Fc region variant with modified FcRn binding properties

Abstract

To provide IgG Fc region variants whose Fc-receptor binding is modified without affecting purification characteristics thereof.SOLUTION: Disclosed is a polypeptide comprising a first polypeptide and a second polypeptide, where the first and second polypeptides respectively comprise, from N terminus to C terminus direction, at least part of an immunoglobulin hinge region containing one or more cysteine residues, immunoglobulin CH2 domain and immunoglobulin CH3 domain, where i) the first and second polypeptides comprise mutations H310A, H433A and Y436A; or ii) the first and second polypeptides comprise mutations L251D, L314D and L432D; or iii) the first and second polypeptides comprise mutations L251S, L314S and L432S.SELECTED DRAWING: None

Description

[Technology Field]

[0001] This specification reports an IgGFc region that has been modified with respect to Fc-receptor binding without impairing its purification characteristics.

[0002] Background of the Invention The demand for cost-effective manufacturing processes has led to the need to optimize downstream purification, including one or more affinity chromatography steps. Larger volumes of material to be processed and more stringent requirements for clean-in-place (CIP) protocols are some of the characteristics that need to be addressed (Hober, S., J. Chrom. B. 848 (2007) 40-47).

[0003] Purification of monoclonal antibodies with selective ligands that have affinity for the Fc region is the most promising methodology for the mass production of therapeutic monoclonal antibodies. In fact, this procedure does not require any interaction with the antigen-specific portion of the antibody, i.e., the Fab domain, and therefore the domain remains unchanged and its properties can be preserved (see Salvalaglio, M., et al., J. Chrom. A 1216 (2009) 8678-8686).

[0004] Affinity purification is used early in a series of purification operations due to its selectivity, thereby reducing the number of subsequent unit operations (see Hober; MacLennan, J., Biotechnol. 13 (1995) 1180; Harakas, NK, Bioprocess Technol. 18 (1994) 259).

[0005] The most commonly used ligands for selective binding to IgG are Staphylococcus protein A and protein G, which can establish highly selective interactions with many IgG Fc regions within a region known as the "consensus binding site" (CBS) (DeLano, WL, et al., Science 287 (2000) 1279) (located in the hinge region between the CH2 and CH3 domains of the Fc region).

[0006] Staphylococcal protein A (SPA) is a cell wall-associated protein domain exposed on the surface of Staphylococcus aureus, a Gram-positive bacterium. SPA has high affinity for IgG from various species, such as human, rabbit, and guinea pig IgG, but only weak interactions with bovine and mouse IgG (see table below) (see Hober; Duhamel, RC, et al., J. Immunol. Methods 31 (1979) 211; Bjork, 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)).

[0007] [Table 1]

[0008] The heavy chain hinge region between the CH2 and CH3 domains of IgG can bind to several proteins other than protein A, including the neonatal Fc receptor (FcRn) (see DeLano and Salvalaglio above).

[0009] SPA CBS contains a hydrophobic pocket on the antibody surface. The residues constituting the IgG CBS are Ile253, Ser254, Met252, Met423, Tyr326, His435, Asn434, His433, Arg255, and Glu380 (numbering of IgG heavy chain residues according to the Kabat EU index numbering system). Charged amino acids (Arg255, Glu380) are located around the hydrophobic knob formed by Ile253 and Ser254. This may lead to the establishment of polar and hydrophilic interactions (see Salvalaglio above).

[0010] Generally, protein A-IgG interactions can be described using two main binding sites: the first is located within the heavy chain CH2 domain and is characterized by hydrophobic interactions between Phe132, Leu136, Ile150 (of protein A) and the hydrophobic knob of IgG composed of Ile253 and Ser254, and a single electrostatic interaction between Lys154 (protein A) and Thr256 (IgG). The second site is located within 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).

[0011] Lindhofer, H., et al. (J. Immunol. 155 (1995) 219-225) reported preferential, species-restricted heavy / light chain pairing in rat / mouse quadromas.

[0012] Jedenberg, L., et al. (J. Immunol. Meth. 201 (1997) 25-34) reported that SPA-binding analysis of two Fc mutants (Fc13 and Fc31, each containing isotype dipeptide substitutions from other isotypes) showed that Fc1 and Fc31 interacted with SPA, but Fc3 and Fc13 did not exhibit detectable SPA binding. They concluded that the resulting SPA binding in the Fc region mutant Fc31 was due to the introduced dipeptide substitutions R435H and F436Y.

[0013] Today, the focus of therapeutic monoclonal antibodies is on the production and use of bispecific or multispecific antibodies that specifically bind to two or more targets (antigens).

[0014] The fundamental challenge in generating multispecific heterodimer IgG antibodies from four antibody chains (two different heavy chains and two different light chains) in a single cell line is the so-called chain association problem (see Klein, C., et al., mAbs 4 (2012) 653-663). The need to use different chains as the left and right arms of a multispecific antibody results in an antibody mixture when expressed in a single cell: the two heavy chains can associate in (theoretically) four different combinations (two of which are the same), and each of these can associate with a light chain in a stochastic manner, and 2 4 This results in a total of 16 theoretically possible chain combinations. Of these 16 theoretically possible combinations, only 10 can actually be found, and of these, only one corresponds to the desired functionally bispecific antibody (De Lau, WB, et al., J. Immunol. 146 (1991) 906-914). The difficulty in isolating this desired bispecific antibody from the complex mixture, and the inherently low yield of 12.5% ​​even at the theoretical maximum, make the production of bispecific antibodies in a single expression cell line extremely challenging.

[0015] To solve the chain association problem and enhance the correct association of two different heavy chains, in the late 1990s, Carter et al. at Genentech invented an approach called "Knobs-into-Holes" (KiH) (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 U.S. Patent No. 7183076). Essentially, this concept relies on modifying the interface between the two CH3 domains of the two heavy chains of the antibody, where the most interaction occurs. A bulky residue is introduced into the CH3 domain of one antibody heavy chain, which behaves like a key ("knob"). A "hole" is formed in the other heavy chain, which can accommodate this bulky residue, mimicking a tablet. The resulting heterodimeric Fc region can be further stabilized by introducing / forming artificial disulfide crosslinks. Notably, all KiH mutations are embedded within the CH3 domain and are not "visible" within the immune system. In addition, the properties and pharmacokinetic (PK) behavior of antibodies with KiH mutations, such as (thermal) stability, FcγR binding, and effector function (e.g., ADCC, FcRn binding), remain unaffected.

[0016] Yields exceeding 97% of correct heavy chain association with heterodimerization can be achieved by introducing six mutations: S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain (see Carter above; residue numbering by Kabat EU index numbering system). Hole-hole homodimers can occur, but knob-knob homodimers are not typically observed. Hole-hole homodimers can be deleted by selective purification procedures or by the procedures summarized below.

[0017] While the problem of random heavy chain association has been addressed, correct light chain association must also be ensured. Similar to the KiH CH3 domain approach, efforts are being made to investigate asymmetric light-heavy chain interactions that could ultimately lead to fully bispecific IgG.

[0018] In recent years, Roche has developed the CrossMab approach, which, when combined with KiH technology, has the potential to enhance correct light chain pairing in bispecific heterodimer IgG antibodies (see Klein; Schaefer. W., et al., Proc. Natl. Acad. Sci. USA 108 (2011) 11187-11192; Cain, C., SciBX 4 (2011) 1-4). This enables the generation of bispecific or multispecific antibodies in a common manner. In this form, one arm of the intended bispecific antibody remains untouched. In the second arm, the entire Fab region, or the VH-VL domain or CH1-CL domain, is exchanged for a domain crossover between the heavy and light chains. As a result, the newly formed "crossed" light chain is no longer associated with the heavy chain Fab region of the other arm of the (normally, i.e., non-crossed) bispecific antibody. Therefore, this minimal change in domain configuration can strengthen the correct "light chain" association (see Schaefer above).

[0019] Zhu et al. introduced several sterically complementary mutations together with disulfide bridges at two VL / VH interfaces of diabody variants. Introduction of the mutations VL Y87A / F98M and VH V37F / L45W at the VL / VH interface of anti-p185HER2 resulted in the recovery of heterodimeric diabody in a yield of over 90% while maintaining the overall yield and affinity compared to the parental diabody (see Zhu supra).

[0020] Researchers both at home and abroad have similarly designed bispecific diabodies by introducing mutations into the VH-VL interface to promote correct light chain association (mainly conversion of the charged residues Q39 in VH and Q38 in VL) (WO 2006 / 106905; Igawa, T., et al., Prot. Eng. Des. Sel. 23 (2010) 667-677).

[0021] WO 2011097603 reports a common light chain mouse.

[0022] WO 2010151792 provides a format of bispecific antibody that offers ease of isolation. This format includes immunoglobulin heavy chain variable domains that are separately modified (i.e., heterodimeric) in the CH3 domain, said separate modification being non-immunogenic or substantially non-immunogenic with respect to the CH3 modification, and at least one of said modifications resulting in a separate affinity for an affinity reagent such as protein A for said bispecific antibody, said bispecific antibody being isolable from disrupted cells, medium or a mixture of antibodies based on its affinity for protein A.

[0023] Neonatal Fc receptors (FcRn) are crucial to the metabolic fate of IgG class antibodies in vivo. FcRn functions to rescue IgG from the lysosomal degradation pathway, resulting in reduced clearance and prolonged half-life. It is a heterodimer protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 moiety of the Fc region of class IgG antibodies. The interaction between class IgG antibodies and FcRn is pH-dependent and occurs in a 1:2 stoichiometric ratio; that is, one IgG antibody molecule can interact with two FcRn molecules via its two heavy-chain Fc region polypeptides (see, e.g., Huber, AH, et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0024] Therefore, the in vitro FcRn binding properties / characteristics of IgG suggest its in vivo pharmacokinetic properties in blood circulation.

[0025] Various 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.

[0026] Various mutations affecting FcRn binding and their associated half-lives in blood circulation are known. Crucial Fc domain residues for mouse Fc domain-mouse FcRn interactions have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (numbered according to the Kabat EU index numbering system) are involved in the aforementioned 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 decisive for the aforementioned interaction between the human Fc region and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819-2885).

[0027] Methods have been developed to increase the binding of the Fc region (and similarly IgG) to FcRn by mutating various amino acid residues within the Fc region: Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and Asn434 (see Kuo, TT, et al., J. Clin. Immunol. 30 (2010) 777-789; Ropeenian, DC, et al., Nat. Rev. Immunol. 7 (2007) 715-725).

[0028] Protein-protein interaction studies have shown that the combination of mutants M252Y, S254T, and T256E enhances FcRn binding, as described by Dall'Acqua et al. (Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524). Studies of the human Fc domain-human FcRn complex have shown that residues I253, S254, H435, and Y436 are decisive for this interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) reported and investigated various mutants at residues 248-259, 301-317, 376-382, and 424-437.

[0029] International Publication No. 2014 / 006217 reports a dimeric protein with a triple mutation. In relation to the pH-dependent binding mechanism, the crystal structure of the FcRn / heterodimer Fc complex at 2.8 angstroms has been reported by Martin, W. et al. (Mol. Cell. 7 (2001) 867-877). US Patent No. 6277375 reports an immunoglobulin-like domain with an extended half-life, as reported in International Publication No. 2013 / 004842. Shields, RL et al. have reported high-resolution mapping of binding sites on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn, and the design of IgG1 mutants with improved binding to Fc gamma R (Biochem. Mol. Biol. 276 (2001) 6591-6604). Delineation of amino acid residues involved in the transcytosis and catabolism of mouse IgG1 has been reported by Medesan, C. et al. (J. Immunol. 158 (1997) 2211-2217). US Publication No. 2010 / 0272720 reports on an antibody fusion protein with a modified FcRn binding site. International Publication No. 2013 / 060867 reports on the production of a heterodimer protein. Qiao, S.-W. et al. have reported on the FcRn dependence of antibody-mediated antigen presentation (Proc. Natl. Acad. Sci. USA 105 (2008) 9337-9342).

[0030] Summary of the Invention This specification reports mutant Fc regions that specifically bind to Staphylococcus protein A but not to human FcRn. These mutant Fc regions contain specific amino acid mutations in the CH2 and CH3 domains. It has been found that when these mutations are used in the whole or knob chain of a heterodimeric Fc region, they enable the purification of the heterodimeric Fc region, i.e., the separation of the heterodimeric Fc region from the homodimeric Fc region.

[0031] One embodiment reported herein is a (dimer) polypeptide, The first polypeptide comprises an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, extending from the N-terminus to the C-terminus; and the second polypeptide comprises an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, extending from the N-terminus to the C-terminus. i) The first and second polypeptides each contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within at least a portion of the immunoglobulin hinge region. It is a polypeptide.

[0032] In one embodiment, the (dimeric) polypeptide does not specifically bind to human FcRn, but specifically binds to Staphylococcus protein A.

[0033] In one embodiment, the (dimer) polypeptide is a homodimer polypeptide.

[0034] In one embodiment, the (dimer) polypeptide is a heterodimer polypeptide.

[0035] In one embodiment, the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V ("hole"), and the second polypeptide further comprises the mutations S354C and T366W ("knob").

[0036] In one embodiment, the first polypeptide further comprises the mutations S354C, T366S, L368A and Y407V ("hole"), and the second polypeptide further comprises the mutations Y349C and T366W ("knob").

[0037] In one embodiment, the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain of the first and second polypeptides are of the human IgG1 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutants L234A and L235A. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutant P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise mutants L234A, L235A, and P329G.

[0038] In one embodiment, the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain of the first and second polypeptides are of the human IgG4 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutants S228P and L235E. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutant P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutants S228P, L235E, and P329G.

[0039] In one embodiment, the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain of the first and second polypeptides are of the human IgG2 subclass. In one embodiment, the first and second polypeptides each further comprise the mutants H268Q, V309L, A330S, and P331S.

[0040] In one embodiment, the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain of the first and second polypeptides are of the human IgG2 subclass. In one embodiment, the first and second polypeptides each further comprise mutants V234A, G237A, P238S, H268A, V309L, A330S, and P331S.

[0041] In one embodiment, the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain of the first and second polypeptides are of the human IgG4 subclass. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutants S228P, L234A, and L235A. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutant P329G. In one embodiment, the first polypeptide and the second polypeptide each further comprise the mutants S228P, L234A, L235A, and P329G.

[0042] In one embodiment, the first and second polypeptides contain the mutation Y436A.

[0043] In one embodiment, the (dimeric) polypeptide is an Fc-domain fusion polypeptide.

[0044] In one embodiment, the (dimeric) polypeptide is a (full-length) antibody.

[0045] 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.

[0046] 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.

[0047] In one embodiment, the (full-length) antibody is a triplicate antibody. In one embodiment, the triplicate antibody is a trivalent triplicate antibody. In one embodiment, the triplicate antibody is a tetravalent triplicate antibody.

[0048] One embodiment reported herein is the use of mutant Y436A to increase the binding of an immunoglobulin Fc region-containing (dimeric) polypeptide to protein A.

[0049] One embodiment reported herein is a (dimer) polypeptide, The first polypeptide comprises an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, extending from the N-terminus to the C-terminus; and the second polypeptide comprises an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, extending from the N-terminus to the C-terminus. The first, second, or first and second polypeptides contain mutation Y436A (numbered according to the Kabat EU index numbering system), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks. It is a polypeptide.

[0050] In one embodiment, the first and second polypeptides contain the mutation Y436A.

[0051] One embodiment reported herein is an antibody, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations S354C, T366W, L234A, L235A and P329G, or ii) The first polypeptide comprises mutations S354C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations Y349C, T366W, L234A, L235A and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0052] One embodiment reported herein is an antibody, The first polypeptide comprises, from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG1, an immunoglobulin CH2 domain of subclass IgG1, and an immunoglobulin CH3 domain of subclass IgG1. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a subclass IgG1 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations S354C, T366W, L234A, L235A and P329G, or ii) The first polypeptide comprises mutations S354C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations Y349C, T366W, L234A, L235A and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0053] One embodiment reported herein is an antibody, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutants Y349C, T366S, L368A, Y407V, S228P, L235E and P329G, and the second polypeptide comprises mutants S354C, T366W, S228P, L235E and P329G, or ii) The first polypeptide comprises mutants S354C, T366S, L368A, Y407V, S228P, L235E and P329G, and the second polypeptide comprises mutants Y349C, T366W, S228P, L235E and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0054] One embodiment reported herein is an antibody, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG4, an immunoglobulin CH2 domain of subclass IgG4, and an immunoglobulin CH3 domain of subclass IgG4. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a subclass IgG4 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutants Y349C, T366S, L368A, Y407V, S228P, L235E and P329G, and the second polypeptide comprises mutants S354C, T366W, S228P, L235E and P329G, or ii) The first polypeptide comprises mutants S354C, T366S, L368A, Y407V, S228P, L235E and P329G, and the second polypeptide comprises mutants Y349C, T366W, S228P, L235E and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0055] One embodiment reported herein is an antibody, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a first scFv. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a second scFv. A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 scFv and the second scFv specifically bind to the second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations S354C, T366W, L234A, L235A and P329G, or ii) The first polypeptide comprises mutations S354C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations Y349C, T366W, L234A, L235A and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0056] One embodiment reported herein is an antibody, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG1, an immunoglobulin CH2 domain of subclass IgG1, an immunoglobulin CH3 domain of subclass IgG1, a peptide linker, and a first scFv. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a second scFv. A third polypeptide comprising a first light chain variable domain and a subclass IgG1 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 scFv and the second scFv specifically bind to the second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations S354C, T366W, L234A, L235A and P329G, or ii) The first polypeptide comprises mutations S354C, T366S, L368A, Y407V, L234A, L235A and P329G, and the second polypeptide comprises mutations Y349C, T366W, L234A, L235A and P329G, i) The first and second polypeptides each further contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each further contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides each further contain mutants L251S, L314S, and L432S (numbered according to the Kabat EU Index Numbering System), The first polypeptide and the second polypeptide are linked by one or more disulfide crosslinks within the hinge region. It is an antibody.

[0057] One embodiment reported herein is a method for producing the (dimeric) polypeptide reported herein, comprising the following steps: a) A step of culturing mammalian cells containing one or more nucleic acids that encode the (dimer) polypeptide, b) A step of recovering the (dimer) polypeptide from the culture medium, and c) A step of purifying the (dimer) polypeptide by protein A affinity chromatography to produce the (dimer) polypeptide. This method includes [something].

[0058] One embodiment reported herein is the use of a combination of mutants H310A, H433A, and Y436A for the separation of heterodimer polypeptides from homodimer polypeptides.

[0059] One embodiment reported herein is the use of a combination of mutations L251D, L314D, and L432D for separating heterodimer polypeptides from homodimer polypeptides.

[0060] One embodiment reported herein is the use of a combination of mutants L251S, L314S, and L432S for the separation of heterodimer polypeptides from homodimer polypeptides.

[0061] One embodiment reported herein is a method for treating a patient with ophthalmic vascular disease, which involves administering a (dimeric) polypeptide or antibody reported herein to a patient requiring such treatment.

[0062] One embodiment reported herein is a (dimeric) polypeptide or antibody reported herein for intravitreal application.

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

[0064] One embodiment reported herein is a (dimeric) polypeptide or antibody for the treatment of ophthalmic vascular disease.

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

[0066] To use antibodies that target / bind to antigens present not only in the eye but also in other parts of the body, it is beneficial that the antibody has a short systemic half-life after crossing the blood-ocular barrier from the eye into the bloodstream, in order to avoid systemic side effects.

[0067] Furthermore, antibodies that specifically bind to receptor ligands are effective in treating eye diseases only if the antibody-antigen complex is removed from the eye, that is, if the antibody functions as a transport medium for the receptor ligand outside the eye, thereby inhibiting receptor signaling.

[0068] The inventors have discovered that antibodies containing an Fc region that does not bind to the human neonatal Fc receptor, i.e., the (dimeric) polypeptide reported herein, can be transported across the blood-ocular barrier. This is surprising because, although binding to FcRn is thought to be necessary for transport across the blood-ocular barrier, the aforementioned antibody does not bind to human FcRn.

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

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

[0071] One aspect reported herein is the use of (dimeric) polypeptides or antibodies reported herein for the treatment of eye diseases, particularly ocular vascular diseases.

[0072] One embodiment reported herein is the use of a (dimeric) polypeptide or antibody, as reported herein, for transporting one or more soluble receptor ligands from the intravitreous space into the blood circulation.

[0073] One embodiment reported herein is a (dimeric) polypeptide or antibody reported herein for use in the treatment of eye diseases.

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

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

[0076] One embodiment reported herein is a (dimeric) polypeptide or antibody for use in treating eye diseases, particularly ocular vascular diseases.

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

[0078] One embodiment reported herein is a method for treating an individual having an ophthalmic vascular disease, comprising the step of administering to the individual an effective amount of a (dimeric) polypeptide or antibody reported herein.

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

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

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

[0082] One embodiment reported herein is a method for transporting a soluble receptor ligand in an individual through the blood-ocular barrier into the intravitreous space or from the eye into the blood circulation, comprising the step of administering an effective amount of a (dimeric) polypeptide or antibody reported herein to the individual to transport the soluble receptor ligand through the blood-ocular barrier from the eye 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 triplicate antibody. In one embodiment, the triplicate antibody is a trivalent triplicate antibody. In one embodiment, the triplicate antibody is a tetravalent triplicate antibody.

[0085] In one embodiment, the (dimeric) polypeptide is CrossMab.

[0086] In one embodiment, the (dimeric) polypeptide is an Fc-domain fusion polypeptide.

[0087] 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.

[0088] 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.

[0089] In one embodiment, the antibody or the Fc region fusion polypeptide is of subclass IgG1. In one embodiment, the antibody or the Fc region fusion polypeptide further comprises mutations L234A and L235A. In one embodiment, the antibody or the Fc region fusion polypeptide further comprises mutation P329G.

[0090] In one embodiment, the antibody or the Fc region fusion polypeptide is of subclass IgG2. In one embodiment, the antibody or the Fc region fusion polypeptide further comprises mutants V234A, G237A, P238S, H268A, V309L, A330S and P331S.

