or Rheo-surgery.

[0318] Late-stage AMD, which causes significant vision loss, has two forms: dry and wet. Central geographic atrophy, the dry form of late-stage AMD, is caused by atrophy of the retinal pigment epithelium beneath the retina, which causes vision loss due to loss of light receptors (rods and cones) in the central part of the eye. Although no treatment is available for this condition, the National Eye Institute and other institutions have shown that vitamin supplementation combined with high-dose antioxidants, lutein and zeaxanthin, slows the progression of dry macular degeneration and improves vision in some patients.

[0319] Retinitis pigmentosa (RP) is a group of inherited eye diseases. As the symptoms of RP progress, night blindness usually precedes tunnel vision by several years or even decades. Many people with RP do not become legally blind until the age of 40 or 50 and retain some vision throughout their lives. Others become completely blind due to RP, in some cases as early as childhood. The progression of RP is different in each case. RP is a type of inherited retinal dystrophy, a genetic disease in which an abnormality of a group of photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) leads to progressive vision loss. Affected individuals first develop defective dark adaptation or night blindness (nyctalopia), followed by contraction of the peripheral visual field (called tunnel vision) and sometimes loss of central vision later in the course of the disease.

[0320] Macular edema occurs when fluid and protein deposits collect on or below the macula (the yellow central area of ​​the retina), causing it to thicken and swell. The swelling can distort a person's central vision because the macula is close to the center of the retina at the back of the eyeball. This area houses tightly packed cones, which provide sharp, clear central vision so that a person sees forms, colors, and details that are perfectly aligned with their line of sight. Cystoid macular edema is a type of macular edema that involves the formation of cysts.

[0321] C. Purification of Antibodies Using Staphylococcal Protein A Affinity Chromatography Column

[0322] In one aspect, a dimeric polypeptide comprising the following polypeptide is provided

[0323] a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising, in the N-terminal to C-terminal direction, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain,

[0324] wherein the first polypeptide, the second polypeptide or the first and second polypeptides comprise the mutation Y436A (numbering according to the Kabat EU index numbering system).

[0325] This dimeric polypeptide has a property resulting from the mutation: improved binding to Staphylococcal protein A.

[0326] Thus, these antibodies can be purified, i.e. separated from unwanted by-products, by using conventional protein A affinity materials, such as MabSelectSure. It is not necessary to use highly complex but class-restricted affinity materials, such as KappaSelect, which can only be used for antibodies containing kappa subclass light chains. In addition, if the light chain subclass is modified / exchanged, no purification method is required (see respectively). Figure 11 and Figure 12 ).

[0327] One aspect as reported herein is a method for producing a dimeric polypeptide as reported herein, said method comprising the steps of

[0328] a) cultivating mammalian cells comprising one or more nucleic acids encoding the dimeric polypeptide as reported herein,

[0329] b) recovering the dimeric polypeptide from the culture medium, and

[0330] c) purifying the dimeric polypeptide using protein A affinity chromatography and thereby producing the dimeric polypeptide.

[0331] One aspect as reported herein is the use of the mutation Y436A to increase the binding of dimeric Fc-region polypeptides to Protein A.

[0332] It has been found that by introducing the mutation Y436A, the binding to Staphylococcal protein A (SPA) can be increased. It is advantageous if additional mutations that reduce the binding to SPA, such as I253A and H310A or H310A and H435A, are introduced (see Figure 15 ).

[0333] The dimeric polypeptides as reported herein are produced by recombinant means. Therefore, one aspect of the present invention is a nucleic acid encoding a dimeric polypeptide as reported herein and another aspect is a cell comprising nucleic acid encoding a dimeric polypeptide as reported herein. The method for recombinant production is widely known in the art and is included in protein expression in prokaryotic and eukaryotic cells, and subsequent separation of the dimeric polypeptide and usually purification to a pharmaceutically acceptable purity. In order to express the dimeric polypeptide as described above in a host cell, the nucleic acid encoding the corresponding first and second polypeptides is inserted into an expression vector by standard methods. Expression is carried out in a suitable prokaryotic or eukaryotic host cell, such as a Chinese hamster ovary celI, a NS0 cell, a SP2 / 0 cell, a HEK293 cell, a COS cell, a PER.C6 cell, a yeast or an Escherichia coli cell, and the dimeric polypeptide is recovered from the cell (cell after culture supernatant or lysis).

[0334] General methods for the recombinant production of antibodies are well known in the art and are described, for example, in the following review articles: Makrides, SC, Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, RJ, Mol. Biotechnol. 16 (2000) 151-160; Werner, RG, Drug Res. 48 (1998) 870-880.

[0335] Thus one aspect as reported herein is a method for producing a dimeric polypeptide as reported herein, said method comprising the steps

[0336] a) transforming a host cell with one or more vectors comprising a nucleic acid molecule encoding a dimeric polypeptide as reported herein,

[0337] b) cultivating the host cell under conditions that allow synthesis of the dimeric polypeptide, and

[0338] c) recovering the dimeric polypeptide from the culture and thereby producing the dimeric polypeptide.

[0339] In one embodiment, the recovery step under c) comprises the use of an immunoglobulin Fc region-specific capture reagent. In one embodiment, such an Fc region-specific capture reagent is used in a bind-elute mode. Examples of such Fc region-specific capture reagents include affinity chromatography columns based on Staphylococcus protein A, which are based on a high-rigidity agarose base matrix that allows high flow rates and low back pressures on a large scale. These are characterized by a ligand that binds to the dimeric polypeptide, i.e., its Fc region. The ligand is attached to the matrix via a long, hydrophilic spacer arm to make it readily available for binding to the target molecule.

[0340] The dimeric polypeptides as reported herein are appropriately separated from culture medium by conventional immunoglobulin purification methods such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. B cells or hybridoma cells can serve as DNA and RNA sources encoding dimeric polypeptides. Conventional methods are used to easily separate and sequence the DNA and RNA encoding monoclonal antibodies. Once separated, this DNA can be inserted into an expression vector, which is then transfected into a host cell (such as HEK293 cells, CHO cells or myeloma cells) that does not produce the dimeric polypeptides, to achieve synthesis of the recombinant monoclonal dimeric polypeptides in the host cell.

[0341] Antibody purification is performed by standard techniques, including alkaline / SDS treatment, CsCl zoning, column chromatography, agarose gel electrophoresis, and other techniques well known in the art for the purpose of eliminating cellular components or other impurities (e.g., other cellular nucleic acids or proteins) (see Ausubel, F. et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Different methods are well established and widely used for protein purification, such as affinity chromatography using microproteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (aminoethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoresis (e.g. gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102).

[0342] One aspect of the present invention is a pharmaceutical formulation comprising a dimeric polypeptide or antibody as reported herein. Another aspect of the present invention is the use of a dimeric polypeptide or antibody as reported herein for the manufacture of a pharmaceutical formulation. Yet another aspect of the present invention is a method for the manufacture of a pharmaceutical formulation comprising a dimeric polypeptide or antibody as reported herein. In another aspect, the present invention provides a formulation, e.g., a pharmaceutical formulation, comprising a dimeric polypeptide or antibody as reported herein formulated with a pharmaceutical carrier.

[0343] The formulations as reported herein can be administered by a variety of methods known in the art. It will be understood by those skilled in the art that the route and / or mode of administration will vary depending on the desired outcome. In order to administer the compounds of the present invention by certain routes of administration, it may be necessary to coat the mixture with a material to prevent its inactivation or to co-administer it with such a material. For example, the compound can be administered to the subject in a suitable carrier such as a liposome or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions on site. The use of such media and agents for pharmaceutically active substances is known in the art.

[0344] Many possible modes of delivery can be used, including but not limited to intraocular application or topical application. In one embodiment, application is intraocular application and includes, but is not limited to, subconjunctival injection, intracranial injection, injection into the anterior chamber through the temporal limbus, intrastromal injection, intracorneal injection, subretinal injection, aqueous humor injection, subfascial injection or sustained delivery device, intravitreal injection (e.g., anterior, middle or posterior vitreous injection). In one embodiment, application is topical and includes but is not limited to eye drops applied to the cornea.

[0345] In one embodiment, the dimeric polypeptide as reported herein or the pharmaceutical formulation as reported herein is administered by intravitreal application, for example by intravitreal injection. This can be performed 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).

[0346] In some embodiments, the therapeutic kit of the present invention may contain one or more doses of a dimeric polypeptide as reported herein in a pharmaceutical formulation as described herein, a suitable device for intravitreal injection of the pharmaceutical formulation, and instructions detailing the applicable subjects and the protocol for implementing the injection. In these embodiments, the formulation is generally administered to a subject in need of treatment by intravitreal injection. This can be performed according to standard procedures 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.

[0347] The formulations may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the above disinfection methods and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the formulations. In addition, prolonged absorption of injectable drug forms can be achieved by the inclusion of substances that delay absorption, such as aluminum monostearate and gelatin.

[0348] Regardless of the route of administration chosen, the compounds as reported herein (which may be used in a suitable hydrated form) and / or the pharmaceutical formulations as reported herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0349] Actual dosage levels of the active ingredients in the pharmaceutical formulations as reported herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response with respect to the particular patient, composition, and mode of administration, while being non-toxic to the patient. The dosage level selected will depend upon a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the compound being administered, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and other factors well known in the medical art.

[0350] The formulation must be sterile and fluid to the extent that it can be delivered by syringe.In addition to water, the carrier is preferably an isotonic buffered saline solution in a preferred embodiment.

[0351] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.

[0352] Preparation can comprise the ophthalmic depot preparation comprising active substance for subconjunctival administration.Ophthalmic depot preparation comprises the microparticle of substantially pure active substance (for example, as the dimeric polypeptide reported herein).The microparticle comprising the dimeric polypeptide reported herein can be embedded in biocompatible pharmaceutically acceptable polymer or lipid encapsulation agent.Depot preparation can be adapted to discharge whole or substantially all active substances over a prolonged period of time.If present, polymer or lipid matrix can be adapted to fully degrade, so that after releasing whole or substantially all active substances, transport out from the application site.Depot preparation can be a liquid preparation, comprising pharmaceutically acceptable polymer and dissolved or dispersed active substance.Once injected, polymer forms a depot at the injection site, for example, formed by gelation or precipitation process.

[0353] Another aspect of the invention is a dimeric polypeptide or antibody as reported herein for use in the treatment of an ocular vascular disease.

[0354] One embodiment of the invention is a dimeric polypeptide or antibody as reported herein for use in the treatment of ocular vascular diseases.

[0355] Another aspect of the present invention is a pharmaceutical formulation for treating ocular vascular diseases.

[0356] Another aspect of the invention is the use of a dimeric polypeptide or antibody as reported herein for the manufacture of a medicament for the treatment of ocular vascular diseases.

[0357] Another aspect of the invention is a method of treating a patient suffering from an ocular vascular disease by administering to a patient in need of such treatment a dimeric polypeptide or antibody as reported herein.

[0358] It is expressly stated here that the term "comprising" as used herein includes the term "consisting of. Thus, all aspects and embodiments containing the term "comprising" are likewise disclosed using the term "consisting of.

[0359] D. Modification

[0360] In yet another aspect, the dimeric polypeptide according to any of the above embodiments may incorporate any of the features described in Sections 1-6 below, alone or in combination:

[0361] 1. Antibody affinity

[0362] In one embodiment, using The surface plasmon resonance assay measures Kd. For example, using -2000 or -3000 (GE Healthcare Inc., Piscataway, NJ) assay was performed at 25°C using an immobilized binding partner CM5 chip with approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The binding partner was diluted to 5 μg / mL (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then loaded at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of the coupled binding partner. After injection of the binding partner, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, a 5% polysorbate 20 (TWEEN-20) containing 0.05% polysorbate 20 was injected at a flow rate of approximately 25 μL / min at 25°C. TM ) surfactant-free PBS (PBST) containing 2-fold serial dilutions (0.78 nM to 500 nM) of the fusion polypeptide or antibody dimer polypeptide. Using a simple one-to-one Langmuir binding model ( The association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) was calculated as k off / k on If the association rate is greater than 10 as shown by the surface plasmon resonance assay above, the 6 M -1 s -1 If the association rate is quantified, the fluorescence quenching technique can be used to determine the association rate, wherein the fluorescence quenching technique is used in a spectrometer such as a spectrophotometer equipped with stop-flow (Aviv Instruments) or an 8000-series SLM-AMINCO with a stirred cuvette. TM The fluorescence quenching technique measures 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 increasing concentrations of antigen as measured in a spectrophotometer (ThermoSpectronic).

[0363] 2. Chimeric and humanized antibodies

[0364] In certain embodiments, the dimeric polypeptide as reported herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in US 4,816,567; and Morrison, SL 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 another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed relative to the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0365] In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies comprise HVR (e.g., CDR) (or portions thereof) derived from non-human antibodies and FR (or portions thereof) derived from one or more variable domains of human antibody sequences. Humanized antibodies optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (e.g., antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0366] Humanized antibodies and methods for making them 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, SV et al., Methods 36 (2005) 25-34 (describing specificity determining region (SDR) grafting); Padlan, EA, Mol. Immunol. 28 (1991) 489-498 (describing "surfacing"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); Osbourn, J. et al., Methods 36 (2005) 61-68; and further described in Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (describing the "guided selection" approach for FR shuffling).

[0367] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (e.g., see Sims, MJ et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from the consensus sequence of the light chain variable region or the heavy chain variable region of a particular subgroup of human antibodies (e.g., see Carter, P. et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Presta, LG et al., J. Immunol. 151 (1993) 2623-2632); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (1996) 22611-22618).

[0368] 3. Human Antibodies

[0369] In certain embodiments, the dimeric polypeptides as reported herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk, MA and van de Winkel, JG, Curr. Opin. Pharmacol. 5 (2001) 368-374 and Lonberg, N., Curr. Opin. Immunol. 20 (2008) 450-459.

[0370] Human antibodies can be prepared by administering an immunogen to a transgenic animal, wherein the transgenic animal has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigen attack. Such animals generally contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci or exist extrachromosomally or randomly integrated into the animal chromosomes. In such transgenic mice, the 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. For example, see also the description of XENOMOUSE TM US 6,075,181 and US 6,150,584 of the technology; description US5,770,429 of the technology; describes KM US 7,041,870 and describes the technology of Veloci Human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0371] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (For example, see Kozbor, D., J. Immunol. 133 (1984) 3001-3005; Brodeur, BR 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 produced by human B-cell hybridoma technology are also 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 hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers, HP and Brandlein, S., Histology and Histopathology 20 (2005) 927-937 and Vollmers, HP and Brandlein, S., Methods and Findings in Experimental and Clinical Pharmacology 27 (2005) 185-191.