[0091] In one embodiment, the antibody or the Fc region fusion polypeptide is of subclass IgG4. In one embodiment, the antibody or the Fc region fusion polypeptide further comprises mutations S228P and L235E. In one embodiment, the antibody or the Fc region fusion polypeptide further comprises mutation P329G. [Brief explanation of the drawing]

[0092] [Figure 1] A schematic diagram illustrating the concept and advantages of anti-VEGF / ANG2 antibodies of IgG1 or IgG4 subclasses associated with IHH-AAA mutations (a combination of mutations I253A, H310A, and H435A (numbered according to the Kabat EU Index Numbering System)). [Figure 2] Small-scale DLS-based viscosity measurement: Extrapolated viscosity at 150 mg / mL in 200 mM arginine / succinate buffer (pH 5.5) (comparison of anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutation) with reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutation)). [Figure 3] Temperature-dependent DLS agglutination (including DLS agglutination onset temperature) in 20 mM histidine buffer and 140 mM NaCl (pH 6.0) (comparison of the anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutation) reported herein with the reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutation)). [Figure 4] Storage at 40°C and 100 mg / mL for 7 days (decrease in main peak and increase in high molecular weight (HMW)) (compared to the reference antibody VEGF / ANG2-0015 (without such IHH-AAA mutation) of the anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutation) reported herein, which showed less aggregation). [Figure 5A] Steady-state FcRn affinity of VEGF / ANG2-0015 (without IHH-AAA mutation). [Figure 5B] Stationary FcRn affinity of VEGF / ANG2-0016 (with IHH-AAA mutation). [Figure 6] Measurement of Fc-gamma RIIIa interaction between VEGF / ANG2-0015 without IHH-AAA mutation and VEGF / ANG2-0016 with IHH-AAA mutation (both IgG1 subclass with P329G LALA mutation; IgG1 subclass anti-digoxigenin (anti-Dig antibody) and IgG4-based antibodies were used as controls). [Figure 7A] Outline of the principle of a pharmacokinetic (PK) ELISA assay for measuring anti-VEGF / ANG2 antibody concentrations in serum and whole eye lysates. [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. [Figure 7C] Serum concentrations after intravitreous administration: Comparison of VEGF / ANG2-0015 without IHH-AAA mutation and VEGF / ANG2-0016 with IHH-AAA mutation. [Figure 7D] Concentrations of VEGF / ANG2-0016 (with IHH-AAA mutation) in ophthalmic lysates in the right and left eyes (compared to intravenous application in the right eye only): Significant concentrations were detected only in the right eye after intravitreal application; after intravenous application, the concentration of VEGF / ANG2-0016 (with IHH-AAA mutation) could not be detected in ophthalmic lysates due to its short serum half-life. [Figure 7E] Concentrations of VEGF / ANG2-0015 (without IHH-AAA mutation) in ocular lysates in the right and left eyes (after intravitreal application to the right eye only, compared to intravenous application): After intravitreal application, the concentration of VEGF / ANG2-0015 was detectable in the right eye (and to some extent in the left eye); this indicates diffusion from the right eye to the serum and then from there to the left eye, which can be explained by the long half-life of VEGF / ANG2-0015 (without IHH-AAA mutation); even after intravenous application, significant concentrations were detected in ocular lysates of both eyes due to the diffusion of serum-stable VEGF / ANG2-0015 (without IHH-AAA mutation) into the eyes. [Figure 8] Antibodies modified for binding to FcRn showed, in SPR analysis, extended (YTE mutation) or shortened (IHH-AAA mutation) in vivo half-life, enhanced (YTE mutation) or decreased binding (IHH-AAA mutation) compared to the reference wild-type (wt) antibody, and extended or decreased retention time in FcRn column chromatography; a) PK data after a single 10 mg / kg iv bolus application to huFcRn transgenic male C57BL / 6J mice + / -276: wt IgG, and AUC data for YTE and IHH-AAA Fc region modified IgG; b) BIAcore sensorgram; c) FcRn affinity column elution; wild-type anti-IGF-1R antibody (reference), YTE-mutant anti-IGF-1R antibody, IHH-AAA-mutant anti-IGF-1R antibody. [Figure 9] Changes in retention time in FcRn affinity chromatography, depending on the number of mutations introduced into the Fc region. [Figure 10] Changes in FcRn binding depend on the asymmetric distribution of mutations introduced into the Fc region. [Figure 11] Elution chromatograms from two consecutive protein A affinity chromatography columns of a bispecific anti-VEGF / ANG2 antibody (VEGF / ANG2-0121) containing the combination of mutations H310A, H433A, and 436A in both heavy chains. [Figure 12] Elution chromatogram from a protein A affinity chromatography column of an anti-IGF-1R antibody (IGF-1R-0045) with mutations H310A, H433A, and Y436A in both heavy chains. [Figure 13] Binding of IgG Fc region-modified anti-VEGF / ANG2 antibody to protein A immobilized on a CM5 chip. [Figure 14] Elution chromatograms of various anti-VEGF / ANG2 antibodies in an FcRn affinity column. [Figure 15] Binding of various fusion polypeptides to Staphylococcus aureus protein A (SPR). [Figure 16] Binding of various anti-VEGF / ANG2 antibodies and anti-IGF-1R antibody mutants to immobilized protein A(SPR). [Figure 17] Comparison of serum concentrations of antibodies IGF-1R 0033, 0035, and 0045 after intravenous administration. [Figure 18] Comparison of concentrations of antibody IGF-1R 0033 in ophthalmic lysates after intravitreal and intravenous administration. [Figure 19] Comparison of concentrations of antibody IGF-1R 0035 in ophthalmic lysates after intravitreal and intravenous application. [Figure 20] Comparison of concentrations of antibody IGF-1R 0045 in ophthalmic lysates after intravitreal and intravenous administration.

[0093] Detailed description of the embodiments of the invention I. Definition The term "approximately" means a range of + / - 20% of the number that follows. In one embodiment, the term "approximately" means a range of + / - 10% of the number that follows. In one embodiment, the term "approximately" means a range of + / - 5% of the number that follows.

[0094] For the purposes of this specification, “acceptor human framework” is a framework containing the amino acid sequence of a light chain variable domain (VL) framework or heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence, or it may contain amino acid sequence variations. In some embodiments, the number of amino acid variations 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 has the same sequence as the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0095] A "affinity-mature" antibody is an antibody that has one or more changes in one or more hypervariable regions (HVRs) (compared to a parent antibody that does not have such changes), and in which such changes result in an improved affinity of the antibody to the antigen.

[0096] The term "modification" refers to the mutation (substitution), insertion (addition), or deletion of one or more amino acid residues in a parent antibody or fusion polypeptide, for example, a fusion polypeptide containing at least an FcRn binding site in the Fc region, in order to obtain a modified antibody or fusion polypeptide. The term "mutation" means that a specified amino acid residue is replaced with a different amino acid residue. For example, mutation L234A means that the amino acid residue lysine at position 234 in the antibody Fc region (polypeptide) is replaced with the amino acid residue alanine (lysine substitution with alanine) (numbered according to the Kabat EU index numbering system).

[0097] For use herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered using the Kabat index numbering system, which is described herein as "Kabat numbering," as found in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specifically, for the κ and λ isotype light chain constant domains CL, the Kabat numbering system from Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pp. 647-660) is used, and for the constant heavy chain domains (CH1, hinge, CH2, and CH3), the Kabat EU index numbering system (see pp. 661-723) is used.

[0098] "Naturally occurring amino acid residues" refers to amino acid residues from the group consisting of alanine (3-letter code: Ala, 1-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).

[0099] The term "amino acid mutation" refers to the substitution of at least one existing amino acid residue with another different amino acid residue (=replacing amino acid residue). The substitution amino acid residue may be a "naturally occurring amino acid residue" and is selected from the group consisting of alanine (3-letter code: Ala, 1-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V). The substitution amino acid residue may also be a "non-naturally occurring amino acid residue." For example, US Patent No. 6586207, 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, PG, ChemBioChem 11 (2002) 1135-1137; Chin, JW, et al., PICAS United States of America 99 (2002) 11020-11024; and Wang, L. and Schultz, PG, Chem. (2002) See 1-10 (all of which are incorporated herein by reference).

[0100] 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 is the insertion of one or two amino acid residues. The inserted amino acid residue can be any amino acid residue, whether naturally occurring or not.

[0101] The term "amino acid deletion" refers to the removal of at least one amino acid residue at a specific position in an amino acid sequence.

[0102] As used herein, the term "ANG-2" refers to human angiopoietin 2 (ANG-2) (or abbreviated as ANGPT2 or ANG2) (SEQ ID NO: 31), as 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 were discovered as ligands for Ties (a family of tyrosine kinases selectively expressed in vascular endothelial cells) (Yancopoulos, GD, et al., Nature 407 (2000) 242-48). There are currently four distinct members in the angiopoietin family. Angiopoietin 3 and 4 (Ang-3 and Ang-4) can represent widely diverged counterparts at the same locus in mouse and human (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 agonist and antagonist, respectively, in tissue culture experiments (see Davis, S., et al., Cell 87 (1996) 1161-1169 for ANG-1; and Maisonpierre, PC, et al., Science 277 (1997) 55-60 for ANG-2). All known angiopoietins primarily bind to Tie2 (SEQ ID NO: 33), while both Ang-1 and Ang-2 bind to Tie2 with an affinity of 3nM(Kd) (Maisonpierre, PC, et al., Science 277 (1997) 55-60).

[0103] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including (but not limited to) monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, tripspecific antibodies), and antibody fragments, as long as they exhibit desired antigen- and / or protein A and / or FcRn-binding activity.

[0104] 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.

[0105] The term “asymmetric Fc region for FcRn binding” refers to an Fc region consisting of two polypeptide chains having different amino acid residues at corresponding positions (determined by the Kabat EU index numbering system), where these differing positions affect the binding of the Fc region to the human neonatal Fc receptor (FcRn). For the purposes of this specification, the difference between the two polypeptide chains in the Fc region of an “asymmetric Fc region for FcRn binding” does not include differences introduced to promote the formation of a heterodimerized Fc region (e.g., for the production of bispecific antibodies). These differences can also be asymmetric, i.e., the two chains differ at non-corresponding amino acid residues according to the Kabat EU index numbering system. These differences promote heterodimerization and decrease homodimerization. An example of such a difference is the so-called “knobs into holes” substitution (see, e.g., U.S. Patent No. 7,695,936 and U.S. Publication No. 2003 / 0078385). The following knob and hole substitutions in the Fc region of subclass IgG1 IgG antibodies on individual polypeptide chains have been found to increase heterodimerization: 1) Y407T on one chain and T366Y on the other; 2) Y407A on one chain and T366W on the other; 3) F405A on one chain and T394W on the other; 4) F405W on one chain and T394S on the other; 5) Y407T on one chain and others 1) T366Y in one chain; 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, of which the last one is particularly suitable. In addition, changes that create new disulfide bridges between two Fc domain polypeptide chains promote heterodimer formation (see, for example, U.S. Public Statement 2003 / 0078385).The following substitutions in the Fc region of IgG antibodies of subclass IgG1, resulting in moderately spaced cysteine ​​residues for the formation of new intrachain disulfide bonds in individual polypeptide chains, have been found to increase heterodimerization: Y349C on one chain and S354C on the other; Y349C on one chain and E356C on the other; Y349C on one chain and E357C on the other; L351C on one chain and S354C on the other; T394C on one chain and E397C on the other; or D399C on one chain and K392C on the other. Further examples of amino acid changes that promote heterodimerization are so-called "charge pair substitutions" (see, e.g., International Publication No. 2009 / 089004). The following charge pair substitutions in the Fc region of subclass IgG1 IgG antibodies in individual polypeptide chains have been found to increase heterodimer formation: 1) K409D or K409E in one chain and D399K or D399R in the other; 2) K392D or K392E in one chain and D399K or D399R in the other; 3) K439D or K439E in one chain and E356K or E356R in the other; 4) K370D or K370E in one chain and E357K or E357R in the other; 5) K409D and K360D in one chain and D399K and E356K in the other; 6) K409D and K370D in one chain and 7) K409D and K392D in one chain, and D399K, E356K 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) K 409D and K392D, 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 in the other chain.

[0106] The term "binding (to an antigen)" means, in an in vitro assay, in one embodiment, the binding of an antibody to its antigen in a binding assay where the antibody binds to a surface and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). The binding is -8 ≤ 10 -13 -10 -8 M, in some embodiments ≤ 10 -13 -10 -9 M of binding affinity (K D ).

[0107] Binding can be examined by a BIAcore assay (GE Healthcare Biosensor AB, Uppsala, Sweden). The binding affinity is defined by terms k a (rate constant for the association of the antibody from the antibody / antigen complex), k d (dissociation constant) and K D (k d / k a ).

[0108] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is from a particular source or species, while the remaining portion of the heavy and / or light chain is from a different source or species.

[0109] The term "CH2 domain" means the portion of the antibody heavy chain polypeptide that extends from approximately 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 E STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAK.

[0110] The term "CH3 domain" refers to the portion of the antibody heavy chain polypeptide that extends approximately from EU position 341 to EU position 446. In one embodiment, the CH3 domain has the amino acid sequence SEQ ID NO: 10: GQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG.

[0111] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0112] The term "comparable length" means that two polypeptides may contain the same number of amino acid residues, or they may differ in length by one or more, but up to 10, amino acid residues. In one embodiment, the (Fc region) polypeptides contain the same number of amino acid residues, or differ by only 1 to 10 amino acid residues. In one embodiment, the (Fc region) polypeptides contain the same number of amino acid residues, or differ by only 1 to 5 amino acid residues. In one embodiment, the (Fc region) polypeptides contain the same number of amino acid residues, or differ by only 1 to 3 amino acid residues.

[0113] "Effector function" refers to the biological activity that may be caused by the Fc region of an antibody, which varies depending on the class of antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0114] The "effective dose" of a drug, such as a prescription medication, refers to the amount that is effective in achieving the desired therapeutic or prophylactic outcome in the required dosage and duration.

[0115] The term "Fc-fusion polypeptide" refers to a fusion of a binding domain (e.g., an antigen-binding domain in a single-chain antibody, or a polypeptide like a receptor ligand) with an antibody Fc region that exhibits desired target, protein A-, and FcRn-binding activity.

[0116] The term "human-derived Fc region" refers to the C-terminal region of a human-derived immunoglobulin heavy chain, containing 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 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, provided that the C-terminal lysine (Lys447) of the Fc region is present or absent.

[0117] The term "FcRn" refers to the human neonatal Fc receptor. FcRn functions to rescue IgG from the lysosomal degradation pathway, resulting in reduced clearance and an extended half-life. FcRn is a heterodimer protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 moiety of the Fc region of IgG. The interaction between IgG and FcRn is strictly pH-dependent and occurs in a 1:2 stoichiometric ratio, with one IgG molecule binding to two FcRn molecules via two heavy chains (Huber, AH, et al., J. Mol. Biol. 230 (1993) 1077-1083). At acidic pH (pH < 6.5), FcRn binding occurs within endosomes, while at the neutral cell surface (pH approximately 7.4), IgG is released. The pH-sensitive nature of the interaction facilitates FcRn-mediated protection of IgG, which is introduced into the cell by binding to receptors in the acidic environment of endosomes, from intracellular degradation. Subsequently, FcRn promotes the recirculation of IgG to the cell surface and the subsequent release of the FcRn-IgG complex into the bloodstream when exposed to the extracellular neutral pH environment.

[0118] The term "FcRn binding region of the Fc domain" refers to the portion of the antibody heavy chain polypeptide extending approximately from EU position 243 to EU position 261, approximately from EU position 275 to EU position 293, approximately from EU position 302 to EU position 319, approximately from EU position 336 to EU position 348, approximately from EU position 367 to EU position 393 and EU position 408, and approximately from EU position 424 to EU position 440. In one embodiment, one or more of the following amino acid residues, according to Kabat's EU numbering, are altered: F243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F27 5, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P29 1, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314 , N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345 , P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbering).

[0119] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR sequence and FR sequence are generally found in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0120] The term "full-length antibody" means an antibody having a structure substantially similar to a native antibody structure containing four polypeptides, or having a heavy chain containing an Fc region as defined herein. A full-length antibody may contain additional domains, such as scFv or scFab, conjugated with one or more of the full-length antibody chains. These conjugates are also included in the term "full-length antibody."

[0121] The term "dimeric polypeptide" refers to a complex comprising at least two polypeptides covalently associated. This complex may also contain further polypeptides covalently or noncovalently associated with other polypeptides. In one embodiment, the dimeric polypeptide comprises two or four polypeptides.

[0122] The term "heterodimer" or "heterodimer" means a molecule comprising two polypeptides (e.g., of comparable length), wherein the two polypeptides have amino acid sequences having at least one different amino acid residue at corresponding positions, and the corresponding positions are determined by the Kabat EU index numbering system.

[0123] The terms "homodimer" and "of a homodimer" refer to a molecule containing two polypeptides of comparable length, wherein the two polypeptides have identical amino acid sequences at corresponding positions, and these corresponding positions are determined by the Kabat EU index numbering system.

[0124] The dimerized polypeptides reported herein may be homodimers or heterodimers, which is determined with respect to the focused mutation or characteristic. For example, with respect to FcRn and / or protein A binding (i.e., the characteristic is focused), the dimerized polypeptide is homodimer with respect to the mutations H310A, H433A, and Y436A (these mutations are focused with respect to the FcRn and / or protein A binding characteristic of the dimerized polypeptide) (i.e., both polypeptides of the dimerized polypeptide contain these mutations), but at the same time, it is also heterodimer with respect to the mutations Y349C, T366S, L368A, and Y407V (these mutations are not focused because they are aimed at heterodimerization of the dimerized polypeptide, not FcRn / protein A binding characteristic) and the mutations S354C and T366W (the first set is contained only in the first polypeptide, but the second set is contained only in the second polypeptide). Furthermore, for example, the dimerized polypeptides reported herein are heterodimers with respect to the mutations I253A, H310A, H433A, H435A, and Y436A (i.e., all of these mutations are directed toward the FcRn and / or protein A binding properties of the dimerized polypeptide), meaning that one polypeptide may contain the mutations I253A, H310A, and H435A, while the other polypeptide may contain the mutations H310A, H433A, and Y436A.

[0125] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which foreign nucleic acids have been introduced, including their offspring. Host cells include “transformed organisms” and “transformed cells,” and include primary transformed cells and their offspring regardless of the number of passages. The nucleic acid content of the offspring does not have to be exactly the same as that of the parent cell and may contain mutations. Offspring of mutants having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0126] A "human antibody" is one that has an amino acid sequence corresponding to an antibody produced by a human or human cell, or an amino acid sequence corresponding to an antibody derived from a non-human source utilizing the human antibody repertoire or other human antibody coding sequences. Humanized antibodies containing non-human antigen-binding residues are specifically excluded from this definition of a human antibody.

[0127] The "Human Consensus Framework" is a framework that indicates the most frequently found amino acid residues when selecting human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Bethesda MD (1991), NIH Publication 91-3242, Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I as described by Kabat et al. In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al.

[0128] The term "derived" means that an amino acid sequence is derived from a parent amino acid sequence by introducing a change at at least one position. Therefore, the derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbered by the Kabat EU index for the antibody Fc region). In one embodiment, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 15 amino acid residues at the corresponding position. In one embodiment, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 10 amino acid residues at the corresponding position. In one embodiment, an amino acid sequence derived from a parent amino acid sequence differs by 1 to 6 amino acid residues at the corresponding position. Similarly, the derived amino acid sequence has high amino acid sequence identity with respect to its parent amino acid sequence. In one embodiment, an amino acid sequence derived from a parent amino acid sequence has 80% or more amino acid sequence identity. In one embodiment, an amino acid sequence derived from a parent amino acid sequence has 90% or more amino acid sequence identity. In one embodiment, an amino acid sequence derived from a parent amino acid sequence has 95% or more amino acid sequence identity.

[0129] The term "human Fc region polypeptide" refers to an amino acid sequence identical to that of a "native" or "wild-type" human Fc region polypeptide. The term "mutant (human) Fc region polypeptide" refers to an amino acid sequence derived from a "native" or "wild-type" human Fc region polypeptide due to at least one "amino acid change." A "human Fc region" consists of two human Fc region polypeptides. Since a "mutant (human) Fc region" consists of two human Fc region polypeptides, it is possible for both to be mutant (human) Fc region polypeptides, or for one to be a human Fc region polypeptide and the other to be a mutant (human) Fc region polypeptide.

[0130] In one embodiment, the human Fc region polypeptide has the amino acid sequence of the human IgG1 Fc region polypeptide of SEQ ID NO: 60, or the human IgG2 Fc region polypeptide of SEQ ID NO: 61, or the human IgG4 Fc region polypeptide of SEQ ID NO: 63, with the mutations reported herein. In one embodiment, the mutant (human) Fc region polypeptide is derived from the Fc region polypeptide of SEQ ID NO: 60, 61, or 63, and has at least one amino acid mutation compared to the Fc region polypeptide of SEQ ID NO: 60, 61, or 63. In one embodiment, the mutant (human) Fc region polypeptide contains / has about 1 to about 10 amino acid mutations, or in one embodiment, about 1 to about 5 amino acid mutations. In one embodiment, the mutant (human) Fc region polypeptide has at least about 80% homology to the human Fc region polypeptide of SEQ ID NO: 60, 61, or 63. In one embodiment, the mutant (human) Fc region polypeptide has at least about 90% homology to the human Fc region polypeptide of SEQ ID NO: 60, 61, or 63. In one embodiment, the mutant (human) Fc region polypeptide has at least about 95% homology to the human Fc region polypeptide of SEQ ID NO: 60, 61, or 63.

[0131] The variant (human) Fc-region polypeptides derived from the human Fc-region polypeptide of SEQ ID NO: 60, 61, or 63 are defined by the amino acid changes they contain. For example, the term P329G refers to the variant (human) Fc-region polypeptide derived from the human Fc-region polypeptide of SEQ ID NO: 60, 61, or 63, which involves a mutation from proline to glycine at amino acid position 329.