[0372] It is also possible to produce human antibodies by separating the Fv clone variable domain sequences selected from the phage display library derived from people. This type of variable domain sequence can then be combined with the required human constant domains. The following description is used to select the technology of human antibodies from the antibody library.

[0373] 4. Library-derived Antibodies

[0374] In certain embodiments, the dimeric polypeptides as reported herein are library-derived antibodies. Library-derived antibodies can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom, HR 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, JD et al., J. Mol. Biol. 222 (1992) 581-597; Marks, JD and Bradbury, A., Methods in Molecular Biology 248 (2003) 161-175; Sidhu, SS et al., J. Mol. Biol. 338 (2004) 299-310; Lee, CV et al., J. Mol. Biol. 340 (2004) 1073-1093; Fellouse, FA, Proc. Natl. Acad. Sci. USA 101 (2004) 12467-12472 and Lee, CV et al., J. Immunol. Methods 284 (2004) 119-132.

[0375] In certain phage display methods, the VH gene library and the VL gene library are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter, G. et al., Ann. Rev. Immunol. 12 (1994) 433-455. Phage generally display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immune sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. 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 additionally against a wide range of self-antigens without any immunization, as described in Griffiths, AD et al., EMBO J. 12 (1993) 725-734. Finally, natural libraries can also be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve in vitro rearrangement, as described in Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388. Patent publications describing human antibody phage libraries include, for example, US 5,750,373 and US 2005 / 0079574, US 2005 / 0119455, US 2005 / 0266000, US 2007 / 0117126, US 2007 / 0160598, US 2007 / 0237764, US 2007 / 0292936 and US 2009 / 0002360.

[0376] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0377] 5. Multispecific Antibodies

[0378] In certain embodiments, the dimeric polypeptides as reported herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity for at least two different sites. In certain embodiments, one binding specificity is for a first antigen and another binding specificity is for a different second antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of the same antigen. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing at least one antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0379] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, WO 93 / 08829 and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-knob" engineering (see, for example, US 5,731,168). Multispecific antibodies can also be produced by engineering electrostatic steering effects to produce antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking 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 produce bispecific antibodies (e.g., see Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology to produce bispecific antibody fragments (e.g., see Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M. et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0380] Also included herein are engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies" (see, eg, US 2006 / 0025576).

[0381] Antibodies or fragments herein also include "dual function Fab" or "DAF" (see, eg, US 2008 / 0069820).

[0382] The antibodies or fragments herein also include the 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.

[0383] 6. Antibody variants

[0384] In certain embodiments, the dimeric polypeptides as reported herein are antibodies. In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. 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 the amino acid sequence and / or replacing residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can be produced to obtain the final construct, as long as the final construct has desired characteristics, such as antigen binding.

[0385] a) Substitution variants, insertion variants, and deletion variants

[0386] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in the table below. More significant changes are described below in the table below under the heading "Exemplary Substitutions" and with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the target antibody and the product can be screened for desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0387] surface

[0388]

[0389]

[0390] Amino acids can be grouped according to common side chain properties:

[0391] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu; Ile;

[0392] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

[0393] (3) Acidic: Asp, Glu;

[0394] (4) Basic: His, Lys, Arg;

[0395] (5) Residues that affect chain direction: Gly, Pro;

[0396] (6) Aromatic: Trp, Tyr, Phe.

[0397] Non-conservative substitutions will exchange a member of one of these classes for a member of another class.

[0398] A substitution variant type involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) and / or will have certain biological properties that are substantially retained relative to the parent antibody in terms of certain biological properties (e.g., increased affinity, reduced immunogenicity). Exemplary substitution variants are affinity matured antibodies, which can be conveniently produced, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0399] Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196) and / or residues that contact antigen, and the resulting variant VH or VL is tested for binding affinity. This has been described, for example, in Hoogenboom, HR et al., cited in Methods in Molecular Biology 178 (2002) 1-37 describes the construction of a secondary library and the reselection thereof to achieve affinity maturation. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then produced. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity relates to a scheme guided by HVR, in which several HVR residues (e.g., 4-6 residues at a time) are randomly grouped. HVR residues involved in antigen binding can be particularly identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0400] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes that do not substantially reduce binding affinity may be made in HVRs (e.g., conservative substitutions as provided herein). Such changes may, for example, be outside the antigen contact residues in HVRs. In certain 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.

[0401] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham, BC and Wells, JA, Science 244 (1989) 1081-1085. In this method, a residue or target residue group (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. This type of contact residue and adjacent residue can be targeted or eliminated as replacement candidates. Variants can be screened to determine whether they contain desired properties.

[0402] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., an enzyme for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0403] b) Glycosylation variants

[0404] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. The generation or deletion of glycosylation sites in an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0405] In the case where the antibody comprises an Fc region, the sugar connected thereto can be changed. Natural antibodies produced by mammalian cells generally comprise branched biantennary oligosaccharides, which are generally linked to Asn297 of the CH2 domain of the Fc region by means of N-links. See, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. Oligosaccharides can include various sugars, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose connected to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with improved certain properties.

[0406] In one embodiment, an antibody variant having a sugar structure is provided, wherein the sugar structure lacks fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined in the following manner: relative to the sum of all sugar structures (e.g., complex structures, hybrid structures, and high mannose structures) connected to Asn297 as measured by MALDI-TOF mass spectrometry, the average amount of fucose at Asn297 inside the sugar chain is calculated, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 can also be located near ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, for example, US 2003 / 0157108; US 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-lacking" 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 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 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 defucosylated antibodies include Lec13 CHO cells deficient 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 CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (e.g., see Yamane-Ohnukii, N et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y. et al., Biotechnol. Bioeng. 94 (2006) 680-688; and WO 2003 / 085107).

[0407] Antibody variants can also be provided with bisecting oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the antibody Fc region is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; US 6,602,684; and US 2005 / 0123546. Antibody variants are also provided in which at least one galactose residue in the oligosaccharide is attached to the Fc region. Such antibody variants can have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964 and WO 1999 / 22764.

[0408] c) Fc region variants

[0409] In certain embodiments, one or more additional amino acid modifications may be introduced into a dimeric polypeptide as reported herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution / mutation) at one or more amino acid positions.

[0410] In certain embodiments, the present invention contemplates dimeric polypeptides that possess some, but not all, effector functions, making them advantageous candidates for applications where the in vivo half-life of the dimeric polypeptide is important and certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. 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 dimeric polypeptide antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, 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 evaluating ADCC activity of a molecule of interest are described in US 5,500,362 (see, e.g., 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); US 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. C1q binding assays can also be performed to confirm that the dimeric polypeptide cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 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).

[0411] Dimeric polypeptides with reduced effector function include those with substitutions of one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( US 6,737,056 ). Such Fc region variants include Fc regions with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc region mutants in which residues 265 and 297 are substituted with alanine ( US 7,332,581 ).

[0412] Certain antibody variants with improved or diminished FcR binding have been described (see, eg, US 6,737,056; WO 2004 / 056312, and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0413] In certain embodiments, the dimeric polypeptide variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 of the Fc region) (EU numbering of residues).

[0414] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or impaired) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0415] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), are described in US 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein, wherein the substitutions improve binding of the Fc region to FcRn. Such Fc region variants include those having a substitution 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 (e.g., substitution of Fc region residue 434) ( US 7,371,826 ).

[0416] See also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821 and WO 94 / 29351 for other examples of Fc region variants.

[0417] d) Cysteine ​​engineered antibody variants

[0418] In certain embodiments, it may be desirable to produce a cysteine ​​engineered dimeric polypeptide, for example, similar to a "thio MAb," wherein one or more residues of the antibody are replaced with cysteine ​​residues. In a specific embodiment, the replaced residues appear at accessible sites of the dimeric polypeptide. By replacing these residues with cysteine, reactive sulfhydryl groups are placed at accessible sites of the dimeric polypeptide and can be used to conjugate the dimeric polypeptide to other moieties, such as drug moieties or linker-drug moieties to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered dimeric polypeptides can be produced, for example, as described in US 7,521,541.

[0419] e) Derivatives

[0420] In certain embodiments, the dimeric polypeptides as reported herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the dimeric polypeptides 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, polyvinyl pyrrolidone, poly 1,3-dioxolane, poly 1,3,6-trifluoromethyl ether, polyvinyl alcohol, polyvinyl pyrrolidone ... In some embodiments, the present invention provides a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention. The dimeric polypeptide of the present invention can be a kind of dimeric polypeptide of the present invention.

[0421] In another embodiment, a conjugate of a dimeric polypeptide as reported herein and a non-protein portion that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein portion is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can have any wavelength, and includes but is not limited to a wavelength that does not harm ordinary cells, but heats the non-protein portion to a temperature that kills cells adjacent to the dimeric polypeptide-non-protein portion.

[0422] f) Heterodimerization

[0423] There are several approaches to modify CH3 to enhance heterodimerization, which are fully described in, for example, 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, WO 2013096291. Generally, in all such approaches, the first and second CH3 domains are engineered in a complementary manner such that each CH3 domain (or heavy chain comprising it) cannot homodimerize with itself, but is forced to heterodimerize with the other engineered complementary CH3 domain (such that the first and second CH3 domains heterodimerize and no homodimers are formed between the two first CH3 domains or between the two second CH3 domains). These various approaches to improving heavy chain heterodimerization are contemplated as different alternatives for combining with heavy-light chain modifications in the multispecific antibodies of the present invention (VH and VL exchange / replacement in one binding arm and introduction of oppositely charged amino acid substitutions at the CH1 / CL interface) to reduce Bence-Jones-type side products of light chain mispairing.

[0424] In a preferred embodiment of the present invention (in which case the multispecific antibody comprises a CH3 domain in the heavy chain), the CH3 domain of the multispecific antibody of the present invention can be altered using the "knob-in-knob" technique, described in detail with several examples in, for example, WO 96 / 027011; Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681; and WO 98 / 050431. In this approach, the interaction surfaces of the two CH3 domains are altered to increase 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 "knob." The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0425] Thus, in one embodiment of the present invention, the multispecific antibody (comprising a CH3 domain in each heavy chain and) is characterized in that

[0426] The first CH3 domain of the first heavy chain of the antibody under a) and the second CH3 domain of the second heavy chain of the antibody under b) each meet at an interface that comprises the initial interface between the antibody CH3 domains.

[0427] wherein the interface is altered to promote formation of multispecific antibodies, wherein the alteration is characterized by

[0428] i) altering the CH3 domain of one heavy chain,

[0429] Thus, within the multispecific antibody, the initial interface of the CH3 domain of one heavy chain meets the initial interface of the CH3 domain of the other heavy chain,

[0430] The amino acid residue is replaced with one having a larger side chain bulk, thereby generating a protrusion within the interface of the CH3 domains of one heavy chain that can fit into a cavity within the interface of the CH3 domains of the other heavy chain.

[0431] and

[0432] ii) altering the CH3 domain of the other heavy chain in this way,

[0433] Thus, within the multispecific antibody, the initial interface of the second CH3 domain meets the initial interface of the first CH3 domain,

[0434] The amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain, within which the protrusion within the interface of the first CH3 domain can be arranged.

[0435] Preferably, the amino acid residue with a larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0436] Preferably, the amino acid residue with a smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V).

[0437] In one aspect of the invention, the two CH3 domains are further altered by introducing cysteine ​​(C) as an amino acid in the corresponding position of each CH3 domain such that a disulfide bond can be formed between the two CH3 domains.

[0438] In a preferred embodiment, the multispecific antibody comprises an amino acid T366W mutation in the first CH3 domain of the "knot" and amino acid T366S, L368A, Y407V mutations in the second CH3 domain of the "buckle." Additional interchain disulfide bonds between CH3 domains can also be used (Merchant, AM et al., Nature Biotech 16 (1998) 677-681), for example by introducing an amino acid Y349C mutation into the CH3 domain of the "buckle" and an amino acid E356C mutation or an amino acid S354C mutation into the CH3 domain of the "knot."

[0439] In a preferred embodiment, the multispecific antibody (which comprises a CH3 domain in each heavy chain) comprises amino acid S354C, T366W mutations in one of the two CH3 domains and amino acid Y349C, T366S, L368A, Y407V mutations 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).

[0440] Other techniques for modifying CH3 to enhance heterodimerization are contemplated 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, WO 2013 / 096291.

[0441] In one embodiment, the heterodimerization scheme described in EP 1 870 459A1 can be used alternatively. This method is based on the introduction of oppositely charged charged amino acid substitutions / mutations at specific amino acid positions in the CH3 / CH3 domain interface between the two heavy chains. A preferred embodiment of the multispecific antibody is the amino acid R409D, K370E mutation in the first CH3 domain (of the multispecific antibody) and the amino acid D399K, E357K mutation in the second CH3 domain of the multispecific antibody (according to Kabat numbering).

[0442] In another embodiment, the multispecific antibody comprises an amino acid T366W mutation in the CH3 domain of the "knob" and amino acid T366S, L368A, Y407V mutations in the CH3 domain of the "buckle" and additionally comprises amino acid R409D, K370E mutations in the CH3 domain of the "knob" and amino acid D399K, E357K mutations in the CH3 domain of the "buckle".

[0443] In another embodiment, the multispecific antibody comprises amino acid S354C, T366W mutations in one of the two CH3 domains and amino acid Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains, or the multispecific antibody comprises amino acid Y349C, T366W mutations in one of the two CH3 domains and amino acid S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains and additionally amino acid R409D, K370E mutations in the CH3 domain of the "knob" and amino acid D399K, E357K mutations in the CH3 domain of the "buckle."

[0444] In one embodiment, the heterodimerization scheme described in WO 2013 / 157953 can alternatively be used. In one embodiment, the first CH3 domain comprises an amino acid T366K mutation and the second CH3 domain polypeptide comprises an amino acid L351D mutation. In another embodiment, the first CH3 domain comprises an amino acid L351K mutation. In another embodiment, the second CH3 domain comprises an additional amino acid mutation selected from Y349E, Y349D, and L368E (preferably L368E).