[0132] For all positions considered in this invention, numbering is based on the Kabat EU index numbering system.

[0133] The human IgG1Fc region polypeptide has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 60)

[0134] Fc region polypeptides derived from human IgG1Fc regions with mutations L234A and L235A have the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 64)

[0135] Fc region polypeptides derived from the human IgG1Fc region with mutations Y349C, T366S, L368A, and Y407V have the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 65)

[0136] The Fc region polypeptide derived from the human IgG1Fc region with mutations S354C and T366W has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 66)

[0137] Fc region polypeptides derived from the human IgG1Fc region with mutations L234A, L235A, and Y349C, T366S, L368A, and Y407V have the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 67)

[0138] Fc region polypeptides derived from human IgG1Fc regions with mutations L234A, L235A, S354C, and T366W have the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 68)

[0139] The Fc region polypeptide derived from the human IgG1Fc region with mutation P329G has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 69)

[0140] Fc region polypeptides derived from the human IgG1Fc region with mutations L234A, L235A, and P329G have the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 70)

[0141] Fc region polypeptides derived from the human IgG1Fc region with mutations P239G and Y349C, T366S, L368A, and Y407V have the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 71)

[0142] The Fc region polypeptide derived from the human IgG1Fc region with mutations P329G and S354C, T366W has the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 72)

[0143] Fc region polypeptides derived from human IgG1Fc regions with mutations L234A, L235A, P329G, and Y349C, T366S, L368A, Y407V have the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 73)

[0144] Fc region polypeptides derived from the human IgG1 Fc region with mutations L234A, L235A, P329G and S354C, T366W have the following amino acid sequences: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 74)

[0145] The human IgG4Fc region polypeptide has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 63)

[0146] The Fc region polypeptide derived from the human IgG4Fc region with mutations S228P and L235E has the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 75)

[0147] Fc region polypeptides derived from the human IgG4Fc region with mutations S228P, L235E, and P329G have the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 76)

[0148] The Fc region polypeptide derived from the human IgG4Fc region with mutations S354C and T366W has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 77)

[0149] Fc region polypeptides derived from the human IgG4Fc region with mutations Y349C, T366S, L368A, and Y407V have the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 78)

[0150] Fc region polypeptides derived from the human IgG4Fc region with mutations S228P, L235E, S354C, and T366W have the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 79)

[0151] Fc region polypeptides derived from the human IgG4Fc region with mutations S228P, L235E, and Y349C, T366S, L368A, and Y407V have the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 80)

[0152] The Fc region polypeptide derived from the human IgG4Fc region with mutation P329G has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 81)

[0153] Fc region polypeptides derived from the human IgG4Fc region with mutations P239G and Y349C, T366S, L368A, and Y407V have the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 82)

[0154] The Fc region polypeptide derived from the human IgG4Fc region with mutations P329G and S354C, T366W has the following amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 83)

[0155] Fc region polypeptides derived from the human IgG4Fc region with mutations S228P, L235E, P329G and Y349C, T366S, L368A, Y407V have the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 84)

[0156] Fc region polypeptides derived from the human IgG4Fc region with mutations S228P, L235E, P329G and S354C, T366W have the following amino acid sequence: ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID 85)

[0157] The alignments of various human Fc regions are shown below (Kabat EU Index Numbering System): [Table 2]

[0158] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human HVR and amino acid residues derived from a human FR. In certain embodiments, a humanized antibody contains at least one, typically two, substantially all of the variable domains, where all or substantially all of the HVR (e.g., CDR) corresponds to that of a non-human antibody, and all or substantially all of the FR corresponds to that of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0159] As used herein, the terms “hypervariable region” or “HVR” refer to each region of an antibody variable domain whose sequence is hypervariable (“complementarity-determining region” or “CDR”), and which forms a structurally distinct loop (“hypervariable loop”) and / or contains an antigen contact residue (“antigen contact”). Generally, an antibody contains six HVRs; three are located in the VH region (H1, H2, H3) and three are located in the VL region (L1, L2, L3). As used herein, HVR includes the following: (a) Hypervariable loops appearing at amino acid residues 26–32 (L1), 50–52 (L2), 91–96 (L3), 26–32 (H1), 53–55 (H2), and 96–101 (H3) (Chothia, C. and Lesk, AM, J. Mol. Biol. 196 (1987) 901–917); (b) CDRs appearing at amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35B (H1), 50–65 (H2), and 95–102 (H3) (Kabat, 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). (c) Antigen contacts appearing at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b) and / or (c) containing 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).

[0160] Unless otherwise specified, in this specification, HVR residues and other residues (e.g., FR residues) in variable domains are numbered according to the Kabat EU index numbering system (Kabat et al.).

[0161] As used herein, the term "IGF-1R" refers to any native IGF-1R from any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," i.e., untreated IGF-1R, as well as any form of IGF-1R resulting from intracellular processing. The term also encompasses naturally occurring variants of IGF-1R, such as splice variants or allele variants. The amino acid sequence of human IGF-1R is shown in SEQ ID NO: 11.

[0162] The “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.

[0163] "Isolated" antibodies are those separated from components in their natural environment. In some embodiments, antibodies are purified to a purity of 95% or greater (determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., size exclusion chromatography, ion exchange, or reverse-phase HPLC)). For an overview of antibody purity assessment methods, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0164] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components in their natural environment. Isolated nucleic acids also include nucleic acid molecules found in cells, such as those that normally contain the nucleic acid molecule but are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0165] "Isolated nucleic acid encoding an anti-IGF-1R antibody" refers to one or more nucleic acid molecules encoding the antibody heavy chain and light chain (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).

[0166] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting this population are identical and / or bind to the same epitope (excluding potentially mutant antibodies, e.g., those containing naturally occurring mutations or those arising during the manufacture of monoclonal antibody preparations (generally present in trace amounts)). Typically, in contrast to polyclonal antibody preparations, which contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation directs toward a single determinant on an antigen. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including (but not limited to) hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0167] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, a native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0168] The term "package insert" is used to refer to the instruction manual that is typically included in the market packaging of a therapeutic product and contains information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or precautions for use of the therapeutic product.

[0169] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after the sequences have been aligned and gaps introduced where necessary to obtain the maximum possible sequence identity percentage (conservative substitutions are not considered part of the sequence identity). Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the sequence comparison computer program ALIGN-2 is used to generate the amino acid sequence identity % value. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been registered with the U.S. Copyright Office (Washington DC, 20559) under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0170] When using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence A to a given amino acid sequence B, or with respect to a given amino acid sequence B (this can also be rephrased as a given amino acid sequence A having or containing a certain amino acid sequence identity percentage to, with, or with respect to a given amino acid sequence B) is calculated as follows: Fraction X / Y×100 In the formula, X is the number of amino acid residues that the sequence alignment program ALIGN-2 scored as an identical match in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if 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 is not equal to the amino acid sequence identity % of B to A. Unless otherwise specifically stated, all amino acid sequence identity % values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0171] The term "pharmaceutical prescription" refers to a preparation in which the biological activity of the active ingredient contained therein can be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject receiving the prescription.

[0172] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation 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.

[0173] As used herein, the term "peptidolinker" means, in one embodiment, a peptide having an amino acid sequence of synthetic origin. In one embodiment, the peptidolinker is a peptide having an amino acid sequence of at least 30 amino acids in length, and in another embodiment, 32 to 50 amino acids in length. In one embodiment, the peptidolinker is a peptide having an amino acid sequence of 32 to 40 amino acids in length. In one embodiment, the peptidolinker is (GxS)n(G=glycine, S=serine, (x=3, n=8,9 or 10) or (x=4 and n=6,7 or 8)), in one embodiment, x=4, n=6 or 7, and in another embodiment, x=4, n=7. In one embodiment, the peptidolinker is (G4S)6G2.

[0174] As used herein, the term “recombinant antibody” means all antibodies (chimeric, humanized, and human) prepared, expressed, produced, or isolated by recombinant means. This includes antibodies isolated from host cells such as NS0 or CHO cells, or from animals transgenic to human immunoglobulin genes (e.g., mice), or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant antibodies have rearranged variable and constant regions. Recombinant antibodies can be subjected to in vivo somatic hypermutation. Therefore, the amino acid sequences of the VH and VL regions of recombinant antibodies are derived from, or related to, human germline VH and VL sequences, but may not naturally exist in the in vivo human antibody germline repertoire.

[0175] As used herein, “treatment” (and its grammatical variations such as “to treat” or “to treat”) refers to a clinical intervention that seeks to alter the natural course of the treated individual, and may be performed for preventive purposes or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, reducing symptoms, minimizing the direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and achieving remission or improving prognosis. In some embodiments, antibodies or Fc region fusion polypeptides reported herein are used to delay the onset or progression of disease.

[0176] As used in this application, the term "valency" refers to the presence of a specific number of binding sites in the (antibody) molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites in the (antibody) molecule, respectively. The bispecific antibodies reported herein are in one preferred embodiment, "bivalent."

[0177] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain that is involved in the binding of an antibody to its antigen. The variable domains of the antibody heavy and light chains (VH and VL, respectively) generally have similar structures, and each domain contains four framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt, TJ et al. Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), page 91). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by using the VH or VL domain from the antibody that binds to that antigen to screen for complementary libraries of VL or VH domains, respectively (see, for example, Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0178] 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 hemangioma hyperplasia, macular telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and retinal degeneration (see, for example, Garner, A., Vascular diseases, In: Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, GK, (eds.), 2nd edition, Marcel Dekker, New York (1994), pp.1625-1710).

[0179] As used herein, the term "vector" refers to a nucleic acid molecule that has the ability to propagate another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, as well as vectors that have been incorporated into the genome of a host cell into which they are introduced. Certain vectors have the ability to direct the expression of the nucleic acid to which they are operationally linked. Such vectors are referred to herein as "expression vectors."

[0180] 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 related 121, 189, and 206 vascular endothelial cell growth factor isoforms (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)). (as described in 20017-20024); refers to the naturally occurring alleles and processed forms of those 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 isolated from several sources and containing several isoforms. VEGF exhibits highly specific pro-mitotic activity in endothelial cells.

[0181] As used herein, the term "with mutation IHH-AAA" refers to the combination of mutations I253A (Ile253Ala), H310A (His310Ala), and H435A (His435Ala); as used herein, the term "with mutation HHY-AAA" refers to the combination of mutations H310A (His310Ala), H433A (His433Ala), and Y436A (Tyr436Ala); and as used herein, the term "with mutation YTE" refers to the combination of mutations M252Y (Met252Tyr), S254T (Ser254Thr), and T256E (Thr256Glu) (within the constant heavy chain region of the IgG1 or IgG4 subclass), where the numbering is according to the Kabat EU index numbering system.

[0182] As used herein, the term "with mutation P329GLALA" refers to the combination of mutations L234A (Leu235Ala), L235A (Leu234Ala), and P329G (Pro329Gly) in the constant heavy chain region of the IgG1 subclass, where the numbering is according to the Kabat EU Index Numbering System. As used herein, the term "with mutation SPLE" refers to the combination of mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region of the IgG4 subclass, where the numbering is according to the Kabat EU Index Numbering System. As used herein, the term "with mutation SPLE and P329G" refers to the combination of mutations S228P (Ser228Pro), L235E (Leu235Glu), and P329G (Pro329Gly) in the constant heavy chain region of the IgG4 subclass, where the numbering is based on the Kabat EU index numbering system.

[0183] II. Compositions and Methods In one embodiment, the present invention is based on the discovery that certain mutations or combinations of mutations that affect the binding of the immunoglobulin Fc region to the neonatal Fc receptor (FcRn), i.e., reduce or eliminate the binding of the Fc region to FcRn, do not simultaneously eliminate the binding of the Fc region to Staphylococcus protein A. This has a significant impact on the purification methods that can be employed, for example, by eliminating the need for specific, species-restricted affinity chromatography materials (such as KappaSelect, which binds only to antibodies containing kappa light chains). Thus, the combinations of mutations reported herein make it possible to maintain binding to Staphylococcus protein A while simultaneously reducing or even eliminating binding to FcRn.

[0184] In one embodiment, the present invention is based in part on the discovery that by using various mutations in the Fc region of each heavy chain, it is possible to provide heterodimer molecules such as bispecific antibodies, in which binding to FcRn is reduced or even eliminated on the one hand, but on the other hand, binding ability to Staphylococcus protein A is maintained. This binding to Staphylococcus protein A can be used to separate heterodimer molecules from homodimer by-products. For example, by using a knobs-into-hole approach, combining mutations I253A, H310A, and H435A in one heavy chain Fc region with mutations H310A, H433A, and Y436A in the other heavy chain Fc region, a heterodimer Fc region can be obtained that does not bind to FcRn (the mutations in both sets are silent with respect to human FcRn) but maintains binding to Staphylococcus protein A (the heavy chain Fc region with mutations I253A, H310A, and H435A does not bind to FcRn and does not bind to Staphylococcus protein A, while the heavy chain Fc region with mutations H310A, H433A, and Y436A does not bind to FcRn but still binds to Staphylococcus protein A). Therefore, the homodimeric hole-hole byproduct no longer binds to Staphylococcus protein A and can be removed using standard protein A affinity chromatography. Thus, by combining the knobs-into-holes approach with the mutations I253A, H310A, and H435A in the hole chain and the mutations H310A, H433A, and Y436A in the knob chain, the purification / isolation of heterodimeric knobs-into-holes products from homodimeric hole-hole byproducts can be accelerated.

[0185] In one embodiment, the present invention is based on the discovery that antibodies for intravitreal application that do not have FcRn binding are beneficial in part, because these antibodies can cross the blood-ocular barrier, do not have substantially prolonged or shortened half-lives in the eye, and are rapidly removed from the blood circulation, thus causing no or very limited systemic side effects outside the eye. The antibodies of the present invention are useful, for example, in the diagnosis or treatment of ocular vascular diseases.

[0186] The present invention is based on the discovery that, at least in part, by using various mutations within each Fc region polypeptide of the Fc region, it is possible to provide a tailor-made heterodimer molecule having FcRn-bonds, such as a bispecific antibody, and thereby provide an antibody with a tailor-made systemic half-life.

[0187] Mutations I253A, H310A, H435A, or combinations of L251D, L314D, L432D, or L251S, L314S, L432S result in loss of binding to protein A, while mutations I253A, H310A, H435A, or combinations of H310A, H433A, Y436A, or combinations of L251D, L314D, L432D result in loss of binding to the human neonatal Fc receptor.

[0188] The table below provides an exemplary overview of amino acid residues within the Fc region that are involved in or have been altered to modify the interaction.

[0189] [Table 3]

[0190] The modifications reported herein alter the binding specificity for one or more Fc receptors, such as human FcRn. Simultaneously, some mutations that alter binding to human FcRn do not alter binding to Staphylococcus protein A.

[0191] In one embodiment, the combination of mutations reported herein alters, or substantially alters, the serum half-life of the dimer polypeptide compared to the corresponding dimer polypeptide lacking this combination of mutations. In one embodiment, the combination of mutations does not further alter, or substantially alter, the binding of the dimer polypeptide to Staphylococcus protein A compared to the corresponding dimer polypeptide lacking this combination of mutations.

[0192] A. Neonatal Fc-receptor (FcRn) The neonatal Fc receptor (FcRn) is crucial to the metabolic fate of IgG class antibodies in vivo. FcRn functions to rescue wild-type IgG from the lysosomal degradation pathway, resulting in reduced clearance and prolonged half-life. It is a heterodimer protein composed of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein (α-FcRn) and a 15 kDa β2-microglobulin (β2m). FcRn binds with high affinity to the CH2-CH3 moiety of the Fc region of class IgG antibodies. The interaction between class IgG antibodies and FcRn is pH-dependent and occurs in a 1:2 stoichiometric ratio; that is, one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc region polypeptides (see, for example, Huber, AH, et al., J. Mol. Biol. 230 (1993) 1077-1083).

[0193] Therefore, the in vitro FcRn binding properties of IgG suggest its in vivo pharmacokinetic properties in blood circulation.

[0194] The interaction between FcRn and the Fc region of IgG class antibodies involves various amino acid residues in the heavy chain CH2 and CH3 domains. The amino acid residues that interact with FcRn are located approximately between EU positions 243 and 261, between EU positions 275 and 293, between EU positions 302 and 319, between EU positions 336 and 348, between EU positions 367 and 393, EU position 408, and between EU positions 424 and 440. More specifically, the interaction between the Fc region and FcRn involves the following amino acid residues according to Kabat's EU numbering: F243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F 275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P 291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L3 14, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E3 45, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W3 81, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 are involved.

[0195] Site-directed mutagenesis studies have shown that the definitive binding sites in the Fc region of IgG are histidine 310, histidine 435, and isoleucine 253, and to a lesser degree, histidine 433 and tyrosine 436 (see, for example, 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).

[0196] Methods have been developed to increase the binding of IgG to FcRn by mutating IgG at various amino acid residues: threonine 250, methionine 252, serine 254, threonine 256, threonine 307, glutamic acid 380, methionine 428, histidine 433, and asparagine 434 (see Kuo, TT, et al., J. Clin. Immunol. 30 (2010) 777-789).

[0197] In some cases, antibodies with a short half-life in blood circulation are desirable. For example, intravitreal drugs should have a long half-life in the patient's eye and a short half-life in blood circulation. Such antibodies also have the advantage of increased exposure at the disease site, such as within the eye.

[0198] Various mutations affecting FcRn binding and their associated half-lives in blood circulation are known. Crucial Fc domain residues for mouse Fc domain-mouse FcRn interactions have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering by Kabat) are involved in the aforementioned 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 decisive for the aforementioned interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819-2885). Protein-protein interaction studies have shown that residues M252Y, S254T, and T256E enhance FcRn binding, as reported by Dall'Acqua et al. (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 decisive for the aforementioned interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437. Exemplary mutants and their effects on FcRn binding are listed in the table below.

[0199] [Table 4]

[0200] It was found that a single mutation in one Fc region polypeptide was sufficient to significantly weaken binding. The more mutations introduced into the Fc region, the weaker the binding to FcRn. However, a single asymmetric mutation is not sufficient to completely inhibit FcRn binding. Both mutations are necessary to completely inhibit FcRn binding.

[0201] The table below shows the results of symmetry manipulation of the IgG1 Fc region that affects FcRn binding (matching of mutations and retention times on the FcRn-affinity chromatography column).

[0202] [Table 5]

[0203] A retention time of less than 3 minutes corresponds to no bonding, which is because the substance is present in the pass-through fraction (void peak).

[0204] The single H310A mutation is the least influential symmetric mutation in removing any FcRn binding.

[0205] Symmetric single mutations I253A and H435A result in a relative retention time change of 0.3–0.4 minutes. This is generally recognized as undetectable binding.

[0206] The single mutation Y436A results in a detectable interaction strength with an FcRn affinity column. While not limited to this theory, this mutation may have a distinguishable effect on the in vivo FcRn-mediated half-life compared to zero interactions such as those seen with the combination of mutations I253A, H310A, and H435A (IHH-AAA mutation).

[0207] The results obtained for the symmetrically modified anti-HER2 antibody are shown in the table below (see International Publication No. 2006 / 031370 for reference).

[0208] [Table 6]

[0209] The effects of asymmetric mutagenesis affecting FcRn binding in the Fc region have been illustrated for bispecific antibodies assembled using the knobs-into-holes technique (see, for example, U.S. Patent No. 7,695,936 and U.S. Publication No. 2003 / 0078385; "hole chain" mutation: S354C / T366W, "knob chain" mutation: Y349C / T366S / L368A / Y407V). The effect of asymmetrically introduced mutations on FcRn binding can be easily measured using FcRn affinity chromatography (see Figure 9 and the table below). Antibodies that elute later from the FcRn affinity column, i.e., those with longer retention times on the FcRn affinity column, have longer in vivo half-lives, and vice versa.

[0210] [Table 7]

[0211] The effects of asymmetric mutagenesis affecting FcRn binding in the Fc region have been further illustrated with monospecific anti-IGF-1R antibodies assembled using knobs-into-holes technology to enable asymmetric mutagenesis (see, e.g., U.S. Patent No. 7,695,936, U.S. Publication No. 2003 / 0078385; “hole chain” mutation: S354C / T366W, “knob chain” mutation: Y349C / T366S / L368A / Y407V). The effect of asymmetrically introduced mutations on FcRn binding can be readily measured using FcRn affinity chromatography (see table below). Antibodies that elute later from the FcRn affinity column, i.e., those with longer retention times on the FcRn affinity column, have longer in vivo half-lives, and vice versa.

[0212] [Table 8]

[0213] Asymmetric IHH-AAA and LLL-DDD mutations (LLL-DDD mutations = combinations of mutations L251D, L314D, and L432D) show weaker binding than the corresponding parental or wild-type antibodies.

[0214] Asymmetric HHY-AAA mutations (a combination of mutations H310A, H433A, and Y436A) result in Fc regions that no longer bind to human FcRn but maintain binding to protein A (see Figures 11, 12, 13, and 14).

[0215] The effects of introducing asymmetric mutations that affect FcRn binding in the Fc region are further illustrated for a monospecific anti-IGF-1R antibody, a bispecific anti-VEGF / ANG2 antibody (VEGF / ANG2), and a full-length antibody (fusion) with a fusion to the C-terminus of both heavy chains assembled using the knobs-into-holes technology that enables the introduction of asymmetric mutations (see, e.g., U.S. Patent No. 7,695,936, U.S. Published Patent Application No. 2003 / 0078385; "hole chain" mutations: S354C / T366W, "knob chain" mutations: Y349C / T366S / L368A / Y407V). The effects of the introduced mutations on FcRn binding and Protein A binding can be readily measured using FcRn affinity chromatography, Protein A affinity chromatography, and SPR-based methods (see the table below).

[0216]

Table 9

[0217] One aspect reported herein is an antibody or Fc region fusion polypeptide comprising a mutant human IgG class Fc region reported herein.