[0445] In one embodiment, the heterodimerization scheme described in WO 2012 / 058768 may alternatively be used.In one embodiment, the first CH3 domain comprises amino acid L351Y, Y407A mutations and the second CH3 domain comprises amino acid T366A, K409F mutations. In a further embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390 or K392, e.g., selected 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 another embodiment, the first CH3 domain comprises amino acid mutations L351Y and Y407A and the second CH3 domain comprises amino acid mutations T366V and K409F. In another embodiment, the first CH3 domain comprises amino acid mutations Y407A and the second CH3 domain comprises amino acid mutations T366A and K409F. In another embodiment, the second CH3 domain comprises further amino acid mutations K392E, T411E, D399R, and S400R.

[0446] In one embodiment, the heterodimerization scheme described in WO 2011 / 143545 may alternatively be used, for example with amino acid modifications at positions selected from 368 and 409.

[0447] In one embodiment, the heterodimerization scheme described in WO 2011 / 090762 can alternatively be used, which also uses the knot-in-knot technology described above. In one embodiment, the first CH3 domain comprises the amino acid T366W mutation and the second CH3 domain comprises the amino acid Y407A mutation. In one embodiment, the first CH3 domain comprises the amino acid T366Y mutation and the second CH3 domain comprises the amino acid Y407T mutation.

[0448] In one embodiment, the multispecific antibody is of the IgG2 isotype and may alternatively use the heterodimerization approach described in WO 2010 / 129304.

[0449] In one embodiment, the heterodimerization scheme described in WO 2009 / 089004 can be used alternatively. In one embodiment, the first CH3 domain comprises an amino acid replacement 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 an amino acid replacement 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 another embodiment, the first CH3 domain further comprises an amino acid replacement 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 with a negatively charged amino acid, such as glutamic acid (E), or aspartic acid (D).

[0450] In one embodiment, the heterodimerization scheme described in WO 2007 / 147901 may alternatively be used.In one embodiment, the first CH3 domain comprises amino acid K253E, D282K and K322D mutations and the second CH3 domain comprises amino acid D239K, E240K and K292D mutations.

[0451] In one embodiment, the heterodimerization approach described in WO 2007 / 110205 may alternatively be used.

[0452] E. Recombinant Methods and Compositions

[0453] Antibodies can be produced, for example, using recombinant methods and compositions, as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding a dimeric polypeptide as reported herein is provided. This nucleic acid can encode the amino acid sequence of a first polypeptide constituting the dimeric polypeptide and / or the amino acid sequence of a second polypeptide constituting the dimeric polypeptide. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In such an embodiment, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding the amino acid sequence of a first polypeptide constituting the dimeric polypeptide and the amino acid sequence of a second polypeptide constituting the dimeric polypeptide, or (2) a first vector comprising a nucleic acid encoding the amino acid sequence of a first polypeptide constituting the dimeric polypeptide, and a second vector comprising a nucleic acid encoding the amino acid sequence of a second polypeptide constituting the dimeric polypeptide. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for producing a dimeric polypeptide as reported herein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the dimeric polypeptide as provided above under conditions suitable for expression of the dimeric polypeptide, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0454] In order to recombinantly produce the dimeric polypeptides as reported herein, nucleic acids encoding the dimeric polypeptides (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding variant Fc region polypeptides and antibody heavy and light chains).

[0455] Suitable host cells for cloning or expressing vectors encoding dimeric polypeptides include prokaryotes or eukaryotic cells as described herein. For example, dimeric polypeptides can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, for example, see US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, KA, cited in Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli). After expression, the dimeric polypeptide can be separated from the bacterial cell paste in a soluble fraction and the polypeptide can be further purified.

[0456] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning hosts or expression hosts for vectors encoding dimeric polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of dimeric polypeptides with partially or completely human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0457] Suitable host cells for expressing glycosylated dimer polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0458] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing plant antibodies produced in transgenic plants). TM technology).

[0459] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension culture can be useful. Other examples of mammalian host cell lines are monkey kidney CV1 line (COS-7) transformed by SV40; human embryonic kidney line (such as HEK293 or 293 cells described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse supporting cells (such as TM4 cells described in, for example, Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT 060562); such as described in, for example, Mather, JP et al., Annals of TRI cells as described in NY Acad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other mammalian host cell lines that can be used 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, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (Ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0460] F. Combination therapy

[0461] In certain embodiments, a dimeric polypeptide as reported herein or a pharmaceutical formulation as reported herein is administered alone (without additional therapeutic agents) to treat one or more ocular vascular diseases as described herein.

[0462] In other embodiments, the dimeric polypeptide antibody or pharmaceutical formulation as reported herein is administered in combination with one or more additional therapeutic agents and methods to treat one or more ocular vascular diseases described herein.

[0463] In other embodiments, the dimeric polypeptide or pharmaceutical formulation as reported herein is formulated in combination with one or more additional therapeutic agents and administered to treat one or more ocular vascular diseases as described herein.

[0464] In certain embodiments, the combination therapies provided herein comprise the sequential administration of a dimeric polypeptide or pharmaceutical formulation as reported herein and one or more additional therapeutic agents to treat one or more ocular vascular diseases as described herein.

[0465] Additional therapeutic agents include, but are not limited to, tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (anti-VEGF PEGylated aptamer), sequamide, RETAANE™ (anecortave acetate depot suspension; Alcon, Inc.), Compretin A4 prodrug (CA4P), MACUGEN™, MIFEPREX™ (mifepristone-ru486), subfascial triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide, Prinomastat (AG3340 - synthetic matrix metalloproteinase inhibitor, Pfizer), fluclosan (including fluocinolone acetonide intraocular implant, Bausch & Lomb / Control Delivery Systems), VEGFR inhibitors (Sugen), VEGF-Trap (Regeneron / Aventis), VEGF receptor tyrosine kinase inhibitors such as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787) and SU11248 (sunitinib), linomide and inhibitors of integrin vβ3 function and angiostatin.

[0466] Other drug therapies that can be used in combination with the dimeric polypeptides or pharmaceutical formulations as reported herein include, but are not limited to, VISUDYNE™ in combination with non-thermal laser, PKC412, Endovion (NeuroSearch A / S), neurotrophic factors including, by way of example, glial-derived neurotrophic factor and ciliary neurotrophic factor, diatazem, dorzolamide, Phototrop, 9-cis-retinal, ophthalmic drugs including phospholine iodide or icosate or carbonic anhydrase inhibitors (including Echo Therapies), AE-941 (AEterna Laboratories, Inc.), Sirna-027 (Sima Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals / Gilead Sciences), neurotrophic factors (including, by way of example only, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (e.g., by EntreMed, Inc.(Genentech), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch / Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-β2 (Genzyme / Celtrix), tyrosine kinase inhibitors (Allergan, SUGEN, Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Biotech), and 1,2-dimethylthiocyanate (DMSO). Pharmaceuticals), cyclosporine A, limited retinal transposition, photodynamic therapy (including, by way of example only, receptor-targeted PDT, Bristol-Myers Squibb, Co.; porfil sodium for injection combined with PDT; verteporfin, QLT Inc.; rotaporfin combined with PDT, Miravent Medical Technologies; talaporfin sodium combined with PDT, Nippon Petroleum; motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, by way of example, products tested by Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laser photocoagulation, occult laser surgery, macular hole surgery, macular translocation surgery, implantable microscopes, Phi-Motion angiography (also known as Micro-Laser Therapy and Feeder Vessel Treatment), proton beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, submacular surgery, transpupillary thermal therapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal purification therapy (also known as membrane differential filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy directed against the hypoxia response element, Oxford Biomedica; Lentipak, Genetix; PDEF Gene Therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, Cell Genesys, Inc.), and acupuncture.

[0467] Any anti-angiogenic agent may be used in combination with the dimeric polypeptides or pharmaceutical formulations as reported herein, including but not limited to those listed by Carmeliet and Jain (Nature 407 (2000) 249-257). In certain embodiments, the anti-angiogenic agent is another VEGF antagonist or VEGF receptor antagonist such as a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a low molecular weight inhibitor of VEGFR tyrosine kinase, and any combination thereof, and these include anti-VEGF aptamers (e.g., pegaptanib), soluble recombinant decoy receptors (e.g., VEGFTrap). In certain embodiments, anti-angiogenic drugs include corticosteroids, angiostatic steroids, anecortave acetate, angiostatin, endostatin, small interfering RNA that reduces the expression of VEGFR or VEGF ligands, VEGFR followed by tyrosine kinase inhibitor blockade, MMP inhibitors, IGFBP3, SDF-1 blocking agents, PEDF, γ-secretase, delta-like ligand 4, integrin antagonists, HIF-1α blockade, protein kinase creatine kinase 2 blockade, and the use of vascular endothelial cadherin (CD-144) to inhibit stem cells (i.e., endothelial progenitor cells) homing to new vascularization sites and stromal-derived factor (SDF)-1 antibodies. Small molecule RTK inhibitors targeting VEGF receptors can also be used, including PTK787. Drugs that are not necessarily VEGF compounds that are active for neovascularization and include anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolismus, cyclospohne, anti-TNF drugs, anti-complement drugs and non-steroidal anti-inflammatory drugs. Drugs that have a neuroprotective effect and can potentially reduce the progression of dry macular degeneration, such as the class of drugs known as "neurosteroids", can also be used. These include drugs such as dehydroepiandrosterone (DHEA) (trade names: Prastera (R) and Fidelin (R)), dehydroepiandrosterone sulfate and pregnenolone sulfate. Any AMD (age-related macular degeneration) therapeutic agent can be used in combination with the dimeric polypeptides or pharmaceutical preparations as reported herein, including but not limited to verteporfin in combination with PDT, pegaptanib sodium, zinc or antioxidants alone or in any combination.

[0468] G Pharmaceutical Preparations

[0469] Pharmaceutical preparations of the dimeric polypeptides as reported herein in the form of lyophilized formulations or aqueous solutions are prepared by mixing such dimers having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. (ed.) (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides. ; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinyl pyrrolidone); 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; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers 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 US 2005 / 0260186 and US 2006 / 0104968. In one aspect, sHASEGPs are combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0470] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0471] The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in amounts effective for the intended purpose.

[0472] The active ingredient can be embedded in microcapsules (e.g., hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared, for example, by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0473] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles (eg, films, or microcapsules).

[0474] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0475] H. Methods of Treatment and Compositions

[0476] Any dimeric polypeptide as reported herein may be used in therapeutic methods.

[0477] In one aspect, a dimeric polypeptide as reported herein is provided for use as a medicament. In other aspects, a dimeric polypeptide for use in the treatment of an ocular vascular disease is provided. In certain embodiments, a dimeric polypeptide for use in a method of treatment is provided. In certain embodiments, the present invention provides a dimeric polypeptide for use in a method of treating an individual suffering from an ocular vascular disease, the method comprising administering to the individual an effective amount of a dimeric polypeptide as reported herein. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., a therapeutic agent as described in Section D above. In other embodiments, the present invention provides a dimeric polypeptide for use in inhibiting angiogenesis in the eye. In certain embodiments, the present invention provides a dimeric polypeptide for use in a method of inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of a dimeric polypeptide to inhibit angiogenesis. The "individual" according to any of the above embodiments is, in a preferred embodiment, a human.

[0478] In yet another aspect, the present invention provides a use of a dimeric polypeptide in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in the treatment of an ocular vascular disease. In yet another embodiment, the medicament is for use in a method for treating an ocular vascular disease, the method comprising administering an effective amount of the medicament to an individual suffering from the ocular vascular disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., a therapeutic agent as described above. In yet another embodiment, the medicament is for use in a method for inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of an angiogenesis-inhibiting agent. The "individual" according to any of the above embodiments may be a human.

[0479] In yet another aspect, the present invention provides a method for treating an ocular vascular disease. In one embodiment, the method comprises administering to an individual suffering from such an ocular vascular disease an effective amount of a dimeric polypeptide as reported herein. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent as described below. The "individual" according to any of the above embodiments may be a human.

[0480] In yet another aspect, the present invention provides a method for inhibiting angiogenesis in the eye of a subject. In one embodiment, the method comprises administering to the subject an effective amount of a dimeric polypeptide as reported herein to inhibit angiogenesis. In one embodiment, the "subject" is a human.

[0481] In yet another aspect, the invention provides a pharmaceutical formulation comprising any dimeric polypeptide as reported herein, e.g., for use in any of the aforementioned methods of treatment. In one embodiment, the pharmaceutical formulation comprises any dimeric polypeptide as reported herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any dimeric polypeptide as reported herein and at least one additional therapeutic agent, e.g., as described below.

[0482] The dimeric polypeptides as reported herein can be used alone or in combination with other drugs in therapy.For example, the dimeric polypeptides as reported herein can be co-administered with at least one additional therapeutic agent.

[0483] As reported herein, the dimeric polypeptide (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal and (if local treatment is required) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, rapid bolus administration and pulse infusion.

[0484] As reported herein, the dimeric polypeptides will be formulated, administered, and used in a manner consistent with good medical practice. Factors to be considered in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of drug delivery, the method of administration, the administration plan, and other factors known to medical practitioners. The dimeric polypeptide does not need to be formulated with one or more drugs currently used to prevent or treat the condition in question, but is optionally formulated with them. The effective amount of such other drugs depends on the amount of dimeric polypeptide present in the formulation, the type of disease or therapy, and other factors discussed above. These drugs are typically used in the same dosage as described herein and using the same route of administration as described herein, or in about 1% to 99% of the dosage described herein, or in any dosage and any route determined to be suitable empirically / clinically.