[0218] The Fc region (dimeric polypeptide) reported herein confers that feature to the molecule when included in an Fc region fusion polypeptide or a full-length antibody. The fusion partner can be any molecule that has biological activity and whose in vivo half-life can be shortened or extended, i.e., whose in vivo half-life is clearly defined and tailored for the intended use.

[0219] The Fc region fusion polypeptide may include, for example, a TNFR-Fc region fusion polypeptide (TNFR = human tumor necrosis factor receptor), an IL-1R-Fc region fusion polypeptide (IL-1R = human interleukin-1 receptor), 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), and may contain a receptor protein that binds to a target, such as a mutant (human) IgG class Fc region and ligand reported herein.

[0220] The Fc region fusion polypeptide may include, for example, the variant (human) IgG class Fc region reported herein, and antibody fragments that bind to a target, such as antibody Fab fragments, scFvs (see, for example, Nat. Biotechnol. 23 (2005) 1126-1136) or domain antibodies (dAbs) (see, for example, International Publication No. 2004 / 058821 and International Publication No. 2003 / 002609).

[0221] The Fc region fusion polypeptide may, for example, include the variant (human) human IgG class Fc region and receptor ligand (either naturally occurring or artificial) reported herein.

[0222] Antibodies, such as full-length antibodies or CrossMabs, may contain the variant (human) human IgG class Fc region reported herein.

[0223] B. Ocular vascular disease Ocular vascular disorders are any pathological conditions characterized by the altered or unregulated proliferation and invasion of new blood vessels into the structure of ocular tissues (such as the retina or cornea).

[0224] In one embodiment, ocular vascular diseases are selected from the group consisting of: exudative age-related macular degeneration (exudative AMD), dry age-related macular degeneration (dry AMD), diabetic macular edema (DME), cystoid macular edema (CME), nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), cystoid macular edema, vasculitis (e.g., central retinal vein occlusion), papilledema, retinitis, conjunctivitis, uveitis, choroiditis, multifocal choroiditis, ocular histoplasma, blepharitis, dry eye (Sjögren's disease), and other eye diseases (the eye disease or disorder is associated with ocular neovascularization, vascular leakage, and / or retinal edema).

[0225] Antibodies comprising dimerized polypeptides reported herein are useful in the prevention and treatment of exudative AMD, dry AMD, CME, DME, NPDR, PDR, blepharitis, dry eye, and uveitis, in one preferred embodiment, exudative AMD, dry AMD, blepharitis, and dry eye, in another preferred embodiment, CME, DME, NPDR, and PDR, in another preferred embodiment, blepharitis, and dry eye, in particular exudative AMD and dry AMD, and especially in the prevention and treatment of exudative AMD.

[0226] In some embodiments, the ophthalmic vascular disease is selected from the group consisting of exudative age-related macular degeneration (exudative AMD), macular edema, retinal vein occlusion, retinopathy of prematurity, and diabetic retinopathy.

[0227] Other conditions associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, limbal keratitis, pterygium keratitis, keratosis sicca, Sjögren's disease, rosacea, firectenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infection, herpes zoster infection, protozoal infection, Kaposi's sarcoma, Mohren's ulcer, Terien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, multiple trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson disease, bullous pemphigoid, radial keratotomy, and corneal transplant rejection.

[0228] Diseases associated with retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis / vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including macular edema), Eels' disease, Behçet's disease, infections causing retinitis or choroiditis, presumptive ocular histoplasmosis, Best's disease, myopia, optic nerve pit, Stargardt disease, squamous cell carcinoma, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications.

[0229] Other diseases include, but are not limited to, those associated with rubeosis (neovascularization of the horns) and those caused by abnormal proliferation of fibrovascular tissue or fibrous tissue (including all forms of proliferative vitreoretinopathy).

[0230] Retinopathy of prematurity (ROP) is an eye disorder that affects infants born prematurely. It is thought to be caused by the disordered growth of retinal blood vessels, which can lead to scarring and retinal detachment. ROP can be mild and resolve spontaneously, but in severe cases it can lead to blindness. As such, all premature infants are at risk for ROP, and very low birth weight is an additional risk factor. Both oxygen toxicity and relative hypoxia can contribute to the development of ROP.

[0231] Macular degeneration is a medical condition primarily found in older adults in which the central lining of the eye (commonly known as the macula of the retina) thins, atrophies, and, in some cases, hemorrhages. This can lead to loss of central vision, which is associated with the inability to see, read, or recognize fine details or faces. According to the American Academy of Ophthalmology, it is the leading cause of central vision loss (blindness) in the United States today in people over 50 years of age. Some macular dystrophy affecting younger people is sometimes referred to as macular degeneration, but this term generally refers to age-related macular degeneration (AMD or ARMD).

[0232] Age-related macular degeneration (AMD) begins with characteristic yellow deposits called drusen in the macula (the central region of the retina that provides detailed central vision, also known as the fovea), between the retinal pigment epithelium and the choroid beneath. Most people with these early changes (also called age-related macular degeneration) have good vision. Those with drusen may progress to progressive AMD. This risk is considerably higher if the drusen are large, numerous, and associated with disruption in the submacular pigment cell layer. Large, soft drusen are associated with elevated cholesterol deposits and may respond to cholesterol-lowering medications or Rheo procedures.

[0233] Progressive AMD is associated with severe blindness and has two forms: dry and exudative. Central geographical atrophy is the dry form of progressive AMD, resulting from atrophy of the retinal pigment epithelium layer beneath the retina, which leads to vision loss through the loss of photoreceptors (rods and cones) in the central part of the eye. There are no available treatments for this condition, however, studies by the National Eye Institute and others have shown that vitamin supplements with high doses of antioxidants, lutein, and zeaxanthin can slow the progression of dry macular degeneration and improve vision in some patients.

[0234] Retinitis pigmentosa (RP) is a group of genetic eye conditions. In the progression of RP, night blindness generally occurs several years or decades before tunnel vision. Many people with RP do not become legally blind until their 40s or 50s and retain some vision for life. Others, in some cases, go from RP to complete blindness as early as childhood. The progression of RP varies from case to case. RP is a type of hereditary retinal dystrophy, a group of genetic disorders in which abnormalities in the retinal photoreceptors (rods and cones) or retinal pigment epithelium (RPE) cause progressive vision loss. Individuals affected initially experience impaired dark adaptation or night blindness, followed later in the course of the disease by peripheral vision loss (commonly known as tunnel vision) and sometimes central vision loss.

[0235] Macular edema occurs when fluid and protein deposits accumulate above and below the macula (the yellow central region of the retina) of the eye, thickening and swelling the macula. This swelling can distort a person's central vision because the macula is located at the back of the eyeball, near the center of the retina. This region holds tightly packed cones that provide a sharp, clear central vision, allowing a person to see shapes, colors, and details directly in their line of sight. Cystoid macular edema is a type of macular edema that involves cystic formation.

[0236] C. Antibody purification using Staphylococcus aureus protein A affinity chromatography column In one aspect, a dimeric polypeptide, The first polypeptide and the second polypeptide each contain, in the direction from the N-terminus to the C-terminus, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain. Includes, i) The first and second polypeptides each contain mutants H310A, H433A, and Y436A, or ii) The first and second polypeptides each contain mutations L251D, L314D, and L432D, or iii) The first and the second polypeptides each contain mutations L251S, L314S, and L432S, or iv) The first polypeptide contains mutations I253A, H310A, and H435A, and the second polypeptide contains mutations H310A, H433A, and Y436A, or v) The first polypeptide contains mutations I253A, H310A, and H435A, and the second polypeptide contains mutations L251D, L314D, and L432D, or vi) The first polypeptide contains mutations I253A, H310A, and H435A, and the second polypeptide contains mutations L251S, L314S, and L432S, a polypeptide is provided.

[0237] These dimeric polypeptides do not bind to human FcRn due to the mutations, but have the property that the binding to Staphylococcus protein A is maintained.

[0238] Thus, these antibodies can be purified, i.e., separated from unwanted by-products, by using conventional protein A affinity materials such as MabSelectSure. For example, there is no need to use highly refined but species-restricted affinity materials such as KappaSelect, which can only be used for antibodies containing kappa subclass light chains. In addition, there is no need to adopt a purification method even if the light chain subclass is modified / exchanged (see FIGS. 11 and 12, respectively).

[0239] One aspect reported herein is a method for producing the dimeric polypeptides reported herein, comprising the following steps: a) culturing mammalian cells containing one or more nucleic acids encoding the dimeric polypeptide, b) recovering the dimeric polypeptide from the culture medium, and c) A step of purifying the dimerized polypeptide by protein A affinity chromatography to produce the dimerized polypeptide. This method includes [something].

[0240] One aspect reported herein is the use of mutants H310A, H433A, and Y436A for the separation of heterodimer polypeptides from homodimer polypeptides.

[0241] One aspect reported herein is the use of mutants L251D, L314D, and L432D for the separation of heterodimer polypeptides from homodimer polypeptides.

[0242] One embodiment reported herein involves the use of mutants L251S, L314S, and L432S for the separation of heterodimer polypeptides from homodimer polypeptides.

[0243] One embodiment reported herein is the use of the mutations I253A, H310A, and H435A within the first Fc region polypeptide, in combination with the mutations H310A, H433A, and Y436A within the second Fc region polypeptide, to separate a heterodimer Fc region containing the first and second Fc region polypeptides from a homodimer Fc region.

[0244] One embodiment reported herein is the use of mutants I253A, H310A, and H435A within the first Fc region polypeptide, in combination with mutants L251D, L314D, and L432D within the second Fc region polypeptide, to separate a heterodimer Fc region containing the first and second Fc region polypeptides from a homodimer Fc region.

[0245] One embodiment reported herein is the use of mutants I253A, H310A, and H435A within the first Fc region polypeptide, in combination with mutants L251S, L314S, and L432S within the second Fc region polypeptide, to separate a heterodimer Fc region containing the first and second Fc region polypeptides from a homodimer Fc region.

[0246] In one embodiment of the three embodiments described above, the first Fc region polypeptide further comprises the mutations Y349C, T366S, L368A, and Y407V, and the second Fc region polypeptide further comprises the mutations S354C and T366W.

[0247] In one embodiment of the three embodiments described above, the first Fc region polypeptide further comprises the mutations S354C, T366S, L368A and Y407V, and the second Fc region polypeptide further comprises the mutations Y349C and T366W.

[0248] One embodiment reported herein is the use of mutant Y436A to increase the binding of a dimeric Fc domain polypeptide to protein A.

[0249] It was found that introducing the Y436A mutation increased the binding of the Fc region to Staphylococcus protein A (SPA). This is advantageous, for example, when introducing further mutations that reduce SPA binding (e.g., I253A and H310A, or H310A and H435A) (see Figure 15).

[0250] One embodiment reported herein is a dimerized polypeptide, The first polypeptide and the second polypeptide each contain, in the direction from the N-terminus to the C-terminus, at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain. Includes, The first, second, or first and second polypeptides contain mutation Y436A (numbered according to the Kabat EU index numbering system), It is a polypeptide.

[0251] In one embodiment, the first and second polypeptides contain the mutation Y436A.

[0252] One embodiment reported herein is a bispecific antibody that provides ease of isolation / purification, comprising a separately modified immunoglobulin heavy chain Fc region, at least one of which results in i) a distinct affinity of the bispecific antibody for protein A, and ii) a distinct affinity of the bispecific antibody for human FcRn, wherein the bispecific antibody is isolated from disrupted cells, culture medium, or antibody mixture based on its affinity for protein A.

[0253] In one embodiment, the bispecific antibody elutes at a pH value greater than 4.0.

[0254] In one embodiment, a bispecific antibody is isolated using protein A affinity chromatography and a pH gradient or pH step, wherein the pH gradient or pH step includes the addition of a salt. In one specific embodiment, the salt is present at a concentration of about 0.5 M to about 1 M. In one embodiment, the salt is selected from the group consisting of lithium, sodium, and potassium acetates; sodium and potassium bicarbonates; lithium, sodium, and potassium carbonates; lithium, sodium, potassium, and magnesium chlorides; sodium and potassium fluorides; sodium, potassium, and calcium nitrates; sodium and potassium phosphates; and calcium and magnesium sulfates. In one embodiment, the salt is an alkali metal or alkaline earth metal halide salt. In one preferred embodiment, the salt is sodium chloride.

[0255] In one embodiment, the dimerized polypeptide comprises a modified first polypeptide and a second polypeptide that is not modified with respect to protein A and FcRn binding, so as to form a heterodimerized polypeptide, wherein the distinct modification results in a dimerized polypeptide that elutes from the protein A affinity material at a pH 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, or 1.4 pH units higher than the corresponding dimerized polypeptide without the distinct modification. In one embodiment, the distinctly modified dimerized polypeptide elutes at pH 4 or higher, while the unmodified dimerized polypeptide elutes at pH 3.5 or lower. In another embodiment, the distinctly modified dimerized polypeptide elutes at approximately pH 4, while the unmodified dimerized polypeptide elutes at approximately pH 2.8–3.5, 2.8–3.2, or 2.8–3. In these embodiments, “unmodified” in both polypeptides means that there are no modifications H310A, H433A, and Y436A (Kabat EU Index Numbering System).

[0256] For chromatographic runs, particularly for human IgG1 subclass samples, the addition of a 0.5 M–1 M salt (e.g., NaCl) may improve the separation of homodimer and heterodimer polypeptides. Adding salt to the elution solution in a way that increases the pH value may broaden the pH range for elution, for example, by creating a stepwise pH gradient to better separate the two species.

[0257] Accordingly, in one embodiment, a method for separating a bispecific antibody containing a heterodimerized IgGFc region, in which one chain contains a mutation reported herein, comprises the step of employing a pH gradient in the presence of a salt. In one embodiment, the salt is present at a concentration sufficient to maximize the pH difference of elution from the protein A chromatography material between the IgGFc region homodimer and the IgGFc region heterodimer. In one embodiment, the salt is present at a concentration of about 0.5 M to about 1 M. In one embodiment, the salt is a salt of an alkali metal or alkaline earth metal and a halogen. In one embodiment, the salt is a chloride salt of an alkali metal or alkaline earth metal, such as NaCl, KCl, LiCl, CaCl2, or MgCl2. In one embodiment, the pH gradient is about pH 4 to about pH 5. In one embodiment, the gradient is a linear gradient. In one embodiment, the pH gradient is a stepped gradient. In one embodiment, the method comprises applying a solution at about pH 4 to an equilibrated protein A affinity column. In one embodiment, a bispecific antibody containing a heterodimerized IgGFc region relating to the modification reported herein is eluted from a protein A affinity chromatography material into one or more fractions substantially free of non-heterodimerized bispecific antibodies.

[0258] The dimerized polypeptides reported herein are produced by recombinant means. Thus, one aspect of the present invention is a nucleic acid encoding the dimerized polypeptides reported herein, and a further aspect is a cell containing the nucleic acid encoding the dimerized polypeptides reported herein. Recombinant production methods are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by isolation of the dimerized polypeptide and purification to a normally pharmaceutically acceptable purity. For the expression of such dimerized polypeptides in host cells, nucleic acids encoding the first and second polypeptides, respectively, are inserted into an expression vector by standard means. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the dimerized polypeptide is recovered from the cells (culture supernatant or lysed cells).

[0259] The general methods for recombinant antibody production are well-known in the current state of the technology and are described, for example, in the reviews 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; and Werner, RG, Drug Res. 48 (1998) 870-880.

[0260] Therefore, one embodiment reported herein is a method for producing the dimer polypeptide reported herein, a) A step of transforming host cells with one or more vectors containing nucleic acid molecules encoding dimeric polypeptides reported herein. b) A step of culturing the host cells under conditions that enable the production of the dimeric polypeptide, and c) A step of recovering the dimer polypeptide from the culture to produce the dimer polypeptide. This method includes [something].

[0261] In one embodiment, the recovery step in c) includes the use of a capture reagent specific to the immunoglobulin Fc region. In one embodiment, this Fc region-specific capture reagent is used in bind-and-elute mode. An example of such an Fc region-specific capture reagent is, for example, a Staphylococcus protein A-based affinity chromatography column based on a highly rigid agarose-based matrix that allows for high flow rates and low back pressures on a large scale. They feature a dimeric polypeptide, i.e., a ligand that binds to its Fc region. The ligand is attached to the matrix via a long hydrophilic spacer arm to facilitate its use for binding to a target molecule.

[0262] The dimerized polypeptides reported herein can be appropriately isolated from culture media by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. B cells or hybridoma cells may serve as the source of DNA and RNA encoding the dimerized polypeptides. DNA and RNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures. Once isolated, the DNA can be inserted into an expression vector, which is then transfected into host cells (that would not otherwise produce the dimerized polypeptide), such as HEK293 cells, CHO cells, or myeloma cells, to synthesize recombinant monoclonal dimerized polypeptides in the host cells.

[0263] Antibody purification is performed using standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, to remove cellular components or other contaminants, such as other cellular nucleic acids or proteins (see Ausubel, F., et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987)). Various methods are well-established and widely used for protein purification. For example, affinity chromatography using microbial proteins (e.g., protein A or protein G affinity chromatography), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin), and mixed-mode exchange), thiophilic adsorption (e.g., with β-mercaptoethanol or other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-sepharose, aza-alenophilic resin, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II) and Cu(II) affinity materials), size exclusion chromatography, and electrophoresis methods (such as gel electrophoresis and capillary electrophoresis) (Vijayalakshmi, MA, Appl. Biochem. Biotech. 75 (1998) 93-102).

[0264] One aspect of the present invention is a pharmaceutical formulation comprising a dimerized polypeptide or antibody as reported herein. Another aspect of the present invention is the use of a dimerized polypeptide or antibody as reported herein for the manufacture of a pharmaceutical formulation. A further aspect of the present invention is a method for manufacturing a pharmaceutical formulation comprising a dimerized polypeptide or antibody as reported herein. In yet another aspect, the present invention provides a formulation comprising a dimerized polypeptide or antibody as reported herein, formulated together with a pharmaceutical carrier, for example, a pharmaceutical formulation.

[0265] The formulations reported herein can be administered by various methods known in the art. As will be apparent to those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. To administer the compounds of the present invention by a certain route of administration, it may be necessary to coat the compounds with a substance that prevents their inactivation, or to administer such a substance simultaneously. For example, the compounds may be administered to a subject in a suitable carrier, e.g., liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is known in the art.

[0266] Many possible modes of delivery can be used, including but not limited to intraocular or topical application. In one embodiment, the application is intraocular and includes, but is not limited to, subconjunctival injection, anterior chamber injection, injection into the anterior chamber via the temporal limbus, intramatrix injection, intracorneal injection, subretinal injection, aqueous humor injection, sub-Tenon's capsule injection or continuous delivery device, or intravitreal injection (e.g., pre-, middle, or posterior intravitreal injection). In one embodiment, the application is topical and includes, but is not limited to, eye drops to the cornea.

[0267] In one embodiment, the dimeric polypeptides or pharmaceutical formulations reported herein are administered via intravitreous application, for example, by intravitreous injection. This can be carried out 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).

[0268] In some embodiments, the therapeutic kit of the present invention may include one or more doses of the dimeric polypeptides reported herein present in the pharmaceutical formulation described herein, a device suitable for intravitreal injection of the pharmaceutical formulation, and instructions detailing suitable subjects and a protocol for administering the injection. In these embodiments, the formulation is typically administered to a subject in need of treatment via intravitreal injection. This can be carried out 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.

[0269] The formulation may also contain adjuvants (e.g., preservatives, humectants, emulsifiers, and dispersants). Prevention of the presence of microorganisms can be ensured by sterilization procedures (as described above) and by the inclusion of various antimicrobial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It is also desirable to include isotonic agents (e.g., sugars, sodium chloride, etc.) in the formulation. Furthermore, long-term absorption of the injectable pharmaceutical form can be achieved by the inclusion of absorption-delaying agents (e.g., aluminum monostearate and gelatin, etc.).

[0270] Regardless of the selected route of administration, the compounds reported herein (which may be used in a suitable hydrated form) and / or the pharmaceutical formulations reported herein are formulated in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0271] The actual dose levels of the active ingredients in the pharmaceutical formulations reported herein may be varied to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dose level may depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0272] The formulation must be sterile and fluid enough to be delivered by syringe. In addition to water, the carrier is, in a preferred embodiment, isotonic buffered saline.

[0273] Appropriate fluidity can be maintained, for example, by the use of a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols (such as mannitol or sorbitol), and sodium chloride in the composition.

[0274] The formulation may include an ophthalmic depot formulation containing an active agent for subconjunctival administration. The ophthalmic depot formulation contains microparticles of an essentially pure active agent, such as the dimeric polypeptide reported herein. Microparticles containing the dimeric polypeptide reported herein may be embedded in a biocompatible, pharmaceutically acceptable polymer or lipid encapsulation agent. The depot formulation may be adapted to release substantially all of the active substance over a long period of time. The polymer or lipid matrix, if present, may be adapted to be sufficiently degraded and transported away from the injection site after the release of all or substantially all of the active agent. The depot formulation may be a liquid formulation containing a pharmaceutically acceptable polymer and a dissolved or dispersed active agent. Upon injection, the polymer forms a depot at the injection site, for example, by gelation or precipitation.

[0275] Another aspect of the present invention is a dimeric polypeptide or antibody reported herein for use in the treatment of ophthalmic vascular diseases.

[0276] One embodiment of the present invention is a dimeric polypeptide or antibody reported herein for use in the treatment of ophthalmic vascular diseases.

[0277] Another aspect of the present invention is a pharmaceutical formulation for use in the treatment of ophthalmic vascular diseases.

[0278] Another aspect of the present invention is the use of dimeric polypeptides or antibodies reported herein for the manufacture of pharmaceuticals for the treatment of ophthalmic vascular diseases.

[0279] Another aspect of the present invention is a method for treating a patient with ophthalmic vascular disease, which involves administering a dimeric polypeptide or antibody reported herein to a patient requiring such treatment.

[0280] As used herein, the term "including" is hereby expressed as encompassing the term "consisting of". Accordingly, all aspects and embodiments containing the term "including" are similarly disclosed using the term "consisting of".