[0485] For the prevention or treatment of disease, the appropriate dosage of the dimeric polypeptide 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 dimeric polypeptide, the severity and course of the disease, whether the dimeric polypeptide is administered for prevention or treatment purposes, previous therapy, the patient's clinical history and response to the dimeric polypeptide, and the decision of the attending physician. The dimeric polypeptide is appropriately administered to the patient once or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg-10 mg / kg) of dimeric polypeptide can be the starting candidate dosage for administration to the patient, whether or not, for example, by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a common daily dose may be from about 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 inhibitory effect on the symptoms of the disease occurs. An exemplary dosage of the dimeric polypeptide will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, weekly or every 3 weeks (e.g., the patient receives from about 2 to about 20 or, for example, about 6 doses of the dimeric polypeptide). A higher initial loading dose can be administered, followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0486] III. Artifacts

[0487] In another aspect of the present invention, an article of manufacture is provided, comprising a substance as described above that can be used to treat, prevent, and / or diagnose a condition. The article of manufacture comprises a container and either a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating, preventing, and / or diagnosing the condition, either by itself or in combination with another composition, and can have a sterile access port (for example, the container can be an intravenous bag or a vial with a stopper pierceable by a hypodermic needle). At least one active substance in the composition is a dimeric polypeptide as described herein. The label or package insert indicates that the composition is used to treat a selected condition. Alternatively, the article of manufacture may comprise (a) a first container containing a composition comprising a dimeric polypeptide as described herein; and (b) a second container containing a composition comprising another cytotoxic agent or therapeutic agent. In this embodiment of the invention, the article of manufacture may also comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0488] It will be appreciated that any of the aforementioned articles of manufacture may comprise an immunoconjugate as reported herein instead of or in addition to the dimeric polypeptide as reported herein. Specific implementation plan

[0489] 1. A dimeric polypeptide comprising

[0490] a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising, in the N-terminal to C-terminal direction, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain,

[0491] in

[0492] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0493] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0494] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0495] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0496] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0497] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0498] 2. The dimeric polypeptide according to item 1, characterized in that the dimeric polypeptide does not specifically bind to human FcRn and specifically binds to Staphylococcal protein A.

[0499] 3. The dimeric polypeptide according to any one of items 1 to 2, characterized in that the dimeric polypeptide is a homodimeric polypeptide.

[0500] 4. The dimeric polypeptide according to any one of items 1 to 2, characterized in that the dimeric polypeptide is a heterodimeric polypeptide.

[0501] 5. The dimeric polypeptide according to any one of items 1 to 4, characterized in that i) the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W.

[0502] 6. The dimeric polypeptide according to any one of items 1 to 5, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of human IgG1 subclass.

[0503] 7. The dimeric polypeptide according to any one of items 1 to 6, characterized in that the first polypeptide and the second polypeptide further comprise the mutations L234A and L235A.

[0504] 8. A dimeric polypeptide according to any one of items 1 to 5, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain belong to the human IgG2 subclass, optionally with the mutations V234A, G237A, P238S, H268A, V309L, A330S and P331S.

[0505] 9. The dimeric polypeptide according to any one of items 1 to 5, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of human IgG4 subclass.

[0506] 10. Dimeric polypeptide according to any one of items 1 to 5 and 9, characterized in that the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.

[0507] 11. The dimeric polypeptide according to any one of items 1 to 10, characterized in that the first polypeptide and the second polypeptide further comprise the mutation P329G.

[0508] 12. The dimeric polypeptide according to any one of items 1 to 11, characterized in that the dimeric polypeptide is an Fc region fusion polypeptide.

[0509] 13. The dimeric polypeptide according to any one of items 1 to 11, characterized in that the dimeric polypeptide is a (full length) antibody.

[0510] 14. Dimeric polypeptide according to any one of items 1 to 11 and 13, characterized in that the (full length) antibody is a monospecific antibody.

[0511] 15. Dimeric polypeptide according to any one of items 1 to 11 and 13 to 14, characterized in that the monospecific antibody is a monovalent monospecific antibody.

[0512] 16. Dimeric polypeptide according to any one of items 1 to 11 and 13 to 15, characterized in that the monospecific antibody is a bivalent monospecific antibody.

[0513] 17. Dimeric polypeptide according to any one of items 1 to 11 and 13, characterized in that the (full length) antibody is a bispecific antibody.

[0514] 18. Dimeric polypeptide according to any one of items 1 to 11 and 13 and 17, characterized in that the bispecific antibody is a bivalent bispecific antibody.

[0515] 19. Dimeric polypeptide according to any one of items 1 to 11 and 13 and 17 to 18, characterized in that the bispecific antibody is a tetravalent bispecific antibody.

[0516] 20. Dimeric polypeptide according to any one of items 1 to 11 and 13, characterized in that the (full length) antibody is a trispecific antibody.

[0517] 21. Dimeric polypeptide according to any one of items 1 to 11 and 13 and 20, characterized in that the trispecific antibody is a trivalent trispecific antibody.

[0518] 22. Dimeric polypeptide according to any one of items 1 to 11 and 13 and 20 to 21, characterized in that the trispecific antibody is a tetravalent trispecific antibody.

[0519] 23. A dimeric polypeptide comprising

[0520] a first polypeptide and a second polypeptide, each of the first polypeptide and the second polypeptide comprising, in the N-terminal to C-terminal direction, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain,

[0521] wherein the first polypeptide, the second polypeptide or the first and second polypeptides comprise the mutation Y436A (numbering according to the EU index).

[0522] 24. Dimeric polypeptide according to item 23, characterized in that the first and the second polypeptide comprise the mutation Y436A.

[0523] 25. The dimeric polypeptide according to any one of items 23 to 24, characterized in that the dimeric polypeptide does not specifically bind to human FcRn and specifically binds to Staphylococcal protein A.

[0524] 26. The dimeric polypeptide according to any one of items 23 to 25, characterized in that the dimeric polypeptide is a homodimeric polypeptide.

[0525] 27. The dimeric polypeptide according to any one of items 23 to 25, characterized in that the dimeric polypeptide is a heterodimeric polypeptide.

[0526] 28. The dimeric polypeptide according to any one of items 23 to 27, characterized in that

[0527] a) the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W, and / or

[0528] b) i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0529] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0530] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0531] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0532] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0533] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0534] 29. The dimeric polypeptide according to any one of items 23 to 28, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of human IgG1 subclass.

[0535] 30. Dimeric polypeptide according to any one of items 23 to 29, characterized in that the first polypeptide and the second polypeptide further comprise the mutations L234A and L235A.

[0536] 31. A dimeric polypeptide according to any one of items 23 to 28, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain belong to the human IgG2 subclass, which optionally has the mutations V234A, G237A, P238S, H268A, V309L, A330S and P331S.

[0537] 32. The dimeric polypeptide according to any one of items 23 to 28, characterized in that the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of human IgG4 subclass.

[0538] 33. Dimeric polypeptide according to any one of items 23 to 28 and 32, characterized in that the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.

[0539] 34. The dimeric polypeptide according to any one of items 23 to 33, characterized in that the first polypeptide and the second polypeptide further comprise the mutation P329G.

[0540] 35. The dimeric polypeptide according to any one of items 23 to 34, characterized in that the dimeric polypeptide is an Fc region fusion polypeptide.

[0541] 36. The dimeric polypeptide according to any one of items 23 to 34, characterized in that the dimeric polypeptide is a (full length) antibody.

[0542] 37. Dimeric polypeptide according to any of items 23 to 34 and 36, characterized in that the (full length) antibody is a monospecific antibody.

[0543] 38. Dimeric polypeptide according to any one of items 23 to 34 and 36 to 37, characterized in that the monospecific antibody is a monovalent monospecific antibody.

[0544] 39. Dimeric polypeptide according to any one of items 23 to 34 and 36 to 38, characterized in that the monospecific antibody is a bivalent monospecific antibody.

[0545] 40. Dimeric polypeptide according to any of items 23 to 34 and 36, characterized in that the (full length) antibody is a bispecific antibody.

[0546] 41. Dimeric polypeptide according to any one of items 23 to 34 and 36 and 40, characterized in that the bispecific antibody is a bivalent bispecific antibody.

[0547] 42. Dimeric polypeptide according to any one of items 23 to 34 and 36 and 40 to 41, characterized in that the bispecific antibody is a tetravalent bispecific antibody.

[0548] 43. Dimeric polypeptide according to any of items 23 to 34 and 36, characterized in that the (full length) antibody is a trispecific antibody.

[0549] 44. Dimeric polypeptide according to any one of items 23 to 34 and 36 and 43, characterized in that the trispecific antibody is a trivalent trispecific antibody.

[0550] 45. Dimeric polypeptide according to any one of items 23 to 34 and 36 and 43 to 44, characterized in that the trispecific antibody is a tetravalent trispecific antibody.

[0551] 46. ​​A dimeric polypeptide comprising

[0552] The first polypeptide comprises, from N-terminal to C-terminal, a first heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass,

[0553] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass,

[0554] The third polypeptide comprises, in an N-terminal to C-terminal direction, a first light chain variable domain and a light chain constant domain,

[0555] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0556] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to the first antigen,

[0557] wherein the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the second antigen,

[0558] wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0559] wherein the first and second polypeptides further comprise mutations L234A, L235A and P329G, and

[0560] in

[0561] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0562] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0563] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0564] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0565] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0566] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0567] 47. A dimeric polypeptide comprising

[0568] The first polypeptide comprises, from N-terminal to C-terminal direction, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass,

[0569] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass,

[0570] The third polypeptide comprises, from N-terminal to C-terminal, the first light chain variable domain of the IgG1 subclass and the immunoglobulin CH1 domain,

[0571] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0572] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to the first antigen,

[0573] wherein the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the second antigen,

[0574] wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0575] wherein the first and second polypeptides further comprise mutations L234A, L235A and P329G, and

[0576] in

[0577] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0578] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0579] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0580] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0581] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0582] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0583] 48. A dimeric polypeptide comprising

[0584] The first polypeptide comprises, from N-terminal to C-terminal, a first heavy chain variable domain, an immunoglobulin CH1 domain of the IgG4 subclass, an immunoglobulin hinge region of the IgG4 subclass, an immunoglobulin CH2 domain of the IgG4 subclass, and an immunoglobulin CH3 domain of the IgG4 subclass,

[0585] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG4 subclass, an immunoglobulin hinge region of the IgG4 subclass, an immunoglobulin CH2 domain of the IgG4 subclass, and an immunoglobulin CH3 domain of the IgG4 subclass,

[0586] The third polypeptide comprises, in an N-terminal to C-terminal direction, a first light chain variable domain and a light chain constant domain,

[0587] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0588] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to the first antigen,

[0589] wherein the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the second antigen,

[0590] wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0591] wherein the first and second polypeptides further comprise the mutations S228P, L235E and P329G, and

[0592] in

[0593] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0594] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0595] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0596] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0597] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0598] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0599] 49. A dimeric polypeptide comprising

[0600] The first polypeptide comprises, from N-terminal to C-terminal direction, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of the IgG4 subclass, an immunoglobulin CH2 domain of the IgG4 subclass, and an immunoglobulin CH3 domain of the IgG4 subclass,

[0601] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG4 subclass, an immunoglobulin hinge region of the IgG4 subclass, an immunoglobulin CH2 domain of the IgG4 subclass, and an immunoglobulin CH3 domain of the IgG4 subclass,

[0602] The third polypeptide comprises, from N-terminal to C-terminal, a first light chain variable domain and an immunoglobulin CH1 domain of the IgG4 subclass,

[0603] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0604] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to the first antigen,

[0605] wherein the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the second antigen,

[0606] wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0607] wherein the first and second polypeptides further comprise the mutations S228P, L235E and P329G, and

[0608] in

[0609] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0610] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0611] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0612] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0613] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0614] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0615] 50. A dimeric polypeptide comprising

[0616] The first polypeptide comprises, from N-terminal to C-terminal direction, a first heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, an immunoglobulin CH3 domain of the IgG1 subclass, a peptide linker, and a first scFv,

[0617] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass, a peptide linker, and a second scFv,

[0618] The third polypeptide comprises, in an N-terminal to C-terminal direction, a first light chain variable domain and a light chain constant domain,

[0619] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0620] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to the first antigen, the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the first antigen, and the first and second scFvs specifically bind to the second antigen,

[0621] wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0622] wherein the first and second polypeptides further comprise mutations L234A, L235A and P329G, and

[0623] in

[0624] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0625] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0626] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0627] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0628] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0629] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0630] 51. A dimeric polypeptide comprising

[0631] The first polypeptide comprises, from N-terminal to C-terminal direction, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, an immunoglobulin CH3 domain of the IgG1 subclass, a peptide linker, and a first scFv,

[0632] The second polypeptide comprises, from N-terminal to C-terminal direction, a second heavy chain variable domain, an immunoglobulin CH1 domain of the IgG1 subclass, an immunoglobulin hinge region of the IgG1 subclass, an immunoglobulin CH2 domain of the IgG1 subclass, and an immunoglobulin CH3 domain of the IgG1 subclass, a peptide linker, and a second scFv,

[0633] The third polypeptide comprises, from N-terminal to C-terminal, a first light chain variable domain and an immunoglobulin CH1 domain of the IgG1 subclass,

[0634] The fourth polypeptide comprises, in an N-terminal to C-terminal direction, a second light chain variable domain and a light chain constant domain,

[0635] wherein the first heavy chain variable domain and the first light chain variable domain form a first binding site that specifically binds to a first antigen, the second heavy chain variable domain and the second light chain variable domain form a second binding site that specifically binds to the first antigen, and the first and second scFvs specifically bind to the second antigen, wherein i) the first polypeptide comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W,

[0636] wherein the first and second polypeptides further comprise mutations L234A, L235A and P329G, and

[0637] in

[0638] i) the first and second polypeptides comprise the mutations H310A, H433A and Y436A, or

[0639] ii) the first and second polypeptides comprise the mutations L251D, L314D and L432D, or

[0640] iii) the first and second polypeptides comprise the mutations L251S, L314S and L432S, or

[0641] iv) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations H310A, H433A and Y436A, or

[0642] v) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251D, L314D and L432D, or

[0643] vi) the first polypeptide comprises the mutations I253A, H310A and H435A and the second polypeptide comprises the mutations L251S, L314S and L432S.

[0644] 52. A method for producing a dimeric polypeptide according to any one of items 1 to 51, comprising the following steps:

[0645] a) cultivating mammalian cells comprising one or more nucleic acids encoding the dimeric polypeptide according to any one of items 1 to 51,

[0646] b) recovering the dimeric polypeptide from the culture medium, and

[0647] c) Purification of the dimeric polypeptide using protein A affinity chromatography.

[0648] 53. Use of the mutation Y436A to increase the binding of a dimeric polypeptide to protein A.

[0649] 54. Use of the mutations H310A, H433A and Y436A to separate heterodimeric polypeptides from homodimeric polypeptides.

[0650] 55. Use of the mutations L251D, L314D, L432D or the mutations L251S, L314S, L432S for separating heterodimeric polypeptides from homodimeric polypeptides.

[0651] 56. Use of the mutations I253A, H310A and H435A in a first polypeptide in combination with the mutations H310A, H433A and Y436A in a second polypeptide for separating heterodimeric polypeptides from homodimeric polypeptides comprising a first and a second polypeptide.

[0652] 57. Use of the mutations I253A, H310A and H435A in a first polypeptide in combination with the mutations L251D, L314D, L432D or the mutations L251S, L314S, L432S in a second polypeptide for separating heterodimeric polypeptides from homodimeric polypeptides comprising a first and a second polypeptide.

[0653] 58. Use according to any one of items 53 to 57, characterized in that i) the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V and the second polypeptide further comprises the mutations S354C and T366W, or ii) the first polypeptide comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W.

[0654] 59. Method of treating a patient suffering from an ocular vascular disease, said method comprising administering to a patient in need of such treatment a dimeric polypeptide according to any one of items 1 to 51.

[0655] 60. Dimeric polypeptide according to any one of items 1 to 51, for intravitreal application.

[0656] 61. A dimeric polypeptide according to any one of items 1 to 51 for use in treating ocular vascular diseases.

[0657] 62. A pharmaceutical formulation comprising the dimeric polypeptide according to any one of items 1 to 51 and optionally a pharmaceutically acceptable carrier.

[0658] 63. Use of the dimeric polypeptide according to any one of items 1 to 51 for transporting a soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation.

[0659] 64. Use of a dimeric polypeptide according to any one of items 1 to 51 for removing one or more soluble receptor ligands from the eye.

[0660] 65. Use of the dimeric polypeptide according to any one of items 1 to 51 for treating eye diseases, especially ocular vascular diseases.

[0661] 66. Use of the dimeric polypeptide according to any one of items 1 to 51 for transporting one or more soluble receptor ligands from the intravitreal cavity into the blood circulation.

[0662] 67. The dimeric polypeptide according to any one of items 1 to 51 for use in treating an eye disease.

[0663] 68. The dimeric polypeptide according to any one of items 1 to 51, for use in transporting a soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation.

[0664] 69. A dimeric polypeptide according to any one of items 1 to 51 for use in removing one or more soluble receptor ligands from the eye.

[0665] 70. The dimeric polypeptide according to any one of items 1 to 51, for use in treating eye diseases, especially ocular vascular diseases.

[0666] 71. The dimeric polypeptide according to any one of items 1 to 51, for use in transporting one or more soluble receptor ligands from the intravitreal cavity into the blood circulation.

[0667] 72. A method of treating a subject suffering from an ocular vascular disease, said method comprising administering to the subject an effective amount of a dimeric polypeptide according to any one of items 1 to 51.

[0668] 73. A method for transporting a soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation in an individual, the method comprising administering to the individual an effective amount of a dimeric polypeptide according to any one of items 1 to 51 to transport the soluble receptor ligand from the eye across the blood-ocular barrier into the blood circulation.

[0669] 74. A method of removing one or more soluble receptor ligands from the eye in an individual, the method comprising administering to the individual an effective amount of a dimeric polypeptide according to any one of items 1 to 51 to remove the one or more soluble receptor ligands from the eye.

[0670] 75. A method for transporting one or more soluble receptor ligands from the vitreous cavity to the blood circulation in an individual, the method comprising administering to the individual an effective amount of a dimeric polypeptide according to any one of items 1 to 51 to transport the one or more soluble receptor ligands from the vitreous cavity to the blood circulation.

[0671] 76. A method for transporting a soluble receptor ligand from the vitreous cavity or the eye into the blood circulation in an individual, the method comprising administering to the individual an effective amount of a dimeric polypeptide according to any one of items 1 to 51 to transport the soluble receptor ligand from the eye into the blood circulation through the blood-eye barrier.

[0672] V. Examples

[0673] The following are examples of methods and compositions of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0674] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0675] method

[0676] Electrospray ionization mass spectrometry (ESI-MS)

[0677] By adding 0.5 μ L N-glycanase plus (Roche) and sodium phosphate buffer (0.1M, pH 7.1) to obtain final sample volume 115 μ L, protein aliquots (50 μ g) are deglycosylated. The mixture is incubated at 37 ° C for 18 hours. Thereafter, for reduction and denaturation, 60 μ L of 0.5 M TCEP (Pierce) and 50 μ L of 8 M guanidine * HCl (Pierce) are added. The mixture is incubated at 37 ° C for 30 minutes. By size exclusion chromatography (agarose gel G-25, equal intensity, 40% acetonitrile, containing 2% formic acid), the sample is desalted. ESI mass spectra (+ve) are 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; multipole 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 cell: 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. Data evaluation used in-house developed software (MassAnalyzer).

[0678] FcRn surface plasmon resonance (SPR) analysis

[0679] The binding properties of wild-type antibodies and mutants to FcRn were analyzed using surface plasmon resonance (SPR) technology using a BIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This system is well-established for studying molecular interactions. It allows continuous real-time monitoring of ligand / analyte binding and, therefore, determination of kinetic parameters under various analytical settings. The SPR technology is based on measuring the refractive index near the surface of a gold-plated biosensor chip. Changes in the refractive index represent changes in mass caused by the interaction of the immobilized ligand on this surface with the analyte injected in solution. If a molecule binds to the immobilized ligand on the surface, its mass increases, and if it dissociates, its 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 amine coupling. The assay was performed at room temperature using PBS, 0.05% Tween 20 pH 6.0 (GE Healthcare Bioscience) as running buffer and dilution buffer. 200nM antibody samples were injected at room temperature at a flow rate of 50 μL / min. The association time was 180 seconds and the dissociation phase took 360 seconds. The chip surface was regenerated by a brief injection of HBS-P, pH 8.0. The SPR data were evaluated by comparing the biological response signal heights 180 seconds after injection and 300 seconds after injection. The corresponding parameters were the highest RU level (180 seconds after injection) and the late stability (300 seconds after the end of the injection).

[0680] Protein A surface plasmon resonance (SPR) analysis

[0681] The assay is based on surface plasmon resonance spectroscopy. 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, therefore, a higher response (e.g., 1 RU is defined as 1 pg / mm 2 ). Thereafter, the sensor chip was regenerated by dissolving the sample-Protein A complex. The obtained response was then evaluated in response units (RU) to evaluate the signal height and dissociation behavior.

[0682] About 3500 response units (RU) of protein A (20 μg / ml) were coupled to a CM5 chip (GE Healthcare) at pH 4.0 by using an amine coupling kit from GE Healthcare.

[0683] The sample and system buffer was HBS-P+ (0.01 M HEPES, 0.15 M NaCl, sterile-filtered 0.005% surfactant P20, pH 7.4). The flow cell temperature was set to 25° C. and the sample compartment temperature was set to 12° C. The system was loaded with running buffer. Subsequently, a 5 nM solution of the sample construct was injected for 120 seconds at a flow rate of 30 μL / min, followed by a 300 second dissociation phase. Subsequently, the sensor chip surface was regenerated by two 30 second long injections of glycine-HCl pH 1.5 at a flow rate of 30 μL / min. Each sample was measured in triplicate.

[0684] Bispecific antibodies and their corresponding sequences

[0685]

[0686]

[0687] As used herein, the term “having the mutation IHH-AAA” refers to a combination of the mutations I253A (Ile253Ala), H310A (His310Ala) and H435A (His435Ala) in the constant heavy chain region of the IgG1 or IgG4 subclass (numbering according to the Kabat EU index numbering system), the term “having the mutation HHY-AAA” as used herein refers to a combination of the mutations H310A (His310Ala), H433A (His433Ala) and Y436A (Tyr436Ala) in the constant heavy chain region of the IgG1 or IgG4 subclass (numbering according to the Kabat EU index numbering system), the term “having the mutation P329G LALA” as used herein refers to a combination of the mutations P329G LALA in the constant heavy chain region of the IgG1 subclass (numbering according to the Kabat EU index numbering system), The term "having the mutation SPLE" as used herein refers to a combination of the mutations L234A (Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly) in the constant heavy chain region of the IgG4 subclass (numbering according to the Kabat EU index numbering system), and the term "having the mutation SPLE" as used herein refers to a combination of the mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region of the IgG4 subclass (numbering according to the Kabat EU index numbering system).

[0688]

[0689]

[0690] Overview

[0691] General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acid residues of the antibody chains are numbered according to the EU numbering and are referred to (Edelman, GM et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0692] Recombinant DNA technology

[0693] Standard methods as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) were used to manipulate DNA. Molecular biology reagents were used according to the manufacturers' instructions.

[0694] gene synthesis

[0695] The gene segments of interest were ordered from Geneart (Regensburg, Germany) according to the instructions given.

[0696] DNA sequencing

[0697] DNA sequences were determined by double-strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

[0698] DNA and protein sequence analysis and sequence data management

[0699] The GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NTI Advance suite version 8.0 were used for sequence generation, mapping, analysis, annotation, and illustration.

[0700] expression vector

[0701] For expression of the described antibodies, expression vectors based on cDNA organization with or without CMV-Intron A promoter or based on genomic organization with CMV promoter are used for transient expression (eg in HEK293-F cells).

[0702] In addition to the antibody expression cassette, the vector also contains:

[0703] an origin of replication that allows the vector to replicate in E. coli,

[0704] a β-lactamase gene that confers ampicillin resistance in Escherichia coli, and

[0705] Dihydrofolate reductase gene from Mus musculus as a selectable marker in eukaryotic cells.

[0706] The transcription unit of an antibody gene contains the following elements:

[0707] A unique restriction site at the 5' end,

[0708] The immediate early enhancer and promoter from human cytomegalovirus,

[0709] In the case of cDNA organization, the subsequent intron A sequence,

[0710] 5' untranslated region of human immunoglobulin genes,

[0711] a nucleic acid encoding an immunoglobulin heavy chain signal sequence,

[0712] Nucleic acids encoding human antibody chains (wild type or with domain swaps), either as cDNA or with an immunoglobulin exon-intron organization in a genomic organization,

[0713] a 3' untranslated region having a polyadenylation signal sequence, and

[0714] Unique restriction site at the 3' end.

[0715] The nucleic acid encoding the antibody chain is generated by PCR and / or gene synthesis and assembled by connecting the corresponding nucleic acid segments, for example, using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequence is verified by DNA sequencing. For transient transfection, vectors are prepared from transformed Escherichia coli cultures (Nucleobond AX, Macherey-Nagel) to obtain larger vectors.

[0716] Cell culture technology

[0717] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (Eds.), John Wiley & Sons, Inc., were used.

[0718] As described below, bispecific antibodies were expressed by transient co-transfection of the corresponding expression vectors in HEK29-F cells grown in suspension.

[0719] Example 1

[0720] Expression and purification

[0721] Transient transfection in the HEK293-F system

[0722] Monospecific and bispecific antibodies were produced by transient transfection with the corresponding vectors (e.g., plasmids encoding heavy and modified heavy chains and corresponding light and modified light chains) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, serum-free FreeStyle TM HEK293-F cells (Invitrogen) grown in suspension in 293 expression medium (Invitrogen) were transfected with the corresponding expression vectors and 293fectin TM For 2 L shake flasks (Corning), HEK293-F cells were transfected at a density of 1*10 6 Cells / mL were seeded in 600 mL and incubated at 120 rpm, 8% CO2. 6The cells of the individual cells / mL cell density are transfected with about 42mL of the following mixture: A) a mixture of 20mL Opti-MEM (Invitrogen) and 600μg of total vector DNA (1μg / mL) encoding heavy chain or modifying heavy chain and corresponding light chain in an equimolar ratio and B) a mixture of 20ml Opti-MEM and 1.2mL 293fectin or fectin (2μL / mL). During the fermentation process, glucose solution is added according to glucose consumption. After 5-10 days, the results contain the supernatant of secretory antibody and the antibody is directly purified from the supernatant or the supernatant is frozen and stored.

[0723] purification

[0724] By using MabSelectSure-Sepharose TM Bispecific antibodies were purified from cell culture supernatants by affinity chromatography using Fabry-Aldrich (for non-IHH-AAA mutants) (GE Healthcare, Sweden) or KappaSelect-Sepharose (for IHH-AAA mutants) (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl-Sepharose (GE Healthcare, Sweden), and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.

[0725] In brief, the cell culture supernatant of sterile filtration is captured on the MabSelectSuRe resin (non-IHH-AAA mutation and wild-type antibody) balanced with PBS buffer (10mM Na hPO , 1mM KH pO , 137mM NaCl and 2.7mM KCl, pH 7.4), washed with equilibrium buffer and eluted with 25mM pH 3.0 sodium citrate. The IHH-AAA mutant is captured on the KappaSelect resin balanced with 25mM Tris, 50mM NaCl, pH 7.2, washed with equilibrium buffer and eluted with 25mM pH 2.9 sodium citrate. The eluted antibody fractions are pooled and neutralized with 2M Tris, pH 9.0. By adding 1.6M ammonium sulfate solution to a final concentration of 0.8M ammonium sulfate and using acetic acid to adjust pH to pH 5.0, the antibody pool is ready for hydrophobic interaction chromatography. The antibody of the present invention is purified by size exclusion chromatography.With 35mM sodium acetate, 0.8M ammonium sulfate, after pH 5.0 balance butyl-agarose resin, antibody is applied to resin, washed with equilibrium buffer and eluted to 35mM sodium acetate pH 5.0 with linear gradient.The fraction containing (monospecific or bispecific) antibody is pooled and used with 20mM histidine, 140mM NaCl, Superdex 200 26 / 60GL (GE Healthcare, Sweden) post of pH 6.0 balance, further passes through size exclusion chromatography purification.The fraction containing (monospecific or bispecific) antibody is pooled, uses Vivaspin ultrafiltration device (Sartorius Stedim Biotech S.A., France) to be concentrated into the concentration of requirement and be stored in-80 ℃.

[0726] Table: Bispecific Antibodies <vegf-ang-2>Antibody yield

[0727]

[0728] After each purification step, microfluidics Labchip technology (Caliper Life Science, USA) was used to analyze purity and antibody integrity by CE-SDS. According to the manufacturer's instructions, HT protein expression kit was used to prepare 5 μL of protein solution for CE-SDS analysis, and HT protein expression chip was used to analyze on the Labchip GXII system. Labchip GX software was used to analyze the data.