[0281] D. Modification In one further embodiment, a dimerized polypeptide according to any of the above embodiments may incorporate any of the features, either alone or in combination, as described in Sections 1-6 below:

[0282] 1. Antibody affinity In one embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of binding partners, with BIACORE®-2000 or BIACORE®-3000 (GE Healthcare Inc., Piscataway, NJ). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, GE Healthcare Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The binding partners are diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl / min to achieve coupling of approximately 10 response units (RUs) of binding partners. Following the injection of the binding partners, 1 M ethanolamine is injected to protect unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78 nM to 500 nM) of the dimer polypeptide-containing fusion polypeptide or antibody are injected into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20®) surfactant at 25°C at a flow rate of approximately 25 μL / min. The association rate (k) is determined by simultaneously fitting association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). on ) and dissociation rate (koff ) calculate the equilibrium dissociation constant (Kd) as the ratio k off / k on Calculate as follows (see, for example, Chen, Y. et al., J. Mol. Biol. 293 (1999) 865-881). In the above surface plasmon resonance assay, if the on-velocity is 10 6 M -1 s -1 If the value exceeds this, the on-rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; fluorescence = 340 nm, bandwidth 16 nm) of a 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C, measured with a spectrometer such as an Aviv Instruments spectrometer with stop-flow or an 8000 series SLM-AMINCO™ spectrometer (ThermoSpectronic) with a stirring cuvette, while increasing the antigen concentration.

[0283] 2. Chimeric antibodies and humanized antibodies In some embodiments, the dimeric polypeptides reported herein are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, the chimeric antibody is a “class-switched” antibody in which the class or subclass has changed from that of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0284] In some embodiments, the chimeric antibody is a humanized antibody. Humanization of a non-human antibody is typically done to reduce its immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains in which the HVR, e.g., CDR (or a portion thereof), is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may also contain at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0285] For humanized antibodies and methods for producing them, see, for example, Almagro, JC and Fransson, J., Front. Biosci. 13 (2008) 1619-1633, as well as, for example, Riechmann, I., et al., Nature 332 (1988) 323-329; Queen, C., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 10029-10033; U.S. Patent Nos. 5821337, 7527791, 6982321 and 7087409; Kashmiri, SV, et al., Methods 36 (2005) 25-34 (describes specificity-determining region (SDR) transplantation); Padlan, EA, Mol. Immunol. 28 (1991) 489-498. Further explanation is provided in the following publications: (Regarding "resurfacing"); Dall'Acqua, WF et al., Methods 36 (2005) 43-60 (Regarding "FR shuffling"); Osbourn, J. et al., Methods 36 (2005) 61-68; and Klimka, A. et al., Br. J. Cancer 83 (2000) 252-260 (Regarding the "guided selection" approach for FR shuffling).

[0286] Human framework regions that can be used for humanization include, but are not limited to, the following: framework regions selected using the "best-fit" method (see, e.g., Sims, MJ, et al., J. Immunol. 151 (1993) 2296-2308); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., 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 (somatic variant) framework regions or human germline framework regions (see, e.g., Almagro, JC and Fransson, J., Front. Biosci. 13 (2008)). See 1619-1633); and framework regions derived from screening FR libraries (e.g., Baca, M. et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok, MJ et al., J. Biol. Chem. 271 (19969 22611-22618)).

[0287] 3. Human antibodies In one embodiment, the dimeric polypeptide reported herein is a human antibody. Human antibodies can be produced using various 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.

[0288] Human antibodies may also be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies containing human variable regions in response to antigen inoculation. Such animals typically contain all or some human immunoglobulin loci that replace endogenous immunoglobulin loci, are extrachromosomal, or are randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci have generally been inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, N., Nat. Biotech. 23 (2005) 1117-1125. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (describes XENOMOUSE® technology); U.S. Patent No. 5,770,429 (describes HUMAB® technology); U.S. Patent No. 7,041,870 (describes KM MOUSE® technology); and U.S. Publication No. 2007 / 0061900 (describes VELOCIMOUSE® technology). Human variable regions derived from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0289] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, 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 via human B-cell hybridoma technology have also been described in Li, J., et al., Proc. Natl. Acad. Sci. USA 103 (2006) 3557-3562. Further methods include, for example, those described in U.S. Patent No. 7189826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, J., Xiandai Mianyixue 26 (2006) 265-268 (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.

[0290] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage-presenting libraries. These variable domain sequences may then be combined with desired human constant domains. A technique for selecting human antibodies from antibody libraries is described below.

[0291] 4. Library-derived antibodies In certain embodiments, the dimerized polypeptides reported herein are library-derived antibodies. Library-derived antibodies can be isolated by screening a combinatorial library for antibodies having the desired activity. For example, a wide variety of methods are known in the art for generating phage-presenting libraries and screening such libraries for antibodies having the desired binding characteristics. Such methods are outlined, for example, in Hoogenboom, HR et al., Methods in Molecular Biology 178 (2001) 1-37, and also in, for example, 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). Further details can be found in 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.

[0292] In one phage presentation method, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described in Winter, G., et al., Ann. Rev. Immunol. 12 (1994) 433-455. The phages typically present antibody fragments as either single-stranded Fv(scFv) fragments or Fab fragments. Libraries derived from immunized sources provide antibodies with high affinity to immunogens without requiring the construction of hybridomas. Alternatively, as described in Griffiths, AD, et al., EMBO J. 12 (1993) 725-734, a naive repertoire can be cloned (e.g., from humans) to provide single-source antibodies against a wide range of non-self and self-antigens without any immunization. Finally, as described in Hoogenboom, HR and Winter, G., J. Mol. Biol. 227 (1992) 381-388, naive libraries can also be synthetically prepared by cloning an unreconstituted V gene segment derived from stem cells and using PCR primers containing random sequences that encode the highly variable CDR3 region and achieve in vitro reconstitution. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0293] In this specification, antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments.

[0294] 5. Multispecific antibodies In some embodiments, the dimeric polypeptide reported herein is a multispecific antibody, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In some embodiments, one binding specificity is for a first antigen and the other is for a second antigen. In some embodiments, a bispecific antibody may bind to two different epitopes of the same antigen. A bispecific antibody can also be used to localize a cytotoxic substance to cells expressing at least one of the antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0295] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, AC, Nature 305 (1983) 537-540, International Publication No. 93 / 08829 and Traunecker, A., et al., EMBO J. 10 (1991) 3655-3659) and "knob-in-hole" operations (see, for example, U.S. Patent No. 5731168). Multispecific antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimer molecules (International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4676980 and Brennan, M. et al., Science 229 (1985) 81-83); producing bispecific antibodies using a leucine zipper (see, e.g., Kostelny, SA, et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology to produce bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv(sFv) dimers (see, e.g., Gruber, M et al., J. Immunol. See 152 (1994) 5368-5374); and it can also be produced by preparing a triplicate antibody, for example, as described in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0296] This also includes engineered antibodies that have three or more functional antigen-binding sites, including "octopus antibody" (see, for example, U.S. Public Notice No. 2006 / 0025576).

[0297] The antibodies or fragments used herein also include "Dual Acting Fabs" or "DAFs" (see, for example, U.S. Publication No. 2008 / 0069820).

[0298] The antibodies or fragments described herein also include the multispecific antibodies described in International Publication Nos. 2009 / 080251, 2009 / 080252, 2009 / 080253, 2009 / 080254, 2010 / 112193, 2010 / 115589, 2010 / 136172, 2010 / 145792, and 2010 / 145793.

[0299] 6. Antibody variants In one embodiment, the dimeric polypeptide reported herein is an antibody. In further embodiments, amino acid sequence variants of the antibody provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct has the desired characteristics (e.g., antigen binding).

[0300] a) Substitution, insertion, and deletion variants In one embodiment, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVR and FR. In the table below, conservative substitutions are shown under the heading "Preferred Substitutions." More substantial changes are shown in the table below under the heading "Exemplary Substitutions," which are further described below in relation to amino acid side chain classes. Amino acid substitutions can be introduced into antibodies of interest, and the products can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0301] [Table 10]

[0302] Amino acids can be classified according to the common characteristics of their side chains. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0303] Non-conservative substitution involves swapping a member of one of these classes with one of another.

[0304] One type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study are modified (e.g., improved) compared to the parent antibody in certain biological properties (e.g., increased affinity, reduced immunogenicity), and / or substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-mature antibody that can be easily generated using phage presentation-based affinity maturation techniques, such as those described herein. In short, one or more HVR residues are mutated, the mutant antibody is made to present on a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0305] Changes (e.g., substitutions) may be induced in HVR to improve antibody affinity, for example. Such changes may be induced in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during somatic cell maturation (see, e.g., Chowdhury, PS, Methods Mol. Biol. 207 (2008) 179-196) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and re-selecting from it is described, for example, in Hoogenboom, HR et al. in Methods in Molecular Biology 178 (2002) 1-37. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of a wide variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody mutants with the desired affinity. Another method for introducing diversity involves an HVR-specific approach that randomizes several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0306] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (to the extent that such changes do not substantially reduce the antibody's ability to bind to the antigen). For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as defined herein) may occur within an HVR. Such changes may be, for example, outside the antigen-contact residue within the HVR. In some embodiments of the variant VH and VL sequences defined earlier, each HVR is either unchanged or contains only one, two, or three amino acid substitutions.

[0307] 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. This method involves identifying a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and substituting them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody's interaction with the antigen is affected. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and their adjacent residues may be targeted or excluded as candidate substitutions. Mutants may be screened to determine whether they possess the desired properties.

[0308] Amino acid sequence insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 residues or more, 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 insertion variants of antibody molecules include fusions of enzymes (e.g., ADEPT) or polypeptides that increase the half-life of the antibody in serum to the N-terminus or C-terminus of the antibody.

[0309] b) Glycosylated mutants In one embodiment, the antibody as defined herein is modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to the antibody can be easily achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0310] If the antibody contains an Fc region, the carbohydrate to which it is attached may be altered. Native antibodies produced by mammalian cells typically contain branched biantennae oligosaccharides, which are generally attached by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, Wright, A. and Morrison, SL, TIBTECH 15 (1997) 26-32. The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennae oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention may be modified to create antibody variants with certain improved properties.

[0311] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the carbohydrate chain at Asn297 relative to the total amount of all carbohydrate structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region, but due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may possess improved ADCC function. See, for example, U.S. Publication No. 2003 / 0157108; U.S. Publication No. 2004 / 0093621. Examples of publications relating to “defucosylated” or “fucose-deficient” antibody variants include U.S. Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Publication No. 2003 / 0115614; U.S. Publication No. 2002 / 0164328; U.S. Publication No. 2004 / 0093621; U.S. Publication No. 2004 / 0132140; U.S. Publication This includes Publication No. 2004 / 0110704; U.S. Publication No. 2004 / 0110282; U.S. Publication No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; International Publication No. 2005 / 035586; International Publication No. 2005 / 035778; International Publication No. 2005 / 053742; International Publication No. 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 protein fucosylation-deficient Lec13 CHO cells (Ripka, J., et al., Arch. Biochem. Biophys. 249 (1986) 533-545; U.S. Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11) and knockout cell lines such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, N., et al., Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y., et al., Biotechnol. Bioeng. 94 (2006) 680-688; and International Publication No. 2003 / 085107).

[0312] For example, antibody variants having bisected oligosaccharides are provided, in which a biantennae-type oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication No. 2003 / 011878; U.S. Patent No. 6602684; and U.S. Publication No. 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publication No. 1997 / 30087; International Publication No. 1998 / 58964; and International Publication No. 1999 / 22764.

[0313] c) Fc region variant In one embodiment, Fc region variants may be generated by introducing one or more further amino acid modifications to the dimeric polypeptide reported herein. The Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4Fc regions) that include amino acid modifications (e.g., substitutions / mutations) at one or more amino acid positions.

[0314] In one embodiment, the present invention intends for a dimeric polypeptide having some, but not all, effector functions. This makes the dimeric polypeptide a desirable candidate for applications where the in vivo half-life of the dimeric polypeptide is important, but certain effector functions (such as CDC and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / deficiency of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that a dimeric polypeptide antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. Monocytes express FcγRI, FcγRII, and FcγRIII, while NK cells, the primary cells for mediating ADCC, express only FcγRIII. FcR expression in 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 the ADCC activity of molecules of interest are described in U.S. Patent No. 5500362 (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); and U.S. Patent No. 5821337 (see Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition to the above, the ADCC activity of the molecule of interest can also be evaluated in vivo in animal models, such as those disclosed in Clynes, R. et al., Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay may be performed to confirm that the dimeric polypeptide cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate complement activation, a CDC assay may 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 determination can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int. Immunol. 18 (2006) 1759-1769).

[0315] Dimeric polypeptides with reduced effector function include those having one or more substitutions at Fc domain residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc domain mutants include Fc domains having two or more substitutions among the amino acids at positions 265, 269, 270, 297, and 327 (including the so-called "DANA" Fc domain mutant (U.S. Patent No. 7,332,581) which has alanine substitutions at residues 265 and 297).

[0316] Certain antibody variants that exhibit improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6737056; International Publication No. 2004 / 056312 and Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604).

[0317] In one embodiment, the dimeric polypeptide variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

[0318] In some embodiments, changes are induced in the Fc region that result in alterations (i.e., improvements or reductions) to C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184.

[0319] Antibodies with improved binding to the neonatal Fc receptor (FcRn) (Guyer, RL et al., J. Immunol. 117 (1976) 587-593 and Kim, JK et al., J. Immunol. 24 (1994) 2429-2434), which have an increased half-life and are responsible for the transfer of maternal IgG to the fetus, are described in U.S. Public Notice No. 2005 / 0014934. These antibodies contain an Fc region that includes one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc region variants include those having substitutions in one or more of the 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, for example, those having a substitution in the Fc region residue 434 (U.S. Patent No. 7,371,826).

[0320] For other examples of Fc region variants, see also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Patent No. 5648260; U.S. Patent No. 5624821; and International Publication No. 94 / 29351.

[0321] d) Cysteine-modified antibody variants In certain embodiments, it may be desirable to create a cysteine-engineered dimerized polypeptide in which one or more residues of the antibody are substituted with cysteine ​​residues, similar to, for example, "thioMAb (thioMAb)". In certain embodiments, the substituted residues are found in an accessible site of the dimerized polypeptide. By substituting these residues with cysteine, a reactive thiol group is placed in the accessible site of the dimerized polypeptide, which can then be used to create an immunoconjugate by conjugating the dimerized polypeptide to another part, such as a drug part or a linker-drug part, as further described herein. In certain embodiments, one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. The cysteine-engineered dimerized polypeptide may be produced, for example, as described in U.S. Patent No. 7521541.

[0322] e) derivatives In some embodiments, the dimerized polypeptides reported herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for derivatization of the dimerized polypeptide 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the dimeric polypeptide may vary, and if two or more polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used in derivatization can be determined based on considerations, including (but not limited to) the specific properties or functions of the dimeric polypeptide to be improved, and whether the dimeric polypeptide derivative will be used therapeutically under certain conditions.

[0323] In another embodiment, a conjugate of a dimerized polypeptide 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 may be of any wavelength and may not harm normal cells, but may include, but is not limited to, wavelengths that heat the non-protein portion to a temperature that kills cells near the dimerized polypeptide-non-protein portion.

[0324] f) Heterodimization Several approaches exist for modifying CH3 to enhance heterodimerization, and these are adequately described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in all such approaches, both the first and second CH3 domains are manipulated in a complementary manner so that each CH3 domain (or the heavy chain containing it) can no longer homodimerize with itself and is forced to heterodimerize with other complementaryly manipulated CH3 domains (in this way, the first and second CH3 domains heterodimerize, and no homodimer is formed between the two first or two second CH3 domains). These various approaches for improved heavy chain heterodimerization are intended as various options to be combined with the heavy-light chain modification in multispecific antibodies according to the present invention (introduction of VH and VL exchange / substitution at one binding arm and substitution with a charged amino acid with the opposite charge at the CH1 / CL interface) to reduce light chains that mispair and become Bensoo-Jones type byproducts.

[0325] In one preferred embodiment of the present invention (where the multispecific antibody contains a CH3 domain in the heavy chain), the CH3 domain of the multispecific antibody according to the present invention can be altered by the “knob-into-holes” technique, which is described in detail with several examples in, for example, International Publication No. 96 / 027011, Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681; International Publication No. 98 / 050431. In this method, the interaction surface of the two CH3 domains is altered to increase heterodimerization of the two heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be a “knob,” while the other is a “hole.” The introduction of disulfide bridges 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.

[0326] Therefore, in one embodiment of the present invention, the multispecific antibody (containing a CH3 domain in each heavy chain, and) further, The first CH3 domain (a) of the first heavy chain of the antibody and the second CH3 domain (b) of the second heavy chain of the antibody each meet at an interface including the original interface between the antibody CH3 domains. The interface has been modified to promote the formation of multispecific antibodies, and this modification is i) The CH3 domain of one of the heavy chains has been altered, As a result, in the multispecific antibody, within the original interface of one heavy chain's CH3 domain that is in contact with the original interface of the other heavy chain's CH3 domain, The amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a projection within the interface of the CH3 domain of one heavy chain that can be positioned within the cavity within the interface of the CH3 domain of the other heavy chain, and ii) The CH3 domain of the other heavy chain has been altered, As a result, in the multispecific antibody, within the original interface of the second CH3 domain that is in contact with the original interface of the first CH3 domain, The amino acid residues are substituted with amino acid residues having smaller side chain volumes, thereby creating a cavity within the interface of the second CH3 domain, into which the protrusions within the interface of the first CH3 domain can be positioned. It is characterized by the following:

[0327] Preferably, the amino acid residue having the larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0328] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0329] In one embodiment of the present invention, both CH3 domains are further modified by the introduction of cysteine ​​(C) as an amino acid at the corresponding position of each CH3 domain, thereby allowing the formation of a disulfide bridge between the two CH3 domains.

[0330] In one preferred embodiment, the multispecific antibody includes an amino acid T366W mutation in the first CH3 domain of the "knob chain" and amino acid T366S, L368A, and Y407V mutations in the second CH3 domain of the "hole chain". Further interchain disulfide crosslinks between the CH3 domains can also be used, for example, by introducing an amino acid Y349C mutation into the CH3 domain of the "hole chain" and an amino acid E356C mutation or an amino acid S354C mutation into the CH3 domain of the "knob chain" (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681).

[0331] In one preferred embodiment, the multispecific antibody (each containing a CH3 domain in its heavy chain) contains 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 interchain disulfide bridges) (Kabat numbering).

[0332] Other techniques for modifying CH3 to enhance heterodimerization are intended as options of the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0333] In one embodiment, the heterodimerization approach described in European Publication No. 1870459A1 can be used instead. This approach is based on the introduction of substitution / mutation with an oppositely charged amino acid at a specific amino acid position within the CH3 / CH3 domain interface between the two heavy chains. One preferred embodiment for 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 (Kabat numbering) in the second CH3 domain of the multispecific antibody.

[0334] In another embodiment, the multispecific antibody includes the amino acid T366W mutation in the CH3 domain of the "knob chain", the amino acid T366S, L368A, Y407V mutations in the CH3 domain of the "hole chain", and additional amino acid R409D;K370E mutations in the CH3 domain of the "knob chain" and amino acid D399K;E357K mutations in the CH3 domain of the "hole chain".

[0335] In another embodiment, the multispecific antibody contains the amino acid S354C, T366W mutation in one of the two CH3 domains and the amino acid Y349C, T366S, L368A, Y407V mutation in the other of the two CH3 domains, or the multispecific antibody contains the amino acid Y349C, T366W mutation in one of the two CH3 domains and the amino acid S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains, as well as an additional amino acid R409D;K370E mutation in the CH3 domain of the "knob chain" and an amino acid D399K;E357K mutation in the CH3 domain of the "hole chain".

[0336] In one embodiment, the heterodimerization approach described in International Publication No. 2013 / 157953 can be used instead. In one embodiment, the first CH3 domain contains an amino acid T366K mutation, and the second CH3 domain polypeptide contains an amino acid L351D mutation. In a further embodiment, the first CH3 domain contains a further amino acid L351K mutation. In a further embodiment, the second CH3 domain contains a further amino acid mutation selected from Y349E, Y349D, and L368E (preferably L368E).

[0337] In one embodiment, the heterodimerization approach described in International Publication No. 2012 / 058768 can be used instead. In one embodiment, the first CH3 domain contains the amino acid L351Y, Y407A mutation, and the second CH3 domain contains the amino acid T366A, K409F mutation. In one further embodiment, the second CH3 domain includes further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, selected from, for example, 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 a further embodiment, the first CH3 domain contains amino acid L351Y, Y407A mutations, and the second CH3 domain contains amino acid T366V, K409F mutations. In a further embodiment, the first CH3 domain contains amino acid Y407A mutations, and the second CH3 domain contains amino acid T366A, K409F mutations. In a further embodiment, the second CH3 domain contains further amino acid K392E, T411E, D399R, and S400R mutations.

[0338] In one embodiment, for example, the heterodimerization approach described in International Publication No. 2011 / 143545, which involves amino acid modification at a position selected from the group consisting of 368 and 409, can be used instead.

[0339] In one embodiment, the heterodimerization approach described in International Publication No. 2011 / 090762, which also uses the knobs-into-holes technique, can be used instead. In one embodiment, the first CH3 domain contains the amino acid T366W mutation, and the second CH3 domain contains the amino acid Y407A mutation. In another embodiment, the first CH3 domain contains the amino acid T366Y mutation, and the second CH3 domain contains the amino acid Y407T mutation.

[0340] In one embodiment, the multispecific antibody is of the IgG2 isotype, and the heterodimerization approach described in International Publication No. 2010 / 129304 can be used instead.

[0341] In one embodiment, the heterodimerization approach described in International Publication No. 2009 / 089004 can be used instead. In one embodiment, the first CH3 domain comprises an amino acid substitution of K392 or N392 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain comprises an amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In one further embodiment, the first CH3 domain further comprises an amino acid substitution of K409 or R409 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In one further embodiment, the first CH3 domain further or alternatively comprises an amino acid substitution of K439 and / or K370 with a negatively charged amino acid (e.g., glutamic acid (E) or aspartic acid (D)).