[0729] Table: Removal of common by-products by different sequences of purification steps as determined by CE-SDS

[0730]

[0731]

[0732] The aggregate content of the antibody samples was analyzed by high performance SEC using a Superdex 200 analytical size exclusion column (GE Healthcare, Sweden) in 2x PBS (20 mM Na2HPO4, 2 mM KH2PO4, 274 mM NaCl and 5.4 mM KCl, pH 7.4) running buffer at 25° C. 25 μg of protein was injected onto the column at a flow rate of 0.75 mL / min and eluted isocratically for 50 minutes.

[0733] Similarly, the anti-VEGF-ANG2 antibodies VEGF / ANG2-0012 and VEGF / ANG2-0201 were prepared and purified with the following yields:

[0734]

[0735] In addition, the anti-VEGF / ANG2 bispecific antibody anti-VEGF / ANG2 CrossMAb IgG4 having an IHH-AAA mutation and a SPLE mutation (SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45), the anti-VEGF / ANG2 OAscFab IgG1 having an IHH-AAA mutation (SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48), the anti-VEGF / ANG2 OAscFab IgG4 having an IHH-AAA mutation and a SPLE mutation (SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51), the anti-VEGF / ANG2 CrossMab IgG1 having an HHY-AAA mutation and a P329G LALA mutation (SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 40, SEQ ID NO: 41), the anti-VEGF / ANG2 CrossMab IgG4 having an HHY-AAA mutation and a SPLE mutation (SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95), and the anti-VEGF / ANG2 CrossMab IgG4 having an HHY-AAA mutation and a SPLE mutation (SEQ ID NO: 96) can be similarly prepared and purified. NO:92, SEQ ID NO:93, SEQ ID NO:44, SEQ ID NO:45), anti-VEGF / ANG2 OAscFab IgG1 with HHY-AAA mutation (SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:48), and anti-VEGF / ANG2 OAscFab IgG4 with HHY-AAA mutation and SPLE mutation (SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:51), and anti-IGF-1R monospecific antibodies anti-IGF-1R wild type (SEQ ID NO:88, SEQ ID NO:89), anti-IGF-1R IgG1 with IHH-AAA mutation (SEQ ID NO:88, SEQ ID NO:90), anti-IGF-1R IgG1 with YTE mutation (SEQ ID NO:88, SEQ ID NO:91), anti-IGF-1R IgG1 wild type with KiH mutation (SEQ ID NO:88, SEQ ID NO:92), can also be prepared and purified similarly. ID NO: 92, SEQ ID NO: 93), anti-IGF-1R IgG1 with KiH mutation and IHH-AAA mutation in the clasp (SEQ ID NO: 88, SEQ ID NO: 94, SEQ ID NO: 95), anti-IGF-1R IgG1 with KiH mutation and HHY-AAA mutation in the clasp (SEQ ID NO: 88, SEQ ID NO: 94, SEQ ID NO: 95),NO:96, SEQ ID NO:97), anti-IGF-1RIgG1 with KiH mutation and YTE mutation (SEQ ID NO:88, SEQ ID NO:98, SEQ ID NO:99), anti-IGF-1RIgG1 with KiH mutation and DDD mutation (SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101), and anti-IGF-1RIgG1 with HHY-AAA mutation (SEQ ID NO:88, SEQ ID NO:112).

[0736] Example 2

[0737] Analytics and Scalability

[0738] Viscosity measurements based on small-scale DLS:

[0739] Viscosity measurements were performed essentially as described in (He, F. et al., Analytical Biochemistry 399 (2009) 141-143). Briefly, samples were concentrated to various protein concentrations in 200 mM arginine succinate, pH 5.5, after which polystyrene latex beads (300 nm diameter) and polysorbate 20 (0.02% v / v) were added. The samples were transferred by centrifugation through a 0.4 μm filter plate into an optical 384-well plate 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 can be 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).

[0740] To allow comparison of various samples at the same concentration, viscosity-concentration data were fit to the Mooney equation (Equation 1) (Mooney, M., Colloid. Sci., 6 (1951) 162-170; Monkos, K., Biochem. Biophys. Acta 304 (1997) 1339) and data interpolated accordingly.

[0741]

[0742] (S: hydrodynamic interaction parameter of protein; K: self-assembly factor; Φ: volume fraction of dissolved protein)

[0743] Figure 2 Results are shown in FIG: VEGF / ANG2-0016 having an IHH-AAA mutation in the Fc region showed lower viscosity at all measured temperatures compared to VEGF / ANG2-0015 without an IHH-AAA mutation in the Fc region.

[0744] DLS aggregation onset temperature

[0745] The sample was prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred by centrifugation through a 0.4 μm filter plate into an optical 384-well plate and 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. Figure 3 The results are displayed in . Figure 3 , aggregation of VEGF / ANG2-0015 without the IHH-AAA mutation and VEGF / ANG2-0016 with the IHH-AAA mutation in the Fc region is shown. VEGF / ANG2-0016 showed an aggregation onset temperature of 61°C, while VEGF / ANG2-0015 without the IHH-AAA mutation showed an onset temperature of 60°C.

[0746] DLS Schedule

[0747] Samples were prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred by centrifugation through a 0.4 μm filter plate into an optical 384-well plate, and covered with paraffin oil. While the samples were maintained at a constant temperature of 50°C for up to 145 hours, the hydrodynamic radius was repeatedly measured by dynamic light scattering. In this experiment, the tendency of naturally unfolded proteins to aggregate at elevated temperatures will result in an increase in average particle size over time. This DLS-based method is very sensitive to aggregates, as these contribute disproportionately to the scattered light intensity. Even after 145 hours at 50°C (a temperature close to the aggregation onset temperature, see above), an increase in average particle size of less than 0.5 nm was observed for VEGF / ANG2-0015 and VEGF / ANG2-0016.

[0748] Store at 100 mg / mL at 40°C for 7 days

[0749] The samples were concentrated to a final concentration of 100 mg / mL in 200 mM arginine succinate pH 5.5, sterile filtered and stored statically at 40°C for 7 days. The content of high molecular weight species and low molecular weight species (HMW and LMW, respectively) was determined by size exclusion chromatography before and after storage. The differences in HMW content and LMW content between the stored samples and the samples measured immediately after preparation are reported as "HMW increase" and "LMW increase", respectively. The following table and Figure 4 Results are shown in Figure 3, which show that VEGF / ANG2-0015 (without the IHH-AAA mutation) exhibited a greater decrease in the main peak and a higher increase in HMW compared to VEGF / ANG2-0016 (with the IHH-AAA mutation). Surprisingly, VEGF / ANG2-0016 (with the IHH-AAA mutation) exhibited a lower tendency to aggregate compared to VEGF / ANG2-0015 (without the IHH-AAA mutation).

[0750] Table: Δ Main peak, HMW peak and LMW peak after 7 days at 40℃

[0751]

[0752] Use at 25℃ Functional analysis of the anti-VEGF / ANG2 bispecific antibody was assessed by surface plasmon resonance (SPR) using a T100 or T200 instrument (GE Healthcare). The system is well established for studying molecular interactions. SPR-technology is based on measuring the refractive index near the surface of a gold-plated biosensor chip. The change in refractive index represents the mass change caused by the interaction of the immobilized ligand on the surface with the analyte injected in the solution. If the molecule binds to the immobilized ligand on the surface, the mass increases, whereas if the analyte dissociates from the immobilized ligand (reflecting the dissociation of the complex), the mass decreases. SPR allows continuous real-time monitoring of ligand / analyte binding and therefore determines the association rate constant (ka), dissociation rate constant (kd) and equilibrium constant (KD).

[0753] Example 3

[0754] Binding to VEGF, ANG2, FcγR, and FcRn

[0755] VEGF isoform kinetic affinity, including assessment of species cross-reactivity

[0756] Approximately 12,000 resonance units (RU) of the capture system (10 μg / mL goat anti-human F(ab)'2; Order code: 28958325; GE Healthcare Bio-Sciences AB, Sweden) were coupled to a CM5 chip (GE Healthcare BR-1005-30) using an amine coupling kit supplied by GE Healthcare at pH 5.0. The sample and system buffer was PBS-T (10 mM phosphate buffered saline containing 0.05% Tween 20) pH 7.4. The flow cell was set to 25°C and the sample block was set to 12°C and filled twice with running buffer. The bispecific antibody was captured by injecting a 50 nM solution in the solution for 30 seconds at a flow rate of 5 μL / min. Binding was measured by injecting various concentrations of human hVEGF121, mouse mVEGF120, or rat rVEGF164 in solution at a flow rate of 30 μL / min for 300 seconds (starting at 300 nM, 1:3 dilution). The dissociation phase was monitored for 1200 seconds and initiated by switching the sample solution to running buffer. The surface was regenerated by washing with glycine pH 2.1 solution at a flow rate of 30 μL / min for 60 seconds. The bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human F(Ab')2 surface. A blank injection (= double reference) was also subtracted. To calculate the apparent K D The Langmuir 1:1 model was used for the kinetic parameters and the results are shown below.

[0757] ANG2 solution affinity, including assessment of species cross-reactivity

[0758] Solution affinity measures the affinity of an interaction by determining the concentration of free interaction partner in the equilibrium mixture. The solution affinity assay involves mixing an anti-VEGF / ANG2 antibody maintained at a constant concentration with a varying concentration of ligand (=ANG2). 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 using an amine coupling kit supplied by GE Healthcare at pH 5.0. The sample and system buffer is HBS-P, pH 7.4. The flow cell is set to 25°C, and the sample block is set to 12°C and primed twice with running buffer. To generate a calibration curve, increasing concentrations of ANG2 are injected into a BIAcore flow cell containing immobilized anti-VEGF / ANG2 antibody. The amount of bound ANG2 is measured as resonance units (RU) and plotted against concentration. Solutions of each ligand (11 concentrations, ranging from 0 to 200 nM for the anti-VEGF / ANG2 antibody) were incubated with 10 nM ANG2 and allowed to equilibrate at room temperature. Free ANG2 concentrations were determined from calibration curves generated before and after measuring the responses of solutions with known amounts of ANG2. Using model 201, a four-parameter fit was established using XLfit4 (IDBS Software), using free ANG2 concentration as the y-axis and the inhibitory antibody concentration as the x-axis. 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. Bulk refractive index differences were corrected by subtracting the responses obtained from a blank-coupled surface. The results are shown below.

[0759] FcRn steady-state affinity

[0760] For FcRn measurements, steady-state affinity was used to compare bispecific antibodies. Human FcRn was diluted in coupling buffer (10 μg / mL, sodium acetate pH 5.0) and fixed to a C1 chip (GE Healthcare BR-1005-35) using the BIAcore wizard to a final response of 200 RU using the directional immobilization method. The flow cell was set to 25°C and the sample block was set to 12°C and filled twice with running buffer. The sample and system buffer was 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. With the flow rate set to 30 μL / min and a binding time of 180 seconds, different samples were injected continuously onto the chip surface. The surface was regenerated by injecting PBS-T pH 8 at a flow rate of 30 μL / min for 60 seconds. By deducting the response values ​​obtained from the blank surface, the bulk refractive index differences were corrected. Buffer injections were also deducted (= double reference). To calculate the steady-state affinity, the method from the BIA-Evaluation software was used. In short, the RU values ​​were plotted against the analyzed concentrations to generate a dose-response curve. Based on a 2-parameter fit, an upper asymptote was calculated, allowing the determination of the half-maximal RU value and thus the affinity. The results are shown in Figure 5 and the table below. Similarly, the affinity for cynomolgus monkey, mouse and rabbit FcRn can be determined.

[0761] FcγRIIIa measurement

[0762] For FcγRIIIa measurements, a direct binding assay was used. Approximately 3,000 resonance units (RU) of the capture system (1 μg / ml Penta-His; Qiagen) were coupled to a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was HBS-P + pH 7.4. The flow cell was set to 25°C and the sample block was set to 12°C and filled twice with running buffer. The FcγRIIIa-His receptor was captured by injecting a 100 nM solution at a flow rate of 5 μL / min for 60 seconds. Binding was measured by injecting 100 nM of the bispecific antibody or monospecific control antibody (anti-digoxigenin antibody for IgG1 and IgG4 subclass antibodies) at a flow rate of 30 μL / min for 180 seconds. The surface was regenerated by washing with a glycine pH 2.5 solution at a flow rate of 30 μL / min for 120 seconds. Because FcγRIIIa binding differs from the Langmuir 1:1 model, only binding / no binding is determined with this assay. In a similar manner, FcγRIa binding and FcγRIIa binding can be determined. Figure 6 The results shown in Figure 2 show that when this was followed up by introducing the mutation P329G LALA, no additional FcγRIII binding could be detected.

[0763] Evaluation of independent VEGF binding and ANG-2 binding of anti-VEGF / ANG2 antibodies

[0764] Approximately 3,500 resonance units (RU) of the capture system (10 μg / mL goat anti-human IgG; GE Healthcare Bio-Sciences AB, Sweden) were coupled to a CM4 chip (GE Healthcare BR-1005-34) using an amine coupling kit supplied by GE Healthcare at pH 5.0. The sample and system buffer was PBS-T (10 mM phosphate buffered saline containing 0.05% Tween 20) at pH 7.4. The temperature of the flow cell was set to 25°C and the temperature of the sample block was set to 12°C. Prior to capture, the flow cell was filled twice with running buffer.

[0765] The bispecific antibody was captured by injecting a 10 nM solution at a flow rate of 5 μL / min for 60 seconds. The independent binding of each ligand to the bispecific antibody was analyzed by determining the effective binding capacity of each ligand added sequentially or simultaneously (flow rate 30 μL / min):

[0766] 1. Inject human VEGF at a concentration of 200 nM for 180 seconds (to identify single binding interactions of the antigen).

[0767] 2. Human ANG2 was injected at a concentration of 100 nM for 180 seconds (to identify a single binding interaction of the antigen).

[0768] 3. Injection of human VEGF at a concentration of 200 nM for 180 seconds, followed by an additional injection of human ANG2 at a concentration of 100 nM for 180 seconds (to identify ANG2 binding in the presence of VEGF).

[0769] 4. Injection of human ANG2 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 ANG2).

[0770] 5. Coinjection of 200 nM human VEGF and 100 nM human ANG2 for 180 seconds (to identify the binding of VEGF and ANG2 at the same time).

[0771] The surface was regenerated by washing with 3M MgCl2 solution at a flow rate of 30 μL / min for 60 seconds. Bulk refractive index differences were corrected by subtracting the response obtained from the goat anti-human IgG surface.

[0772] If the final signals obtained from Schemes 3, 4, and 5 are equal to or similar to the sum of the final signals from Schemes 1 and 2, the bispecific antibody is capable of binding to both antigens independently of one another. The results are shown in the table below, where both antibodies, VEGF / ANG2-0016 and VEGF / ANG2-0012, were shown to be capable of binding to VEGF and ANG2 independently of one another.

[0773] Evaluation of simultaneous VEGF and ANG-2 binding of anti-VEGF / ANG2 antibodies

[0774] First, approximately 1,600 resonance units (RU) of VEGF (20 μg / mL) were coupled to a CM4 chip (GE Healthcare BR-1005-34) using an amine coupling kit supplied by GE Healthcare at pH 5.0. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline containing 0.05% Tween 20) at pH 7.4. The flow cell was set to 25°C and the sample block to 12°C and filled twice with running buffer. Next, a 50 nM solution of the bispecific antibody was injected at a flow rate of 30 μL / min for 180 seconds. Third, hANG2 was injected at a flow rate of 30 μL / min for 180 seconds. The hANG2 binding response depended on the amount of bispecific antibody bound to VEGF and showed simultaneous binding. The surface was regenerated by washing with a 0.85% H₃PO₄ solution at a flow rate of 30 μL / min for 60 seconds. Simultaneous binding was demonstrated by the additional specific binding signal of hANG2 to the anti-VEGF / ANG2 antibody previously bound to VEGF. For the two bispecific antibodies, VEGF / ANG2-0015 and VEGF / ANG2-0016, simultaneous binding of the anti-VEGF / ANG2 antibody to VEGF and ANG-2 was detected (data not shown).

[0775] Table: Results: Kinetic affinity for VEGF isoforms from different species

[0776]

[0777] Table: Results: Solution affinity for ANG2

[0778]

[0779] Table: Results: Affinity of anti-VEGF / ANG2 antibodies to FcRn

[0780]

[0781]

[0782] Table: Binding results with FcγRI–IIIa

[0783] VEGF / ANG2-0015 VEGF / ANG2-0016 VEGF / ANG2-0012 VEGF / ANG2-0201 FcγRIa Not combined Not combined Combine Combine FcγRIIa Not combined Not combined Not combined Combine FcγRIIIa Not combined Not combined Not combined Combine

[0784] Table: Results: Independent binding effects of anti-VEGF / ANG2 antibodies on VEGF and ANG2

[0785]

[0786] Example 4

[0787] Mass spectrometry

[0788] This section describes the characterization of anti-VEGF / ANG2 antibodies, with an emphasis on correct assembly. The expected primary structure was confirmed by deglycosylation and electrospray ionization mass spectrometry (ESI-MS) of intact or IdeS-digested (IgG-degrading enzyme from Streptococcus pyogenes) anti-VEGF / ANG2 antibodies. IdeS-digestion was performed using 100 μg of purified antibody, which was incubated with 2 μg of IdeS protease (Fabricator) in 100 mmol / L NaH2PO4 / Na2HPO4, pH 7.1 at 37°C for 5 hours. 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 16 hours and then desalted by HPLC on a Sephadex G25 column (GE Healthcare). Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0789] The masses obtained for the IdeS-digested deglycosylated (lower table) or intact deglycosylated (lower table) molecules correspond to the predicted masses from the chromatin fragments derived from two different light chain LC ANG-2 and LC Lucentis and two different heavy chains HC ANG-2 and HC Lucentis The amino acid sequences of the constituent anti-VEGF / ANG2 antibodies were deduced.

[0790] Table: Mass of deglycosylated and IdeS-digested bispecific anti-VEGF / ANG2 antibodies VEGF / ANG2-0201 (without IHH-AAA mutation) and VEGF / ANG2-0012 (with IHH-AAA mutation)

[0791]

[0792] Table: Mass of deglycosylated anti-VEGF / ANG2 antibodies VEGF / ANG2-0016 (with IHH-AAA mutation) and VEGF / ANG2-0015 (without IHH-AAA mutation)

[0793]

[0794] Example 5

[0795] FcRn chromatography

[0796] Coupled to streptavidin agarose gel:

[0797] One gram of Streptavidin Sepharose (GE Healthcare) was added to the biotinylated and dialyzed receptor and incubated for two hours with shaking. The receptor derivatized Sepharose was packed into a 1 mL XK column (GE Healthcare).

[0798] Chromatography using an FcRn affinity column:

[0799] condition:

[0800] Column size: 50mm x 5mm

[0801] Bed height: 5cm

[0802] Loading capacity: 50 μg sample

[0803] Equilibration buffer: 20 mM MES containing 150 mM NaCl, adjusted to pH 5.5

[0804] Elution buffer: 20 mM Tris / HCl containing 150 mM NaCl, adjusted to pH 8.8

[0805] Elution: 7.5CV equilibration buffer, to 100% elution buffer within 30CV, 10CV elution buffer

[0806] Human FcRn affinity column chromatography

[0807] In the table below, the retention times of anti-VEGF-ANG2 antibodies on an affinity column containing human FcRn are given. The data were obtained using the conditions described above.

[0808] Table: Results: Retention time of anti-VEGF / ANG2 antibodies

[0809] Antibody Retention time [minutes] VEGF / ANG2-0015 (without IHH-AAA mutation) 78.5 VEGF / ANG2-0201 (without IHH-AAA mutation) 78.9 VEGF / ANG2-0012 (with IHH-AAA mutation) 2.7 (void peak) VEGF / ANG2-0016 (with IHH-AAA mutation) 2.7 (void peak)

[0810] Example 6

[0811] Pharmacokinetic (PK) properties of antibodies harboring IHH-AAA mutations

[0812] PK data of human FcRn transgenic FcRn mice

[0813] In life stage:

[0814] The study included female C57BL / 6J mice (background); FcRn-deficient mice that were hemizygous for human FcRn (huFcRn, strain 276- / tg)

[0815] Part 1:

[0816] All mice were injected once intravitreally into the right eye with 2 μL / animal of the appropriate solution (i.e., 21 μg compound / animal (VEGF / ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg compound / animal (VEGF / ANG2-0016 (with IHH-AAA mutation)).

[0817] Mice were divided into 2 groups of 6 animals each. Blood samples were taken from Group 1 at 2, 24 and 96 hours after dosing and from Group 2 at 7, 48 and 168 hours after dosing.

[0818] The mice were injected into the vitreous of the right eye using a NanoFil microinjector system for nanoliter injections from World Precision Instruments, Inc., Berlin, Germany. The mice were anesthetized with 2.5% isoflurane, and a Leica MZFL3 microscope with a magnification of 40x and a ring light with a Leica KL 2500 LCD flash was used to observe the mouse eyes. Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

[0819] Blood was collected from each animal via the retroorbital venous plexus of the contralateral eye to determine compound levels in serum.

[0820] After 1 hour at room temperature, at least 50 μL of serum samples were obtained from the blood by centrifugation (9300 x g) at 4°C for 3 minutes. Serum samples were frozen immediately after centrifugation and stored frozen at -80°C until analysis. 96 hours after treatment, the treated eyes of animals in Group 1 were isolated, and 168 hours after treatment, the treated eyes of animals in Group 2 were isolated. Samples were stored frozen at -80°C until analysis.

[0821] Part 2:

[0822] All mice were injected once intravenously via the tail vein with 200 μL / animal of the appropriate solution (i.e., 21 μg compound / animal (VEGF / ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg compound / animal (VEGF / ANG2-0016 (with IHH-AAA mutation)).

[0823] Mice were divided into 2 groups of 5 animals each. Blood samples were taken from group 1 at 1, 24, and 96 hours after dosing and from group 2 at 7, 48, and 168 hours after dosing. Blood was collected from each animal via the retroorbital venous plexus to determine the compound level in serum.

[0824] After 1 hour at room temperature, at least 50 μL of serum samples were obtained from the blood by centrifugation (9300×g) for 3 minutes at 4° C. Serum samples were frozen immediately after centrifugation and stored frozen at −80° C. until analysis.

[0825] Preparation of whole eye lysate (mouse)

[0826] Eye lysates were obtained by physical-chemical disintegration of the whole eye from laboratory animals. For mechanical rupture, 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 wash buffer (Bio-Rad, Bio-Plex cell lysis kit, catalog number (Cat. No.) 171-304011). In subsequent steps, 500 μL of freshly prepared cell lysis buffer was added and a 1.5 mL tissue grinding pestle (Kimble Chase, 1.5 mL pestle, product number 749521-1500) was used to grind the eye. Subsequently, the mixture was frozen and thawed 5 times and ground again. In order to separate the lysate 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 quantitative ELISA.

[0827] analyze

[0828] The concentrations of anti-VEGF / ANG2 antibodies in mouse serum and eye lysates were determined by enzyme-linked immunosorbent assay (ELISA).

[0829] To quantify anti-VEGF / ANG2 antibodies in mouse serum samples and eye lysates, a standard solid-phase serial sandwich immunoassay was performed using biotinylated and digoxigenin-labeled monoclonal antibodies as capture and detection antibodies. To verify the integrity of the analyte's bispecificity, the biotinylated capture antibody recognized the VEGF binding site, while the digoxigenin-labeled detection antibody bound to the ANG2 binding site of the analyte. The bound immune complexes of capture antibody, analyte, and detection antibody were subsequently detected on the solid phase of a streptavidin-coated microtiter plate (SA-MTP) using horseradish peroxidase conjugated to the anti-digoxigenin antibody. After washing unbound material from the SA-MTP and adding ABTS substrate, the signal obtained was proportional to the amount of analyte bound to the SA-MTP solid phase. The measured signal of the sample was then converted to concentration by reference to a simultaneously analyzed calibrator for quantification.

[0830] In the first step, SA-MTP was shaken on a MTP shaker at 500 rpm with 100 μL / well of a 1 μg / mL biotinylated capture antibody solution (mAb <Id <vegf>Coat with M-2.45.51-IgG-Bi (DDS, anti-idiotypic antibody) for 1 hour. Simultaneously, prepare calibrators, QC samples, and samples. Dilute calibrators and QC samples into a 2% serum matrix; dilute samples until the signal is within the linear range of the calibrators.

[0831] After the SA-MTP was coated with the capture antibody, the plate was washed three times with wash buffer and 300 μL / well. Subsequently, 100 μL / well calibrators, QC samples, and samples were aspirated onto the SA-MTP and incubated again at 500 rpm for 1 hour. The analyte is now bound to the solid phase of the SA-MTP via the capture antibody via its VEGF binding site. After incubation and removal of unbound analyte by washing the plate, 100 μL / well primary detection antibody (mAb) at a concentration of 250 ng / mL was added. <Id- <ang2>>M-2.6.81-IgG-Dig (XOSu), anti-idiotypic antibody) was added to the SA-MTP. Again, the plate was 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 each well of the SA-MTP and the plate was incubated again at 500 rpm for 1 hour. After a final wash step to remove excess detection antibody, 100 μL / well of substrate (ABTS) was added. Antibody-enzyme conjugate catalyzed The color reaction of the substrate is followed by measuring the signal using an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)).

[0832] Pharmacokinetic evaluation

[0833] Pharmacokinetic parameters were calculated by non-compartmental analysis using the pharmacokinetic evaluation program WinNonlin™ (Pharsight) version 5.2.1.

[0834] result:

[0835] A) Serum concentration

[0836] table below and Figures 7B to 7C Results of serum concentrations are shown in .

[0837] Table: VEGF / ANG2-0015 (without IHH-AAA mutation): Comparison of serum concentrations after intravitreal and intravenous administration

[0838] Serum concentration after intravitreal administration Serum concentration after intravenous administration ID Average concentration [μg / mL] Average concentration [μg / mL] 1 hour 17.7 2 hours 9.8 7 hours 10.4 12.1 24 hours 6.4 8.3 48 hours 6.5 6.9 96 hours 3.4 4.1 168 hours 2.9 2.7

[0839] Table: VEGF / ANG2-0016 (with IHH-AAA mutation): Comparison of serum concentrations after intravitreal and intravenous administration

[0840]

[0841]

[0842] Table: VEGF / ANG2-0015 (without IHH-AAA mutation) and VEGF / ANG2-0016 (with IHH-AAA mutation): Comparison of serum concentrations after intravitreal administration

[0843]

[0844] Table: VEGF / ANG2-0015 (without IHH-AAA mutation) and VEGF / ANG2-0016 (with IHH-AAA mutation): Comparison of serum concentrations after intravenous administration

[0845]

[0846] result:

[0847] B) Concentrations in eye lysates from left and right eyes

[0848] table below and Figures 7D to 7E Results for concentrations in eye lysates are shown in .

[0849] Table: Concentration of VEGF / ANG2-0015 (without IHH-AAA mutation) in eye lysates after intravitreal application to the right eye

[0850]

[0851] Table: Concentration of VEGF / ANG2-0015 (without IHH-AAA mutation) in eye lysates after intravenous administration

[0852]

[0853] Table: Concentration of VEGF / ANG2-0016 (with IHH-AAA mutation) in eye lysates after intravitreal application to the right eye

[0854]

[0855] Table: Concentration of VEGF / ANG2-0016 (with IHH-AAA mutation) in eye lysates after intravenous administration

[0856]

[0857]

[0858] Summary of results:

[0859] After intravitreal application, the bispecific VEGF / ANG2 antibody VEGF / ANG2-0016 (with the IHH-AAA mutation) as reported herein showed similar concentrations in eye lysates (after 96 and 168 hours) compared to the bispecific anti-VEGF / ANG2 antibody VEGF / ANG2-0015 without the IHH-AAA mutation.

[0860] Furthermore, after intravitreal administration, the bispecific VEGF / ANG2 antibody VEGF / ANG2-0016 (with the IHH-AAA mutation) as reported herein additionally showed faster clearance in serum and a shorter half-life compared to the bispecific VEGF / ANG2 antibody VEGF / ANG2-0015 without the IHH-AAA mutation.