[0342] In one embodiment, the heterodimerization approach described in International Publication No. 2007 / 147901 can be used instead. In one embodiment, the first CH3 domain contains amino acid K253E, D282K and K322D mutations, and the second CH3 domain contains amino acid D239K, E240K and K292D mutations.

[0343] In one embodiment, the heterodimerization approach described in International Publication No. 2007 / 110205 can be used instead.

[0344] E. Recombinant methods and recombinant compositions Antibodies can be produced using recombinant methods and recombinant compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding a dimeric polypeptide reported herein is provided. Such nucleic acid may encode an amino acid sequence comprising the first polypeptide and / or the second polypeptide of the dimeric polypeptide. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed by) (1) a vector comprising nucleic acid encoding an amino acid sequence comprising the first polypeptide of the dimeric polypeptide and an amino acid sequence comprising the second polypeptide of the dimeric polypeptide; (2) a first vector comprising nucleic acid encoding an amino acid sequence comprising the first polypeptide of the dimeric polypeptide and a second vector comprising nucleic acid encoding an amino acid sequence comprising the second polypeptide of the dimeric polypeptide. In one embodiment, the host cell is a eukaryote, for example, Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method is provided for producing the dimeric polypeptide reported herein, comprising the steps of culturing host cells containing nucleic acids encoding the previously defined dimeric polypeptide under conditions suitable for the expression of the dimeric polypeptide, and optionally recovering antibodies from the host cells (or host cell culture medium).

[0345] For recombinant production of the dimerized polypeptides reported herein, the nucleic acid encoding the dimerized polypeptide is isolated, for example, as described above, 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 procedures (e.g., by using oligonucleotide probes that have the ability to specifically bind to genes encoding mutant Fc region polypeptides and the heavy and light chains of antibodies).

[0346] Suitable host cells for cloning or expressing vectors encoding dimeric polypeptides include prokaryotic or eukaryotic cells as described herein. For example, dimeric polypeptides may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the dimeric polypeptides may be isolated from the bacterial cell paste into a soluble fraction and further purified.

[0347] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi and yeasts (including fungal and yeast strains whose glycosylation pathways are "humanized," resulting in the production of dimerized polypeptides with partially or completely human glycosylation patterns) are also suitable cloning or expression hosts for vectors encoding dimerized polypeptides. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414 and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0348] Suitable host cells for glycosylated dimeric polypeptide expression can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used with insect cells, and in particular, for transfection of Spodoptera frugiperda cells.

[0349] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for producing antibodies in transgenic plants).

[0350] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be useful. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7), human fetal kidney cells (e.g., HEK293 or 293 cells described in Raham, FL, et al., J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), and mouse mammary cancer cells (MMT 060562), e.g., Mather, JP et al., Annals NY Acad. Sci. 383. These include TRI cells, MRC 5 cells, and FS4 cells, as described in (1982) 44-68. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of some 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.

[0351] F. Combination Treatment In one embodiment, the dimeric polypeptide or the pharmaceutical formulation reported herein is administered alone (without additional therapeutic agents) for the treatment of one or more ophthalmic vascular diseases described herein.

[0352] In other embodiments, the dimeric polypeptide antibodies or pharmaceutical formulations reported herein are administered in combination with one or more additional therapeutic agents or treatment methods for the treatment of one or more ophthalmic vascular diseases described herein.

[0353] In other embodiments, the dimeric polypeptides or pharmaceutical formulations reported herein are formulated in combination with one or more additional therapeutic agents and administered for the treatment of one or more ophthalmic vascular diseases as described herein.

[0354] In one embodiment, the combination treatment provided herein comprises sequentially administering a dimeric polypeptide antibody or pharmaceutical formulation reported herein, together with one or more additional therapeutic agents, for the treatment of one or more ophthalmic vascular diseases described herein.

[0355] Additional therapeutic agents include tryptophanyl-tRNA synthetase (TrpRS), EyeOOl (anti-VEGF PEGylated aptamer), squalamine, RETAANE (Anecol acetate tablets for depot suspension; Alcon, Inc.), Combretastatin A4 prodrug (CA4P), MACUGEN (Trademark), MIFEPREX (Trademark) (mifepristone-ru486), subtenone triamcinolone acetonide, intravitreal crystalline triamcinolone acetonide, prinomast (AG3340 synthetic matrix metalloproteinase inhibitor, Pfizer), and fluocinolone acetonide (including fluocinone intraocular implants, Bausch & Lomb / Control Delivery). This includes, but is not limited to, integrin v.beta.3 function inhibitors and angiostatin inhibitors, such as VEGF-Trap (Regeneron / Aventis), VEGF receptor tyrosine kinase inhibitors, such as 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(l-methylpiperidine-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindole-5-yloxy)-6-methoxy-7-(3-pyrrolidine-1-ylpropoxy)quinazoline (AZD2171), batalanib (PTK787) and SU11248 (sunitinib), linamide, and integrin v.beta.3 function inhibitors.

[0356] Other pharmaceutical therapies that may be used in combination with the dimeric polypeptides or pharmaceutical formulations reported herein include non-thermal lasers such as VISUDYNE®, PKC412, Endovion (NeuroSearch A / S), neurotrophic factors (including, for example, glial neurotrophic factors and ciliary neurotrophic factors), diatazem, dorzolamide, phototrop, 9-cis-retinal, eye drops (including ultrasound therapy) (including phospholine iodide, ecothiophate, or carbonic anhydrase inhibitors), AE-941 (AEterna Laboratories, Inc.), Sirna-027 (Sima Therapeutics, Inc.), pegaptanib (NeXstar Pharmaceuticals / Gilead Sciences), neurotrophins (including, for example, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), and integrin antagonists (Jerini AG and Abbott). (Including those from laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (as used, e.g., by EntreMed, Inc.), 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 cell ganglion neuroprotective agents (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, limited retinal translocation, photodynamic therapy (e.g., receptor-targeted PDT, Bristol-Myers Squibb, Co.; Porfimer sodium for injection with PDT; Verteporfin, QLT Inc.; Rostaporfin with PDT, Miravent Medical Technologies; Talaporfin sodium with PDT, Nippon Petroleum; Motexafin lutetium, Pharmacyclics, Antisense oligonucleotides (including, for example, products tested by Novagali Pharma SA and ISIS-13650, Isis Pharmaceuticals), laser photocoagulation, drusen laser processing, macular hole surgery, macular translocation surgery, implantable miniature telescopes, phimotion angiography (also known as microlaser therapy and feeder vessel procedures), proton beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, submacular surgery, transpupillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal respiration (also known as differential filtration and rheotherapy), microchip implantation, stem cell therapy, gene exchange therapy, ribozyme gene therapy (including gene therapy for hypoxia-responsive elements, Oxford Biomedica; Lentipak, GENETIX; PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (implantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, Cell Genesys, Inc.)This includes, but is not limited to, acupuncture needles, etc. (including, but also includes, acupuncture needles).

[0357] Any anti-angiogenic agent (including, but not limited to, those enumerated in Carmeliet and Jain, 2000, Nature 407: 249-257) may be used in combination with the dimeric polypeptides or pharmaceutical formulations reported herein. In some embodiments, the anti-angiogenic agent is another VEGF antagonist or VEGF receptor antagonist, e.g., VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecular weight inhibitors of VEGFR tyrosine kinases, and any combination thereof, including anti-VEGF aptamers (e.g., pegaptanib) and soluble recombinant decoy receptors (e.g., VEGF traps). In one embodiment, anti-angiogenic agents include corticosteroids, angiogenesis-inhibiting steroids, anecol tabs acetate, angiostatins, endostatins, small interfering RNAs that reduce the expression of VEGFR or VEGF ligands, post-VEGFR blockade using tyrosine kinase inhibitors, MMP inhibitors, IGFBP3, SDF-1 blockers, PEDF, gamma-secretase, delta-like ligand 4, integrin antagonists, HIF-1 alpha blockade, protein kinase CK2 blockade, and inhibition of stem cell (i.e., endothelial progenitor cell) homing to angiogenesis sites using vascular endothelial cadherin (CD-144) and stromal-derived factor (SDF)-I antibodies. Small molecule RTK inhibitors targeting VEGF receptors (including PTK787) may also be used. While not necessarily anti-VEGF compounds, drugs with angiogenesis-promoting activity can also be used, including anti-inflammatory drugs, m-Tor inhibitors, rapamycin, everolimus, temsirolimus, cyclosporine, anti-TNF agents, anti-complement agents, and nonsteroidal anti-inflammatory drugs. Neuroprotective drugs that can potentially slow the progression of dry macular degeneration (such as a class of drugs called "neurosteroids") can also be used. These include drugs such as dehydroepiandrosterone (DHEA) (brand names: Prastera® and Fidelin®), dehydroepiandrosterone sulfate, and pregnenolone sulfate.Any AMD (age-related macular degeneration) treatment agent may be used in combination with the dimeric polypeptides or pharmaceutical formulations reported herein, including, but not limited to, verteporfin, pegaptanib sodium, zinc, or antioxidants (alone or in combination) in combination with PDT.

[0358] G. Prescription drugs The pharmaceutical formulations of dimerized polypeptides reported herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such dimerized polypeptides of desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.(ed.) (1980)). The pharmaceutically acceptable carriers are generally nontoxic to the recipient at the dosage and concentration used and include buffering agents such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypeptides. Butides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, e.g., poly(vinylpyrrolidone); amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates, e.g., glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers as used herein further include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rhuPH20 (HYLENEX®, Baxter International, Inc.). rhuPH20 and other exemplary sHASEGPs and methods of use are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968.In one embodiment, sHASEGP is used in combination with one or more additional glycosaminoglycansases, such as chondroitinases.

[0359] An example of a lyophilized antibody formulation is described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0360] The formulations described herein may contain two or more active ingredients, preferably those having complementary activities that do not adversely affect each other, as required for the specific indication being treated. Such active ingredients may be present in combination in amounts effective for the intended purpose, as appropriate.

[0361] The active ingredient may be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, each prepared by coacervation technology or interfacial polymerization), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0362] A sustained-release preparation may be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody, wherein the matrix is ​​in the form of a molded body such as a film or microcapsule.

[0363] Formulas used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0364] H. Treatment methods and therapeutic compositions All of the dimeric polypeptides reported herein can be used in therapeutic methods.

[0365] In one embodiment, a dimer polypeptide reported herein is provided for use as a pharmaceutical. In a further embodiment, a dimer polypeptide is provided for use in the treatment of an eye disease. In one embodiment, a dimer polypeptide is provided for use in a treatment method. In one embodiment, the present invention provides a dimer polypeptide for use in a method for treating an individual having an ocular vascular disease, the method comprising administering to the individual an effective amount of the dimer polypeptide 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., as described in Section D). In a further embodiment, the present invention provides a dimer polypeptide for use in inhibiting angiogenesis in the eye. In one embodiment, the present invention provides a dimer polypeptide for use in a method for inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of the dimer polypeptide to inhibit angiogenesis. In any of the above embodiments, “individual” is, in one preferred embodiment, a human.

[0366] In one further embodiment, the present invention provides the use of a dimeric polypeptide in the manufacture or preparation of a pharmaceutical. In one embodiment, the pharmaceutical is for the treatment of an ophthalmic vascular disease. In one further embodiment, the pharmaceutical is for use in a method for treating an ophthalmic vascular disease, comprising the step of administering an effective amount of the pharmaceutical to an individual having the ophthalmic vascular disease. In one such embodiment, the method further comprises the step of administering to the individual an effective amount of at least one additional therapeutic agent (for example, as described above). In one further embodiment, the pharmaceutical is for inhibiting angiogenesis. In one further embodiment, the pharmaceutical is for use in a method for inhibiting angiogenesis in an individual, comprising the step of administering to the individual an effective amount of the pharmaceutical to inhibit angiogenesis. The “individual” in any of the above embodiments may be a human.

[0367] In one further embodiment, the present invention provides a method for treating an ophthalmic vascular disease. In one embodiment, the method comprises administering an effective amount of the dimeric polypeptide reported herein to an individual having such an ophthalmic vascular disease. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as those described below, to the individual. The “individual” in any of the above embodiments may be a human.

[0368] In a further embodiment, the present invention provides a method for inhibiting angiogenesis in the eye of an individual. In one embodiment, the method comprises administering to the individual an amount of the dimeric polypeptide reported herein that is effective in inhibiting angiogenesis. In one embodiment, “individual” is a human.

[0369] In a further embodiment, the present invention provides a pharmaceutical formulation comprising any of the dimerized polypeptides reported herein for use, for example, in any of the therapeutic methods described herein. In one embodiment, the pharmaceutical formulation comprises any of the dimerized polypeptides reported herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the dimerized polypeptides reported herein and at least one additional therapeutic agent, for example, as described below.

[0370] The dimerized polypeptides reported herein can be used therapeutically, either alone or in combination with other active ingredients. For example, the dimerized polypeptides reported herein can be administered concurrently with at least one additional therapeutic agent.

[0371] The dimeric polypeptides (and any additional therapeutic agents) reported herein may be administered by any suitable means, including parenteral administration, intrapulmonary administration, intranasal administration, and, where desirable for local treatment, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any suitable route, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various dosing schedules, including (but not limited to) single doses, multiple doses at various points in time, bolus doses, and pulse infusions, are intended herein.

[0372] The dimerized polypeptides reported herein are prescribed, administered, and given in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to healthcare professionals. Dimerized polypeptides do not necessarily need to be prescribed with one or more active agents currently used to prevent or treat the disorder in question, but this may be done in some cases. The effective amount of such other active agents depends on the amount of dimerized polypeptide present in the prescription, the type of disorder or treatment, and the other factors mentioned above. These are generally used in the same dosage and route of administration as described herein, or at approximately 1–99% of the dosage described herein, or in any dosage and route as determined experimentally / clinically appropriate.

[0373] With regard to the prevention or treatment of disease, the appropriate dosage of the dimerized polypeptide reported herein (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of dimerized polypeptide, the severity and course of the disease, whether the dimerized polypeptide is administered prophylactically or therapeutically, the patient's treatment history, the patient's medical history and response to the dimerized polypeptide, and the discretion of the attending physician. The dimerized polypeptide is administered appropriately to the patient in a single dose or a series of treatments. Depending on the type and severity of the disease, for example, whether by a single or more independent dose or by continuous infusion, a dimerized polypeptide of about 1 μg / kg to 15 mg / kg (e.g., 0.5 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or more, treatment is generally maintained, depending on the condition, until the desired suppression of disease symptoms occurs. One exemplary dosage of the dimer polypeptide ranges from approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, a patient may be administered one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof). Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to 20 doses of the dimer polypeptide, for example, approximately 6 doses). A high initial loading dose may be administered first, followed by one or more lower doses. The progression of this treatment is readily monitored using conventional techniques and assays.

[0374] III. Manufactured products In another embodiment of the present invention, a product is provided comprising a substance useful for treating, preventing and / or diagnosing the aforementioned disorder. The product comprises a container and a label or accompanying documentation on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be formed from a variety of materials, such as glass or plastic. The container may hold a composition effective for treating, preventing and / or diagnosing the aforementioned condition, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured with a subcutaneous needle). At least one active substance in the composition is a dimeric polypeptide reported herein. The label or accompanying documentation indicates that the composition is used for treating a selected condition. Furthermore, the product may comprise (a) a first container containing a composition comprising the dimeric polypeptide reported herein, and (b) a second container containing a composition comprising further cytotoxic substances or other therapeutic agents. The product of this embodiment of the present invention may further include a document indicating that the composition can be used to treat a particular condition. Alternatively, or in addition thereto, the product may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, and syringes.

[0375] It is understood that any of the above-mentioned products may contain, in place of or in addition to, the immunoconjugate reported herein for the dimerized polypeptides reported herein.

[0376] IV. Specific Embodiments 1. A dimerized polypeptide, The first polypeptide and the second polypeptide each comprise at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, in the direction from the N-terminus to the C-terminus. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0377] 2. The dimer polypeptide described in item 1, which does not specifically bind to human FcRn but specifically binds to Staphylococcus protein A.

[0378] 3. A homodimer polypeptide, as described in any one of items 1 to 2.

[0379] 4. A dimer polypeptide described in any one of items 1 to 2, which is a heterodimer polypeptide.

[0380] 5.i) The first polypeptide further comprises mutants Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutants S354C and T366W, or ii) The first polypeptide further comprises mutants S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutants Y349C and T366W, according to any one of claims 1 to 4.

[0381] 6. The polypeptide according to any one of claims 1 to 5, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG1 subclass.

[0382] 7. The dimer polypeptide according to any one of claims 1 to 6, wherein the first polypeptide and the second polypeptide further comprise mutants L234A and L235A.

[0383] 8. The dimer polypeptide according to any one of claims 1 to 5, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG2 subclass and may be accompanied by mutations V234A, G237A, P238S, H268A, V309L, A330S, and P331S.

[0384] 9. The dimer polypeptide according to any one of claims 1 to 5, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG4 subclass.

[0385] 10. The dimer polypeptide according to any one of claims 1 to 5 and 9, wherein the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.

[0386] 11. The dimer polypeptide according to any one of claims 1 to 10, wherein the first polypeptide and the second polypeptide further comprise mutant P329G.

[0387] 12. A dimer polypeptide according to any one of items 1 to 11, which is an Fc region fusion polypeptide.

[0388] 13. A (full-length) antibody, a dimerized polypeptide as described in any one of items 1 to 11.

[0389] 14. A dimerized polypeptide according to any one of items 1 to 11 and 13, wherein the (full-length) antibody is a monospecific antibody.

[0390] 15. The dimer polypeptide according to any one of items 1 to 11 and 13 to 14, wherein the monospecific antibody is a monovalent monospecific antibody.

[0391] 16. A dimerized polypeptide according to any one of items 1 to 11 and 13 to 15, wherein the monospecific antibody is a bivalent monospecific antibody.

[0392] 17. A dimerized polypeptide according to any one of items 1 to 11 and 13, wherein the (full-length) antibody is a bispecific antibody.

[0393] 18. The dimer polypeptide according to any one of items 1 to 11, 13, and 17, wherein the bispecific antibody is a bivalent bispecific antibody.

[0394] 19. A dimerized polypeptide according to any one of items 1 to 11, 13, and 17 to 18, wherein the bispecific antibody is a tetravalent bispecific antibody.

[0395] 20. A dimerized polypeptide according to any one of items 1 to 11 and 13, wherein the (full-length) antibody is a trispecific antibody.

[0396] 21. The dimer polypeptide according to any one of items 1 to 11, 13, and 20, wherein the triple-specific antibody is a trivalent triple-specific antibody.

[0397] 22. The dimer polypeptide according to any one of items 1 to 11, 13, and 20 to 21, wherein the triple-specific antibody is a tetravalent triple-specific antibody.

[0398] 23. Dimeric polypeptide, The first polypeptide and the second polypeptide each comprise at least a portion of an immunoglobulin hinge region containing one or more cysteine ​​residues, an immunoglobulin CH2 domain and an immunoglobulin CH3 domain, in the direction from the N-terminus to the C-terminus. The first, second, or first and second polypeptides contain mutation Y436A (numbered according to the Kabat EU index numbering system), Dimeric polypeptide.

[0399] 24. The dimer polypeptide according to item 23, wherein the first and second polypeptides contain the mutation Y436A.

[0400] 25. A dimer polypeptide according to any one of items 23 to 24, which does not specifically bind to human FcRn but specifically binds to Staphylococcus protein A.

[0401] 26. A homodimer polypeptide, as described in any one of items 23 to 25.

[0402] 27. A heterodimer polypeptide, as described in any one of items 23 to 25.

[0403] 28.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 b)i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D, and L432D, or iii) The first and second polypeptides contain mutants L251S, L314S, and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. A dimerized polypeptide as described in any one of items 23 to 27.

[0404] 29. The dimer polypeptide according to any one of claims 23 to 28, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG1 subclass.

[0405] 30. The dimer polypeptide according to any one of claims 23 to 29, wherein the first polypeptide and the second polypeptide further comprise mutants L234A and L235A.

[0406] 31. The dimer polypeptide according to any one of claims 23 to 28, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG2 subclass and may be accompanied by mutations V234A, G237A, P238S, H268A, V309L, A330S, and P331S.

[0407] 32. The dimer polypeptide according to any one of claims 23 to 28, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain, and the immunoglobulin CH3 domain are of the human IgG4 subclass.

[0408] 33. The dimer polypeptide according to any one of claims 23 to 28 and 32, wherein the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.

[0409] 34. The dimer polypeptide according to any one of items 23 to 33, wherein the first polypeptide and the second polypeptide further comprise mutant P329G.

[0410] 35. A dimer polypeptide according to any one of items 23 to 34, which is an Fc region fusion polypeptide.

[0411] 36. A (full-length) antibody, a dimerized polypeptide as described in any one of items 23 to 34.

[0412] 37. A dimerized polypeptide according to any one of items 23 to 34 and 36, wherein the (full-length) antibody is a monospecific antibody.

[0413] 38. The dimer polypeptide according to any one of claims 23-34 and 36-37, wherein the monospecific antibody is a monovalent monospecific antibody.

[0414] 39. A dimerized polypeptide according to any one of claims 23-34 and 36-38, wherein the monospecific antibody is a bivalent monospecific antibody.

[0415] 40. A dimerized polypeptide according to any one of items 23 to 34 and 36, wherein the (full-length) antibody is a bispecific antibody.

[0416] 41. The dimer polypeptide according to any one of items 23 to 34, 36, and 40, wherein the bispecific antibody is a bivalent bispecific antibody.

[0417] 42. The dimer polypeptide according to any one of items 23-34, 36, and 40-41, wherein the bispecific antibody is a tetravalent bispecific antibody.

[0418] 43. The dimer polypeptide according to any one of items 23 to 34 and 36, wherein the (full-length) antibody is a triplicate antibody.