[0861] Example 7

[0862] Mouse corneal micropocket angiogenesis assay

[0863] To test the anti-angiogenic effects of bispecific anti-VEGF / ANG2 antibodies with the corresponding VEGF-binding VH and VL of SEQ ID NOs: 20 and 21 and the ANG2-binding VH and VL of SEQ ID NOs: 28 and 29 on VEGF-induced angiogenesis in vivo, a mouse corneal angiogenesis assay was performed. In this assay, a VEGF-impregnated Nylaflo disc was implanted into an avascular corneal pocket at a fixed distance relative to the limbal vessels. Blood vessels immediately grew into the cornea, targeting the developing VEGF gradient. 8- to 10-week-old female BALB / c mice were purchased from Charles River, Sulzfeld, Germany. The study protocol was adapted from the method described by Rogers, MS et al., Nat. Protoc. 2 (2007) 2545-2550. Briefly, micropockets approximately 500 μm wide were prepared under a microscope in anesthetized mice using a surgical blade and fine-tipped forceps, extending approximately 1 mm from the limbus to the apex of the cornea. Implant a 0.6 mm diameter disc ( Pall Corporation, Michigan) and smooth the surface of the implanted area. The discs were incubated in the corresponding growth factor or in the vehicle for at least 30 minutes. After 3, 5, and 7 days (or alternatively, only 3, 5, or 7 days), the eyes were photographed and the vascular response was measured. The assay was quantified by calculating the percentage of new vessel area / total corneal area.

[0864] The discs were loaded with 300 ng of VEGF or PBS as a control and implanted for 7 days. Blood vessels growing from the limbus to the disc were 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 (due to intravenous administration, serum-stable VEGF / ANG2-0015 (without the IHH-AAA mutation), which differs from VEGF / ANG2-0016 only by the IHH-AAA mutation and has the same VH and VL binding to VEGF and ANG-2 to mediate efficacy, was used as a surrogate) to test the anti-angiogenic effect on VEGF-induced angiogenesis in vivo. Animals in the control group received vehicle. The application volume was 10 mL / kg.

[0865] Example 8

[0866] Pharmacokinetic (PK) properties of antibodies harboring the HHY-AAA mutation

[0867] PK data of human FcRn transgenic FcRn mice

[0868] In life stage:

[0869] The study included female C57BL / 6J mice (background); mice deficient in mouse FcRn but hemizygous for human FcRn transgenes (huFcRn, strain 276- / tg)

[0870] Part 1:

[0871] All mice were injected once intravitreally into the right eye with the appropriate IGF-1R 0033, IGF-1R 0035, IGF-1R 0045 solution (i.e., 22.2 μg compound / animal for IGF-1R 0033, 24.4 μg compound / animal for IGF-1R 0035, 32.0 μg compound / animal for IGF-1R, and 32.0 μg compound / animal for IGF-1R 0045).

[0872] Thirteen mice were assigned to two groups of six and seven animals each. Blood samples were taken from Group 1 at 2, 24, and 96 hours post-dose and from Group 2 at 7, 48, and 168 hours post-dose.

[0873] The mice were injected into the vitreous of the right eye using a NanoFil microinjector system for nanoliter injections from World Precision Instruments, Inc., Berlin, Germany. The mice were anesthetized with 2.5% isoflurane, and a Leica MZFL3 microscope with a magnification of 40x and a ring light with a Leica KL 2500 LCD flash was used to observe the mouse eyes. Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

[0874] Blood was collected from each animal via the retroorbital venous plexus of the contralateral eye to determine compound levels in serum.

[0875] After 1 hour at room temperature, at least 50 μL of serum samples were obtained from the blood by centrifugation (9300 x g) at 4°C for 3 minutes. Serum samples were frozen immediately after centrifugation and stored frozen at -80°C until analysis. 96 hours after treatment, the treated eyes of animals in Group 1 were isolated, and 168 hours after treatment, the treated eyes of animals in Group 2 were isolated. Samples were stored frozen at -80°C until analysis.

[0876] Part 2:

[0877] All mice were injected once intravenously via the tail vein with the appropriate IGF-1R 0033, IGF-1R 0035, IGF-1R 0045 solution (i.e., 22.2 μg compound / animal for IGF-1R 0033, 24.4 μg compound / animal for IGF-1R 0035, 32.0 μg compound / animal for IGF-1R, and 32.0 μg compound / animal for IGF-1R 0045).

[0878] Twelve mice were divided into two groups of six animals each. Blood samples were taken from group 1 at 1, 24, and 96 hours after dosing and from group 2 at 7, 48, and 168 hours after dosing. Blood was collected from each animal via the retroorbital venous plexus to determine the compound level in serum.

[0879] After 1 hour at room temperature, at least 50 μL of serum samples were obtained from the blood by centrifugation (9300×g) for 3 minutes at 4° C. Serum samples were frozen immediately after centrifugation and stored frozen at −80° C. until analysis.

[0880] Preparation of cell lysis buffer

[0881] 100 μL Factor 1, 50 μL Factor 2 and 24.73 mL Cell Lysis Buffer (all from Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011) were carefully mixed and 125 μL PMSF-solution (174.4 mg phenylmethylsulfonyl fluoride diluted in 2.0 mL DMSO) was added.

[0882] Preparation of whole eye lysate (mouse)

[0883] Eye lysates were obtained by physical-chemical disintegration of the whole eye from laboratory animals. For mechanical rupture, each eye was transferred to a 1.5 mL conical bottom microvial. After thawing, eyes were washed once with 1 mL cell wash buffer (Bio-Rad, Bio-Plex cell lysis kit, catalog number (Cat. No.) 171-304011). In subsequent steps, 500 μL of freshly prepared cell lysis buffer was added and 1.5 mL tissue grinding pestle (VWR Int., product number 431-0098) was used to grind the eye. Subsequently, the mixture was frozen and thawed 5 times and ground again. In order to separate the lysate from the remaining tissue, the sample was centrifuged at 4500 x g for 4 minutes. After centrifugation, the supernatant was collected and stored at -20 ° C until further analyzed in quantitative ELISA.

[0884] Analysis (serum)

[0885] To quantify antibodies in mouse serum samples, a standard solid phase serial sandwich immunoassay was performed using biotinylated and digoxigenylated monoclonal antibodies as capture and detection antibodies. Serum occupied approximately 50% of the total blood sample volume.

[0886] In more detail, the concentration of antibodies in mouse serum samples was determined by a human IgG (Fab)-specific enzyme-linked immunosorbent assay. Streptavidin-coated microtiter plates were incubated with biotinylated anti-human Fab (kappa) monoclonal antibody M-1.7.10-IgG diluted in assay buffer as the capture antibody for 1 hour at room temperature with stirring. After washing three times with phosphate-buffered saline-polysorbate 20 (Tween 20), serum samples were added at various dilutions and then incubated a second time at room temperature for 1 hour. After three repeated washes, bound antibodies were detected by subsequent incubation with anti-human Fab (CH1) monoclonal antibody M-1.19.31-IgG conjugated to digoxigenin, followed by incubation with anti-digoxigenin antibody conjugated to horseradish peroxidase (HRP). ABTS (2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); Roche Diagnostics GmbH, Mannheim, Germany) was used as an HRP substrate to form a colored reaction product. The absorbance of the generated reaction product was read at 405 nm (ABTS; reference wavelength: 490 nm).

[0887] All samples, positive control samples, and negative control samples were analyzed in duplicate and calibrated against the provided antibody standards.

[0888] Analysis (eye lysate)

[0889] based on The concentrations of analytes in mouse eye lysate samples were determined using a qualified electrochemiluminescence immunoassay (ECLIA) method using a ELISA platform (Roche Diagnostics GmbH, Mannheim, Germany) under non-GLP conditions.

[0890] The undiluted supernatant (eye lysate) was incubated with capture and detection molecules at 37°C for 9 minutes. Biotinylated anti-human-Fab (kappa) monoclonal antibody M-1.7.10-IgG was used as the capture molecule and Ruthenium(II) tris(bipyridyl) 3 2+ Labeled anti-human-Fab (CH1) monoclonal antibody M-1.19.31-IgG was used for detection. Streptavidin-coated magnetic microparticles were added and incubated at 37°C for an additional 9 minutes to allow the preformed immune complex to bind due to the biotin-streptavidin interaction. The microparticles were magnetically captured on the electrode and a chemiluminescent signal was generated using the co-reactant tripropylamine (TPA). The obtained signal was measured by a photomultiplier detector.

[0891] Table: Standard Table IGF-1R 0033

[0892]

[0893] Table: Standard Table IGF-1R 0035

[0894]

[0895] Table: Standard Table IGF-1R 0045

[0896]

[0897]

[0898] result:

[0899] A) Serum concentration

[0900] table below and Figure 17 Results of serum concentrations are shown in .

[0901] Table: IGF-1R 0033 (without HHY-AAA mutation): Comparison of serum concentrations after intravitreal and intravenous administration (nd = not determined)

[0902] Serum concentration after intravitreal administration Serum concentration after intravenous administration ID Average concentration [μg / mL] Average concentration [μg / mL] 1 hour nd 34.7 2 hours 5.9 nd 7 hours 11.1 24.7 24 hours 4.4 13.6 48 hours 7.8 12.6 96 hours 2.1 8.9 168 hours 2.9 6.2

[0903] Table: IGF-1R 0035 (with HHY-AAA mutation in one Fc region polypeptide): Comparison of serum concentrations after intravitreal and intravenous administration

[0904]

[0905]

[0906] Table: IGF-1R 0045 (with HHY-AAA mutations in both Fc region polypeptides): Comparison of serum concentrations after intravitreal and intravenous administration (BLQ = below limit of quantitation)

[0907] Serum concentration after intravitreal administration Serum concentration after intravenous administration ID Average concentration [μg / mL] Average concentration [μg / mL] 1 hour nd 40.5 2 hours 13.2 nd 7 hours 9.6 21.7 24 hours 2.2 5.1 48 hours 0.9 0.7 96 hours 0.05 0.03 168 hours 0.01 BLQ

[0908] Table: Comparison of serum concentrations normalized to 1 μg of applied antibody after intravenous administration of antibodies IGF-1R 0033, 0035 and 0045

[0909]

[0910]

[0911] result:

[0912] B) Concentrations in eye lysates from left and right eyes

[0913] table below and Figures 18 to 20 Results for concentrations in eye lysates are shown in .

[0914] Table: Concentration of IGF-1R 0033 (without HHY-AAA mutation) in eye lysates after intravitreal application into right eyes

[0915]

[0916] Table: Concentration of IGF-1R 0033 (without HHY-AAA mutation) in eye lysates after intravenous application (BLQ = below limit of quantification)

[0917]

[0918] Table: Concentration of IGF-1R 0035 (with HHY-AAA mutation in one Fc region polypeptide) in ocular lysates after intravitreal application into right eyes

[0919]

[0920]

[0921] Table: Concentration of IGF-1R 0035 (with HHY-AAA mutation in one Fc region polypeptide) in eye lysates after intravenous administration (BLQ = below limit of quantification)

[0922]

[0923] Table: Concentration of IGF-1R 0045 (with HHY-AAA mutation in both Fc region polypeptides) in ocular lysates after intravitreal application into the right eye

[0924]

[0925] Table: Concentration of IGF-1R 0045 (with HHY-AAA mutation in both Fc region polypeptides) in eye lysates after intravenous administration (BLQ = below limit of quantification)

[0926]

[0927] Table: IGF-1 R0033, 0035 and 0045 concentrations in eye lysates normalized to 1 μg of applied antibody after intravitreal application into right eyes

[0928]

[0929] Summary of results:

[0930] After intravitreal application, the anti-IGF-1R antibodies 0035 and 0045 (with unilateral or bilateral HHY-AAA mutations) as reported herein showed similar concentrations in eye lysates (after 96 and 168 hours) compared to the anti-IGF-1R antibody without the HHY-AAA mutation (IGF-1R 0033).

[0931] Furthermore, after intravitreal administration, the anti-IGF-1R antibodies 0035 and 0045 (with one or both HHY-AAA mutations) as reported herein additionally showed faster clearance and a shorter half-life in serum compared to the anti-IGF-1R antibody without the HHY-AAA mutation (IGF-1R0033).

[0932] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. Sequence Listing <110> Hoffmann-La Roche GmbH <120> FC region variants with improved protein A binding <130> 31954 <150> EP14151322.6 <151> 2014-01-15 <150> EP14165924.3 <151> 2014-04-25 <160> 112 <170> PatentIn version 3.5 <210> 1 <211> 448 <212> PRT <213> Homo sapiens <400> 1 Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 2 <211> 448 <212> PRT <213> Human <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Ile Ile Trp Phe Asp Gly Ser Ser Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 12 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 3 <211> 215 <212> PRT <213> Person <400> 3 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125[[ID=​​130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 4 <211> 215 <212> PRT <213> Human <400> 4 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 118 <212> PRT <213> Human <400> 5 Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ile Ile Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 六十 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr [[ID=2۴]]65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Ser Val Ser Ser 115 <210> 6 <211> 118 <212> PRT <213> Human <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 It should be noted that there is an error in the original text where "六十" in line 20 of the translation should be "60". I have corrected it in the translation above.Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Ile Ile Trp Phe Asp Gly Ser Ser Lys Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 7 <211> 108 <212> PRT <213> Human <400> 7 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Lys Trp Pro Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys 100 105 <210> 8 <211> 108 <212> PRT <213> Human <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 ...

Claims

1. An antibody comprising A first heavy chain polypeptide and a second heavy chain polypeptide, each of which comprises, from the N-terminal to the C-terminal direction, at least a portion of an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain comprising one or more cysteine ​​residues, and the hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain belong to the IgG1 subclass; It is characterized in that The mutations H310A, H433A and Y436A in the first heavy chain polypeptide are combined with the mutations H310A, H433A and Y436A in the second heavy chain polypeptide, and the numbering is based on the EU index numbering of SEQ ID NO: 60 as the first heavy chain polypeptide and as the second heavy chain polypeptide, And wherein the antibody does not specifically bind to human FcRn and specifically binds to Staphylococcal protein A.

2. The antibody according to claim 1, characterized in that the antibody is an anti-IGF-1R antibody.

3. Use of the antibody according to claim 1 or 2 for preparing a medicament for treating ocular vascular diseases.

4. The use according to claim 3, wherein the medicament is for intravitreal administration.

5. A pharmaceutical preparation comprising the antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

6. The use according to claim 3, wherein the medicament is used to transport soluble receptor ligands from the eye through the blood-eye barrier into the blood circulation.

7. The use according to claim 3, wherein the medicament is for removing one or more soluble receptor ligands from the eye.

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