[0419] 44. The dimer polypeptide according to any one of items 23-34, 36, and 43, wherein the triple-specific antibody is a trivalent triple-specific antibody.

[0420] 45. The dimer polypeptide according to any one of items 23-34, 36, and 43-44, wherein the triple-specific antibody is a tetravalent triple-specific antibody.

[0421] 46. ​​Dimeric polypeptide, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide further comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise mutants L234A, L235A, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0422] 47. Dimeric polypeptide, The first polypeptide comprises, from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG1, an immunoglobulin CH2 domain of subclass IgG1, and an immunoglobulin CH3 domain of subclass IgG1. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, and a subclass IgG1 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a subclass IgG1 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise mutants L234A, L235A, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0423] 48. Dimeric polypeptide, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise the mutations S228P, L235E, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0424] 49. Dimeric polypeptide, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG4, an immunoglobulin CH2 domain of subclass IgG4, and an immunoglobulin CH3 domain of subclass IgG4. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG4 immunoglobulin CH1 domain, a subclass IgG4 immunoglobulin hinge region, a subclass IgG4 immunoglobulin CH2 domain, and a subclass IgG4 immunoglobulin CH3 domain, A third polypeptide comprising a first light chain variable domain and a subclass IgG4 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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 second antigen. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise the mutations S228P, L235E, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0425] 50. Dimeric polypeptide, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a first scFv. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a second scFv. A third polypeptide comprising a first light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise mutants L234A, L235A, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0426] 51. Dimeric polypeptide, The first polypeptide comprises, in the direction from the N-terminus to the C-terminus, a first heavy chain variable domain, an immunoglobulin light chain constant domain, an immunoglobulin hinge region of subclass IgG1, an immunoglobulin CH2 domain of subclass IgG1, an immunoglobulin CH3 domain of subclass IgG1, a peptide linker, and a first scFv. A second polypeptide comprising, from the N-terminus to the C-terminus, a second heavy chain variable domain, a subclass IgG1 immunoglobulin CH1 domain, a subclass IgG1 immunoglobulin hinge region, a subclass IgG1 immunoglobulin CH2 domain, a subclass IgG1 immunoglobulin CH3 domain, a peptide linker, and a second scFv. A third polypeptide comprising a first light chain variable domain and a subclass IgG1 immunoglobulin CH1 domain, in the direction from the N-terminus to the C-terminus. A fourth polypeptide comprising a second light chain variable domain and a light chain constant domain, extending from the N-terminus to the C-terminus. Includes, 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. i) The first polypeptide comprises mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises mutations S354C and T366W, or ii) The first polypeptide comprises mutations S354C, T366S, L368A and Y407V, and the second polypeptide comprises mutations Y349C and T366W, The first and second polypeptides further comprise mutants L234A, L235A, and P329G. i) The first and second polypeptides contain mutations H310A, H433A and Y436A, or ii) The first and second polypeptides contain mutations L251D, L314D and L432D, or iii) The first and second polypeptides contain mutations L251S, L314S and L432S, or iv) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations H310A, H433A and Y436A, or v) The first polypeptide contains mutations I253A, H310A and H435A, and the second polypeptide contains mutations L251D, L314D and L432D, or vi) The first polypeptide comprises mutations I253A, H310A and H435A, and the second polypeptide comprises mutations L251S, L314S and L432S. Dimeric polypeptide.

[0427] A method for producing a dimerized polypeptide as described in any one of items 52.1 to 51, comprising the following steps: a) A step of culturing mammalian cells containing one or more nucleic acids encoding a dimeric polypeptide as described in any one of items 1 to 51, b) A step of recovering the dimer polypeptide from the culture medium, and c) Purifying the dimerized polypeptide by protein A affinity chromatography. A method that includes this.

[0428] 53. Use of mutant Y436A to increase the binding of dimeric polypeptides to protein A.

[0429] 54. Use of mutants H310A, H433A, and Y436A for the isolation of heterodimer polypeptides from homodimer polypeptides.

[0430] 55. Use of mutants L251D, L314D, L432D or mutants L251S, L314S, L432S for the isolation of heterodimer polypeptides from homodimer polypeptides.

[0431] 56. Use of heterodimer polypeptides comprising the first and second polypeptides, in combination with mutations I253A, H310A, and H435A in the first polypeptide, and mutations H310A, H433A, and Y436A in the second polypeptide, for the separation of heterodimer polypeptides from homodimer polypeptides.

[0432] 57. Use of heterodimer polypeptides comprising the first and second polypeptides, in combination with mutations I253A, H310A, and H435A in the first polypeptide, or mutations L251D, L314D, L432D, or mutations L251S, L314S, L432S in the second polypeptide, for the separation of heterodimer polypeptides from homodimer polypeptides.

[0433] 58. The use according to any one of claims 53 to 57, wherein the first polypeptide further comprises the mutations Y349C, T366S, L368A and Y407V, and the second polypeptide comprises the mutations S354C and T366W.

[0434] 59. A method for treating a patient with ocular vascular disease, comprising administering a dimerized polypeptide described in any one of items 1 to 51 to a patient requiring such treatment.

[0435] 60. A dimer polypeptide according to any one of items 1 to 51, for intravitreal application.

[0436] 61. A dimerized polypeptide according to any one of items 1 to 51, for use in the treatment of ocular vascular diseases.

[0437] 62. A pharmaceutical formulation comprising a dimeric polypeptide as described in any one of paragraphs 1 to 51 and optionally a pharmaceutically acceptable carrier.

[0438] 63. Use of a dimerized polypeptide according to any one of items 1 to 51 for transporting a soluble receptor ligand across the blood-ocular barrier from the eye into the blood circulation.

[0439] 64. Use of a dimerized polypeptide according to any one of items 1 to 51 for removing 64.1 or more soluble receptor ligands from the eye.

[0440] 65. Use of any one of the dimer polypeptides described in sub-sub

[0441] 66. Use of a dimerized polypeptide according to any one of items 1 to 51 for transporting 66.1 or more soluble receptor ligands from the intravitreous space into the blood circulation.

[0442] 67. A dimerized polypeptide according to any one of items 1 to 51, for use in treating eye diseases.

[0443] 68. A dimerized polypeptide according to any one of items 1 to 51, for use in transporting a soluble receptor ligand from the eye to the blood circulation across the blood-ocular barrier.

[0444] 69. A dimerized polypeptide according to any one of items 1 to 51, for use in the removal of 69.1 or more soluble receptor ligands from the eye.

[0445] 70. A dimerized polypeptide according to any one of items 1 to 51, for use in treating eye diseases, particularly ocular vascular diseases.

[0446] 71. A dimerized polypeptide according to any one of items 1 to 51, for use in transporting 1 or more soluble receptor ligands from the intravitreous space to the blood circulation.

[0447] 72. A method for treating an individual having an ophthalmic vascular disease, comprising the step of administering to the individual an effective amount of a dimerized polypeptide described in any one of items 1 to 51.

[0448] 73. A method for transporting a soluble receptor ligand from the eye to the blood circulation across the blood-ocular barrier in an individual, comprising the step of administering an effective amount of a dimerized polypeptide described in any one of items 1 to 51 to the individual, thereby transporting the soluble receptor ligand from the eye to the blood circulation across the blood-ocular barrier.

[0449] 74. A method for removing one or more soluble receptor ligands from the eye of an individual, comprising the step of administering an effective amount of a dimerized polypeptide described in any one of items 1 to 51 to the individual to remove one or more soluble receptor ligands from the eye.

[0450] 75. A method for transporting one or more soluble receptor ligands from the intravitreous space to the blood circulation in an individual, comprising the step of administering an effective amount of a dimerized polypeptide described in any one of items 1 to 51 to the individual, thereby transporting one or more soluble receptor ligands from the intravitreous space to the blood circulation.

[0451] 76. A method for transporting a soluble receptor ligand to the blood circulation from the vitreous space or the eye across the blood-ocular barrier in an individual, comprising the step of administering an effective amount of a dimerized polypeptide described in any one of items 1 to 51 to the individual, thereby transporting the soluble receptor ligand from the eye across the blood-ocular barrier to the blood circulation. [Examples]

[0452] V. Examples The following are examples of the method and composition of the present invention. Based on the above general description, it will be understood that various other embodiments can be implemented.

[0453] Although the above invention has been described in some detail by description and examples for the sake of clarity, such description and examples should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated herein by reference in their entirety.

[0454] method Electrospray ionization mass spectrometry (ESI-MS) A protein aliquot (50 μg) was deglycosylated by adding 0.5 μL of N-glycanase plus (Roche) and sodium phosphate buffer (0.1 M, pH 7.1) to obtain a final sample volume of 115 μL. This mixture was incubated at 37°C for 18 hours. Subsequently, 60 μL of 0.5 M TCEP (Pierce) dissolved in 4 M guanidine hydrochloride (Pierce) and 50 μL of 8 M guanidine hydrochloride were added for reduction and denaturation. This mixture was incubated at 37°C for 30 minutes. The sample was desalted by size exclusion chromatography (Sepharose G-25, isocratic, 40% acetonitrile with 2% formic acid). ESI mass spectra (+ve) were recorded using a Q-TOF instrument (maXis, Bruker) equipped with a nanoESI source (TriVersa NanoMate, Advion). The MS parameters were set as follows: Movement: Funnel RF, 400 Vpp; ISCID energy, 0 eV; Multipole RF, 400 Vpp; Quadrupole: Ion energy, 4.0 eV; Low mass, 600 m / z; Supply: 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; Movement time: 120 μs; Pre-pulse accumulation, 10 μs; Scan range m / z 600–2000. In-house developed software (MassAnalyzer) was used to evaluate the data.

[0455] FcRn surface plasmon resonance (SPR) analysis The binding characteristics of wild-type and mutant antibodies to FcRn were analyzed using surface plasmon resonance (SPR) technology with a BIAcore T100 instrument (BIAcore AB, Uppsala, Sweden). This method is well-established for studying molecular interactions. It allows for continuous, real-time monitoring of ligand / analyte binding and thus enables the measurement of dynamic parameters in various assay settings. SPR technology is based on measuring the refractive index near the surface of a gold-coated biosensor chip. The change in refractive index suggests a change in surface mass caused by the interaction between the immobilized ligand and the analyte injected into the solution. When a molecule binds to the immobilized ligand on the surface, its mass increases; when it dissociates, its mass decreases. In this assay, FcRn receptors were immobilized on a BIAcore CM5 biosensor chip (GE Healthcare Bioscience, Uppsala, Sweden) by amine coupling to a level of 400 response units (RU). The assay was performed at room temperature using PBS and 0.05% Tween20 pH 6.0 (GE Healthcare Bioscience) as running and dilution buffers. A 200 nM sample was injected at room temperature at a flow rate of 50 μL / min. The binding time was 180 seconds. The dissociation phase lasted 360 seconds. Chip surface regeneration was achieved by short-term injection of HBS-P, pH 8.0. SPR data was evaluated by comparing the height of the biological response signal at 180 seconds and 300 seconds after injection. The corresponding parameters were the maximum RU level (180 seconds after injection) and late-stage stability (300 seconds after injection completion).

[0456] Protein A surface plasmon resonance (SPR) analysis This assay is based on surface plasmon resonance spectroscopy. Protein A is immobilized on the surface of an SPR biosensor. By injecting a sample into the flow cell of the SPR spectrometer, it forms a complex with the immobilized protein A, which results in an increased mass on the sensor chip surface and therefore a higher response (1 RU = 1 pg / mm³). 2 (As defined above). Subsequently, the sensor chip is regenerated by dissociating the sample-protein A complex. Then, the obtained response is evaluated in terms of the signal with a high response unit (RU) and the dissociation behavior.

[0457] Using a GE Healthcare amine coupling kit, approximately 3500 response units (RU) (20 μg / mL) of protein A were coupled onto a CM5 tip (GE Healthcare) at pH 4.0.

[0458] The sample and system buffer was HBS-P+ (0.01M HEPES, 0.15M NaCl, 0.005% surfactant P20, sterile filtration, pH 7.4). The flow cell temperature was set to 25°C, and the sample compartment temperature to 12°C. The system was prepared with running buffer. Next, a 5 nM sample constituent solution was injected at a flow rate of 30 μL / min for 120 seconds, followed by a 300-second dissociation phase. Then, the sensor tip surface was regenerated by two injections of glycine-HCl (pH 1.5) at a flow rate of 30 μL / min for 30 seconds each. Each sample was measured three times.

[0459] [Table 11]

[0460] As used herein, the term "with mutation IHH-AAA" refers to a combination of mutations I253A (Ile253Ala), H310A (His310Ala), and H435A (His435Ala) in the constant heavy chain region of an IgG1 or IgG4 subclass (numbered according to the Kabat EU index numbering system), and as used herein, the term "with mutation HHY-AAA" refers to a combination of mutations H310A (His310Ala), H433A (His433Ala), and Y436A (Tyr436Ala) in the constant heavy chain region of an IgG1 or IgG4 subclass (Kabat EU index numbering system). The term "with mutation P329GLALA" as used herein refers to the combination of mutations L234A (Leu234Ala), L235A (Leu235Ala), and P329G (Pro329Gly) in the constant heavy chain region of the IgG1 subclass (numbered according to the Kabat EU Index Numbering System), and the term "with mutation SPLE" as used herein refers to the combination of mutations S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy chain region of the IgG4 subclass (numbered according to the Kabat EU Index Numbering System).

[0461] [Table 12]

[0462] general General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given below: 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 chain are numbered and referenced according to EU numbering (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)).

[0463] Recombinant DNA technology DNA was manipulated using 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). Molecular biological reagents were used according to the manufacturer's instructions.

[0464] gene synthesis The desired gene segment was ordered from Geneart (Regensburg, Germany) according to the given specifications.

[0465] DNA sequencing The DNA sequence was determined by double-strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0466] DNA and protein sequence analysis and sequence data management. GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 were used for sequence creation, mapping, analysis, annotation, and visualization.

[0467] Expression vector For the expression of the described antibodies, an expression plasmid for transient expression (e.g., in HEK293-F) was used, based on either cDNA organization with or without the CMV-intron A promoter, or genomic organization with the CMV promoter.

[0468] The vector, in addition to the antibody expression cassette, included the following: - The origin of replication that enables replication of this plasmid in E. coli, - The β-lactamase gene that confers ampicillin resistance in E. coli, and - The dihydrofolate reductase gene from mice as a selectable marker in eukaryotic cells.

[0469] The transcription unit of an antibody gene consists of the following elements: -5' terminus specific restriction site, - Early enhancers and promoters from human cytomegalovirus, -In the case of cDNA organization, the intron A sequence follows. - The 5' untranslated region of the human antibody gene, - Nucleic acids encoding immunoglobulin heavy chain signal sequences, - Nucleic acids encoding human antibody chains (wild-type or with domain exchange) as cDNA or in genomic organization, with immunoglobulin exon-intron organization. -3' untranslated region with polyadenylated signal sequence, and The -3' end is a specific restriction site.

[0470] Nucleic acids encoding antibody chains were generated by PCR and / or gene synthesis and assembled using known recombinant methods and by linking matched nucleic acid segments (e.g., using specific restriction sites in each vector). Subcloned nucleic acid sequences were validated by DNA sequencing. For transient transfection, larger quantities of vectors were prepared by vector preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

[0471] Cell culture technology Standard cell culture techniques were used 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.

[0472] The bispecific antibodies were expressed by transient, simultaneous transfection of their respective expression vectors in HEK29-F cells grown in the suspension described below.

[0473] Example 1 Expression and Purification Transient transfection in the HEK293-F system Monospecific and bispecific antibodies were generated by transient transfection with their respective vectors (e.g., encoding heavy chain and modified heavy chain, and corresponding light chain and modified light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen), grown in suspension in serum-free FreeStyle® 293 expression medium (Invitrogen) in either a shaking flask or a stirred fermenter, were transfected with their respective expression vectors and a mixture of 293 fectin® or fectin (Invitrogen). In a 2L shaking flask (Corning), HEK293-F cells were transfected at a rate of 1 × 10⁶ cells per 600mL. 6 Cells were seeded at a density of individual cells / mL and incubated at 120 rpm in 8% CO2. The following day, cells were subjected to approximately 1.5 × 10⁶ cells using a mixture of approximately 42 mL of Opti-MEM (Invitrogen) containing A) 20 mL of total vector DNA (1 μg / mL) encoding either the heavy chain or modified heavy chain and the corresponding light chain in equimolar ratios, and B) 20 mL of Opti-MEM containing 1.2 mL of 293 phenitin or phenitin (2 μl / mL). 6 Cells were transfected at a cell density of individual cells / mL. Glucose solution was added during the fermentation process according to glucose consumption. The supernatant containing the secreted antibodies was collected after 5-10 days, and the antibodies were purified directly from the supernatant, or the supernatant was frozen and stored.

[0474] purification Bispecific antibodies were purified from cell culture supernatant by affinity chromatography using MabSelectSure-Sepharose™ (for non-IHH-AAA mutants) (GE Healthcare, Sweden) or kappaSelect-Agarose (for IHH-AAA mutants) (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl Sepharose (GE Healthcare, Sweden), and Superdex 200 size exclusion chromatography (GE Healthcare, Sweden).

[0475] Briefly, sterile filtered cell culture supernatant was captured on MabSelectSuRe resin (non-IHH-AAA mutant and wild-type antibodies) equilibrated with PBS buffer (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 3.0. The IHH-AAA mutant was captured on kappaSelect resin equilibrated with 25 mM Tris, 50 mM NaCl, pH 7.2, washed with equilibration buffer, and eluted with 25 mM sodium citrate at pH 2.9. The eluted antibody fraction was pooled and neutralized with 2 M Tris, pH 9.0. For hydrophobic interaction chromatography, the antibody pool was prepared to a final concentration of 0.8 M ammonium sulfate by adding 1.6 M ammonium sulfate solution, and the pH was adjusted to pH 5.0 using acetic acid. After equilibrating the butyl Sepharose resin with 35 mM sodium acetate, 0.8 M ammonium sulfate, and pH 5.0, the antibody was added to the resin, washed with equilibrium buffer, and eluted using a linear gradient down to 35 mM sodium acetate and pH 5.0. The antibody (single-specific or bi-specific) fraction was pooled and further purified by size exclusion chromatography using a Superdex 200 26 / 60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, and pH 6.0. The antibody (single-specific or bi-specific) fraction was pooled and concentrated to the required concentration using a Vivaspin ultrafiltration system (Sartorius Stedim Biotech SA, France), and stored at -80°C.

[0476] [Table 13]

[0477] Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (Caliper Life Science, USA) after each purification step. 5 μl of protein solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according to the manufacturer's instructions and analyzed using the LabChip GXII system with the HT Protein Express Chip. Data were analyzed using LabChip GX Software.

[0478] [Table 14]

[0479] The aggregate content of antibody samples was analyzed at 25°C using a high-performance SEC (Secondary Analysis Cell) with a Superdex 200 analytical size exclusion column (GE Healthcare, Sweden) in 2×PBS (20 mM Na2HPO4, 2 mM KH2PO4, 274 mM NaCl and 5.4 mM KCl, pH 7.4) running buffer. 25 μg of protein was injected into the column at a flow rate of 0.75 ml / min and eluted isocratically for 50 minutes.

[0480] Similarly, the anti-VEGF / ANG2 antibodies VEGF / ANG2-0012 and VEGF / ANG2-0201 were prepared and purified, and the yields were as follows:

[0481] [Table 15]

[0482] Furthermore, there are anti-VEGF / ANG2 bispecific antibodies, including anti-VEGF / ANG2 CrossMAb IgG4 with IHH-AAA mutation and SPLE mutation (SEQ ID NO: 42, 43, 44, 45), anti-VEGF / ANG2 OAscFab IgG1 with IHH-AAA mutation (SEQ ID NO: 46, 47, 48), anti-VEGF / ANG2 OAscFab IgG4 with IHH-AAA mutation and SPLE mutation (SEQ ID NO: 49, 50, 51), anti-VEGF / ANG2 CrossMab IgG1 with HHY-AAA mutation and P329G LALA mutation (SEQ ID NO: 90, 91, 40, 41), anti-VEGF / ANG2 CrossMab IgG4 with HHY-AAA mutation and SPLE mutation (SEQ ID NO: 92, 93, 44, 45), and anti-VEGF / ANG2 OAscFab with HHY-AAA mutation. IgG1 (SEQ ID NO: 94, 95, 48), and anti-VEGF / ANG2 OAscFab IgG4 with HHY-AAA mutations and SPLE mutations (SEQ ID NO: 96, 97, 51), as well as anti-IGF-1R monospecific antibodies, wild-type anti-IGF-1R (SEQ ID NO: 88, 89), anti-IGF-1R IgG1 with IHH-AAA mutation (SEQ ID NO: 88, 90), anti-IGF-1R IgG1 with YTE mutation (SEQ ID NO: 88, 91), wild-type anti-IGF-1R IgG1 with KiH mutation (SEQ ID NO: 88, 92, 93), anti-IGF-1R IgG1 with KiH mutation and IHH-AAA mutation in the hole chain (SEQ ID NO: 88, 94, 95), and anti-IGF-1R with KiH mutation and HHY-AAA mutation in the hole chain IgG1 (SEQ ID NO: 88, 96, 97), anti-IGF-1R IgG1 with KiH mutations and YTE mutations (SEQ ID NO: 88, 98, 99), anti-IGF-1R IgG1 with KiH mutations and DDD mutations (SEQ ID NO: 88, 100, 101), and anti-IGF-1R with HHY-AAA mutationsIgG1 (SEQ ID NO: 88, SEQ ID NO: 112) can be prepared and purified in the same manner.

[0483] Example 2 Analysis & developability Small-scale DLS-based viscosity measurement Viscosity measurements were essentially performed as described in (He, F. et al., Analytical Biochemistry 399 (2009) 141-143). Briefly, the sample was concentrated to various protein concentrations in 200 mM arginine succinate, pH 5.5, and then polystyrene latex beads (300 nm in diameter) and polysorbate 20 (0.02% v / v) were added. The sample was transferred to an optical 384-well plate by centrifugation through a 0.4 μm filter 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 could 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).

[0484] To enable comparison of various samples at the same concentration, viscosity-concentration data was fitted using Mooney's equation (Equation 1) (Mooney, Colloid Sci, 1951; Monkos, Biochem.Biophys.Acta 1997), and data was interpolated as appropriate. [Math 1] (S: Hydrodynamic interaction parameter of the protein; K: Self-compression factor; Φ: Volume fraction of dissolved protein)

[0485] The results are shown in Figure 2: VEGF / ANG2-0016, which has an IHH-AAA mutation in the Fc region, exhibits lower viscosity at all measured temperatures compared to VEGF / ANG2-0015, which does not have an IHH-AAA mutation in the Fc region.

[0486] DLS agglomeration start temperature The sample was prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine hydrochloride, 140 mM NaCl, pH 6.0, and transferred to an optical 384-well plate by centrifugation through a 0.4 μm filter plate, then covered with paraffin oil. The hydrodynamic radius was repeatedly measured by dynamic light scattering while the sample was heated from 25 °C to 80 °C at a rate of 0.05 °C / min. The aggregation onset temperature was defined as the temperature at which the hydrodynamic radius began to increase. The results are shown in Figure 3. Figure 3 shows the aggregation of VEGF / ANG2-0015 without the IHH-AAA mutation compared to VEGF / ANG2-0016 with the IHH-AAA mutation in the Fc portion. 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.

[0487] DLS time elapsed The sample was prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine hydrochloride, 140 mM NaCl, pH 6.0, transferred to an optical 384-well plate by centrifugation through a 0.4 μm filter plate, and covered with paraffin oil. The hydrodynamic radius was repeatedly measured by dynamic light scattering while the sample was maintained at a constant temperature of 50°C for up to 145 hours. In this experiment, the native, unfolded protein aggregation tendency at high temperatures can lead to an increase in average particle size over time. This DLS-based method is very sensitive to aggregates because they contribute excessively to the scattered light intensity. At 50°C (a temperature close to the aggregation onset temperature, see above), even after 145 hours, the increase in average particle size observed for both VEGF / ANG2-0015 and VEGF / ANG2-0016 was only slightly less than 0.5 nm.

[0488] Store at 40°C for 7 days at 100 mg / mL. The sample was concentrated to a final concentration of 100 mg / mL in 200 mM arginine succinate, pH 5.5, filtered sterile, and stored at 40°C for 7 days in a static state. The content of high molecular weight and low molecular weight species (HMW and LMW, respectively) was determined by size exclusion chromatography before and after storage. The difference in HMW and LMW content between the stored sample and the sample measured immediately after preparation was reported as "increased HMW" and "increased LMW," respectively. The results are shown in the table and Figure 4 below, which indicate that VEGF / ANG2-0015 (without IHH-AAA mutation) showed a higher decrease in the main peak and a higher increase in HMW compared to VEGF / ANG2-0016 (with IHH-AAA mutation). Surprisingly, VEGF / ANG2-0016 (with IHH-AAA mutation) showed a lower tendency to aggregate compared to VEGF / ANG2-0015 (without IHH-AAA mutation).

[0489] [Table 16]

[0490] Functional analysis of anti-VEGF / ANG2 bispecific antibodies was evaluated at 25°C by surface plasmon resonance (SPR) using a BIAcore® T100 or T200 instrument (GE Healthcare). The BIAcore® system is well-established for testing molecular interactions. SPR technique is based on measuring the refractive index near the surface of a gold-coated biosensor chip. The change in refractive index indicates the mass change on the surface caused by the interaction between the immobilized ligand and the analyte injected into the solution. When a molecule binds to the ligand immobilized on the surface, the mass increases; conversely, when the analyte dissociates from the immobilized ligand (reflecting the dissociation of the complex), the mass decreases. SPR enables continuous real-time monitoring of ligand / analyte binding and, therefore, the determination of the association rate constant (ka), dissociation rate constant (kd), and equilibrium constant (KD).

[0491] Example 3 Binding with VEGF, ANG2, Fc gamma R, and FcRn Dynamic affinity of VEGF isoforms, including evaluation of species cross-reactivity. A capture system with approximately 12,000 resonance units (RUs) (10 μg / ml goat anti-human F(ab)'2; order code: 28958325; GE Healthcare Bio-Sciences AB, Sweden) was coupled to a CM5 tip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. 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 preparation was carried out with two runs of running buffer. The bispecific antibody was captured by injecting a 50 nM solution at a flow rate of 5 μL / min over 30 seconds. The association of human hVEGF121, mouse mVEGF120, or rat rVEGF164 at various concentrations (starting at 300 nM and diluted 1:3) in solution was measured by injecting at a flow rate of 30 μL / min for 300 seconds. The dissociation phase was monitored for up to 1200 seconds and induced by switching from the sample solution to the running buffer. The surface was regenerated by washing with a glycine pH 2.1 solution at a flow rate of 30 μL / min for 60 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from the goat anti-human F(ab')2 surface. Blank injections were also subtracted (= double reference). The Langmuir 1:1 model was used to calculate apparent KD and other dynamical parameters. The results are shown below.

[0492] ANG2 solution affinity, including evaluation of species cross-reactivity. Solution affinity measures the affinity of an interaction by determining the concentration of the free interaction partner in the equilibrium mixture. The solution affinity assay involves mixing anti-VEGF / ANG2 antibody (maintained at a constant concentration) with ligand (=ANG2) at various concentrations. The antibody with the maximum possible resonance units (e.g., 17,000 resonance units (RU)) was immobilized on the surface of 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 to 12°C, and preparation was carried out with two runs of running buffer. To create a calibration curve, ANG2 was injected into a BIAcore flow cell containing immobilized anti-VEGF / ANG2 antibody, increasing in concentration. The amount of bound ANG2 was determined by resonance units (RU) and plotted against concentration. Each ligand solution (11 concentrations of anti-VEGF / ANG2 antibody, ranging from 0 to 200 nM) was incubated with 10 nM ANG2 and allowed to reach equilibrium at room temperature. The free ANG2 concentrations generated before and after measuring the response of solutions containing known amounts of ANG2 were determined from the calibration curve. Four-parameter fitting was set using XLfit4 (IDBS Software) with Model 201, where the free ANG2 concentration was the y-axis and the concentration of the antibody used for inhibition was the x-axis. Affinity was calculated by determining the inflection point of this curve. The surface was regenerated by a single wash with 0.85% H3PO4 solution at a flow rate of 30 μL / min for 30 seconds. The difference in bulk refractive index was corrected by subtracting the response obtained from the blank coupling surface. The results are shown below.

[0493] FcRn's steady-state affinity For FcRn measurement, bispecific antibodies were compared using steady-state affinity. Human FcRn was diluted in coupling buffer (10 μg / ml, sodium acetate, pH 5.0) and immobilized on a C1-Chip (GE Healthcare BR-1005-35) using the BIAcore wizard target immobilization procedure to achieve a final response of 200 RU. The flow cell was set to 25°C and the sample block to 12°C, and preparation was carried out with two runs of 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. The flow rate was set to 30 μL / min, and various samples were continuously injected onto the chip surface, with an association time of 180 seconds. The surface was regenerated with PBS-T pH8 injected at a flow rate of 30 μL / min for 60 seconds. Differences in bulk refractive index were corrected by subtracting the response obtained from the blank surface. Buffer injection was also subtracted (= double reference). To calculate steady-state affinity, a method from BIA-Evaluation software was used. Briefly, the RU value was plotted against the analyzed concentration to generate a dose-response curve. Based on two-parametric fitting, the upper asymptote was calculated, allowing for the determination of the maximum half-volume RU value and affinity. The results are shown in Figure 5 and the table below. Similarly, affinity with cynomolgus monkey, mouse, and rabbit FcRn can be determined.

[0494] Fc Gamma RIIIa Measurement A direct binding assay was used for Fc gamma RIIIa measurement. A capture system with approximately 3000 resonance units (RU) (1 μg / ml Penta-His;Quiagen) was coupled to a CM5 tip (GE Healthcare BR-1005-30) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffers were HBS-P+ pH 7.4. The flow cell was set to 25°C and the sample block to 12°C, and preparation was carried out with two runs of running buffer. The Fc gamma RIIIa-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 a 100 nM bispecific antibody or monospecific control antibody (anti-digoxigenin antibody against 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. Since Fc gamma-RIIIa binding differs from the Langmuir 1:1 model, only binding / non-binding was determined using this assay. Fc gamma-RIa and Fc gamma-RIIa binding can be determined in a similar manner. The results are shown in Figure 6, where the introduction of mutant P329G LALA resulted in no further detection of binding to Fc gamma-RIIIa.

[0495] Independent evaluation of VEGF and ANG2 binding with anti-VEGF / ANG2 antibodies. A capture system with approximately 3500 resonance units (RU) (10 μg / mL goat anti-human IgG; GE Healthcare Bio-Sciences AB, Sweden) was coupled to a CM4 tip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. 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 temperature was set to 25°C and the sample block temperature to 12°C. Before capture, the flow cell was prepared with two passes of running buffer.

[0496] 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 active binding capacity for each ligand, either sequentially or simultaneously (at a flow rate of 30 μL / min): 1. Infusion of 200 nM human VEGF over 180 seconds (to identify single antigen binding). 2. Infusion of 100 nM human ANG2 over 180 seconds (to identify single antigen binding). 3. Injection of human VEGF at a concentration of 200 nM over 180 seconds, followed by an additional injection of human ANG2 at a concentration of 100 nM over 180 seconds (to identify ANG2 binding in the presence of VEGF). 4. Injection of 100 nM human ANG2 over 180 seconds, followed by additional injection of 200 nM human VEGF (to identify VEGF binding in the presence of ANG2). 5. Simultaneous injection of 200 nM human VEGF and 100 nM human ANG2 over 180 seconds (to simultaneously identify the binding of VEGF and ANG2).

[0497] The surface was regenerated by washing with a 3M MgCl2 solution at a flow rate of 30 μL / min for 60 seconds. The difference in bulk refractive index was corrected by subtracting the response obtained from the goat anti-human IgG surface.

[0498] If the final signals obtained as a result of approaches 3, 4, and 5 are equal to or similar to the sum of the individual final signals from approaches 1 and 2, then the bispecific antibody can bind to both antigens independently of each other. The results are shown in the table below, where it is shown that both antibodies VEGF / ANG2-0016 and VEGF / ANG2-0012 can bind to VEGF and ANG2 independently of each other.

[0499] Evaluation of simultaneous binding of VEGF and ANG2 with anti-VEGF / ANG2 antibodies. First, VEGF (20 μg / ml) with approximately 1600 resonance units (RUs) was coupled onto a CM4 tip (GE Healthcare BR-1005-34) at pH 5.0 using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was PBS-T (10 mM phosphate-buffered saline with 0.05% Tween 20) at pH 7.4. The flow cell was set to 25°C and the sample block to 12°C, and preparation was carried out with two runs of running buffer. Second, a 50 nM solution of 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 binding response of hANG2 was dependent on the amount of bispecific antibody bound to VEGF and showed co-binding. The surface was regenerated by washing with 0.85% H3PO4 solution at a flow rate of 30 μL / min for 60 seconds. Co-binding is indicated by a signal of additional specific binding of hANG2 to the anti-VEGF / ANG2 antibody that has already bound to VEGF. For both the bispecific antibodies VEGF / ANG2-0015 and VEGF / ANG2-0016, the co-binding of VEGF and ANG2 to the anti-VEGF / ANG2 antibody was detected (data not shown).

[0500] [Table 17]

[0501] [Table 18]

[0502] [Table 19]

[0503] [Table 20]

[0504] [Table 21]

[0505] Example 4 mass spectrometry This section focuses on accurate assembly and describes the characterization of the anti-VEGF / ANG2 antibody. The expected primary structure was confirmed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated, intact, or IdeS-digested (S. pyogenes IgG-degrading enzyme) anti-VEGF / ANG2 antibodies. IdeS digestion was performed using 100 μg of purified antibody, incubated with 2 μg of IdeS protease (Fabricator) (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 using N-glycosidase F, neuraminidase, and O-glycosidase (100 mmol / L NaH2PO4 / Na2HPO4, pH 7.1) (Roche) at 37°C for up to 16 hours, and then desalted by HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0506] The masses obtained for molecules that were deglycosylated by IdeS digestion (see table below) or deglycosylated intact (see table below) were obtained for two different light chain LCs. ANG2 and LC Lucentis , as well as two different heavy chain HC ANG2 and HC LucentisThis corresponds to the predicted mass estimated from the amino acid sequence of the anti-VEGF / ANG2 antibody consisting of [the specified components].

[0507] [Table 22]

[0508] [Table 23]

[0509] Example 5 Fc-Rn chromatography Coupling with streptavidin cephalose: Biotinylated and dialyzed receptors were mixed with 1 g of streptavidin cephalose (GE Healthcare) and incubated with shaking for 2 hours. Receptor-inducible cephalose was then packed into a 1 mL XK column (GE Healthcare).

[0510] Chromatography using FcRn affinity columns: conditions: Column dimensions: 50mm x 5mm Bed height: 5cm Load: 50 μg of sample Equilibrium buffer: 20 mM MES containing 150 mM NaCl, adjusted to pH 5.5. Elution buffer: 20 mM Tris / HCl containing 150 mM NaCl, adjusted to pH 8.8. Elution: From 7.5 CV of equilibrium buffer (30 CV), to 100% elution buffer (10 CV of elution buffer).

[0511] Human FcRn affinity column chromatography The retention times of anti-VEGF / ANG2 antibodies on affinity columns containing human FcRn are shown in the table below. The data were obtained under the conditions described above.

[0512] [Table 24]

[0513] Example 6 Pharmacokinetic (PK) properties of antibodies with IHH-AAA mutations PK data for human FcRn using transgenic Fc-Rn mice Survival phase: The study included female C57BL / 6J mice (background), which are deficient in mouse FcRn but are hemizygous transgenic for human FcRn (huFcRn, strain 276- / tg).

[0514] Part 1: All mice received a single intravitreous injection into the right eye using 2 μL / animal of an appropriate solution (i.e., 21 μg of compound / animal (VEGF / ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg of compound / animal (VEGF / ANG2-0016 (with IHH-AAA mutation))).

[0515] The mice were divided into two groups of six. Blood samples were collected from group 1 at 2, 24, and 96 hours after administration, and from group 2 at 7, 48, and 168 hours.

[0516] Intravitreal injection into the right eye of mice was performed using a NanoFil Microsyringe system for nanoliter injection (World Precision Instruments, Inc., Berlin, Germany). Mice were anesthetized with 2.5% isoflurane, and the mouse eyeballs were visualized using a Leica MZFL3 microscope (40x magnification, with a ring light from a Leica KL2500 LCD). Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

[0517] To measure the levels of compounds in the serum, blood was collected from each animal via the retrobulbar venous plexus of the opposite eye.

[0518] At least 50 μL of serum sample was obtained from blood after being left at room temperature for 1 hour by centrifugation (9300xg) at 4°C for 3 minutes. The serum sample was frozen immediately after centrifugation and stored at -80°C until analysis. The treated eyeballs were isolated 96 hours after treatment for animals in Group 1 and 168 hours after treatment for animals in Group 2. The samples were stored at -80°C until analysis.

[0519] Part 2: All mice were intravenously injected once via the tail vein using 200 μL / animal of an appropriate solution (i.e., 21 μg of compound / animal (VEGF / ANG2-0015 (without IHH-AAA mutation)) or 23.6 μg of compound / animal (VEGF / ANG2-0016 (with IHH-AAA mutation)).

[0520] The mice were divided into two groups of five. Blood samples were collected from group 1 at 1, 24, and 96 hours after administration, and from group 2 at 7, 48, and 168 hours. Blood was collected from each animal via the retrobulbar venous plexus to measure the levels of compounds in the serum.

[0521] After leaving blood at room temperature for 1 hour, at least 50 μL of serum sample was obtained by centrifugation at 4°C for 3 minutes (9300xg). The serum sample was frozen immediately after centrifugation and stored frozen at -80°C until analysis.

[0522] Preparation of whole eyeball lysate (mouse) Eye lysates were obtained by physicochemical degradation of the entire eyeball from experimental animals. For mechanical disruption, each eyeball was transferred to a 1.5 mL cone-bottomed microvial. After freezing and thawing, the eyeballs were washed once with 1 mL of cell washing buffer (Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011). In the next step, 500 μL of freshly prepared cell lysis buffer was added, and the eyeballs were disrupted using a 1.5 mL tissue disruption pestle (Kimble Chase, 1.5 mL pestle, Art. No. 749521-1500). The mixture was then frozen and thawed five times and pulverized again. To separate the lysate from the remaining tissue, the samples were centrifuged at 4500 × g for 4 minutes. After centrifugation, the supernatant was collected and stored at -20°C until further analysis in quantitative ELISA.

[0523] analysis The concentrations of anti-VEGF / ANG2 antibodies in mouse serum and ophthalmic lysates were determined using solid-phase enzyme immunosorbent assay (ELISA).

[0524] To quantify anti-VEGF / ANG2 antibodies in mouse serum samples and ophthalmic lysates, a standard solid-phase serial sandwich immunoassay was performed using biotinylated and digoxigenin-modified monoclonal antibodies as capture and detection antibodies. To verify the completeness of the analyte's bispecificity, the biotinylated capture antibody recognizes the VEGF binding site, while the digoxigenin-modified detection antibody can bind to the ANG2 binding site of the analyte. The bound immunocomplexes of the capture antibody, analyte, and detection antibody on the solid phase of a streptavidin-coated microtiter plate (SA-MTP) were then detected using horseradish peroxidase coupled with an anti-digoxigenin antibody. After washing the unbound material from the SA-MTP and adding the ABTS substrate, the resulting signal was proportional to the amount of analyte bound to the solid phase of the SA-MTP. Next, the measurement signal of the sample was converted to concentration by referring to a calibrator analyzed in parallel to perform quantification.

[0525] In the first step, SA-MTP is mixed with a 100 μL / well biotinylated capture antibody solution (mAb) at a concentration of 1 μg / mL. <Id <vegf>The samples were coated with M-2.45.51-IgG-Bi (DDS, anti-idiotype antibody) on an MTP shaker at 500 rpm for 1 hour. During this time, the calibrator, QC-sample, and sample were prepared. The calibrator and QC-sample were diluted in 2% serum matrix; the sample was diluted until the signal was within the linear range of the calibrator.

[0526] After coating SA-MTP with capture antibody, the plate was washed three times with wash buffer at 300 μL / well. Then, 100 μL / well of calibrator, QC sample, and sample were pipetteed onto the SA-MTP and incubated again at 500 rpm for 1 hour. Thus, the analyte bound to the solid phase of SA-MTP via the capture antibody through its anti-VEGF binding site. After removal of unbound analyte by incubation and washing, 100 μL / well of the first detection antibody (mAb) was added. <Id- <ang2>>M-2.6.81-IgG-Dig(XOSu, anti-idiotype antibody) (concentration 250 ng / mL) was added to SA-MTP. The plate was incubated again 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)) (concentration 50 mU / mL) was added to the wells of SA-MTP, and the plate was incubated again at 500 rpm for 1 hour. After a final washing step to remove excess detection antibody, 100 μL / well of substrate (ABTS) was added. The antibody-enzyme conjugate catalyzes the color reaction of the ABTS® substrate. The signal was then measured at a wavelength of 405 nm (reference wavelength: 490 nm ([405 / 490] nm)) using an ELISA reader.

[0527] Pharmacokinetic evaluation Pharmacokinetic parameters were calculated using non-compartmental analysis with the pharmacokinetic evaluation program WinNonlin® (Pharsight), version 5.2.1.

[0528] result: A) Serum concentration The results regarding serum concentrations are shown in the table and Figures 7B-7C below.

[0529] [Table 25]

[0530] [Table 26]

[0531] [Table 27]

[0532] [Table 28]

[0533] result: B) Concentration in the ocular elution of the left and right eyes The results regarding the concentration in the ocular lysate are shown in the table and Figures 7D to 7E below.

[0534] [Table 29]

[0535] [Table 30]

[0536] [Table 31]

[0537] [Table 32]

[0538] Summary of results: After intravitreous administration, the bispecific anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutation) reported herein exhibits similar concentrations in ophthalmic lysates (at 96 and 168 hours) compared to VEGF / ANG2-0015, a bispecific anti-VEGF / ANG2 antibody without the IHH-AAA mutation.

[0539] Furthermore, after intravitreous administration, the bispecific anti-VEGF / ANG2 antibody VEGF / ANG2-0016 (with IHH-AAA mutation) reported herein exhibits faster clearance and a shorter half-life in serum compared to VEGF / ANG2-0015, a bispecific anti-VEGF / ANG2 antibody without the IHH-AAA mutation.

[0540] Example 7 Mouse corneal micropocket angiogenesis assay To test the anti-angiogenic effects of bispecific anti-VEGF / ANG2 antibodies—VEGF-binding VH and VL (SEQ ID NOs. 20 and 21, respectively) and ANG2-binding VH and VL (SEQ ID NOs. 28 and 29)—on in vivo VEGF-induced angiogenesis, a mouse corneal angiogenesis assay was performed. In this assay, VEGF-soaked Nylaflo discs were embedded in pockets of avascular corneas at a constant distance to the corneal marginal vessels. Vessels immediately grew inward towards the developing VEGF gradient. Female Balb / c mice aged 8–10 weeks were purchased from Charles River (Sulzfeld, Germany). The protocol was modified according to the method described in Rogers, MS, et al., Nat. Protoc. 2 (2007) 2545–2550. In short, a micropocket approximately 500 μm wide is prepared under a microscope in an anesthetized mouse, using a surgical blade and sharp forceps, approximately 1 mm from the corneal margin towards the upper cornea. A 0.6 mm diameter disc (Nylaflo®, Pall Corporation, Michigan) is embedded, and the surface of the embedded area is smoothed. The disc is incubated in the corresponding growth factor or excipient for at least 30 minutes. Photographs of the eye are taken at 3, 5, and 7 days (or alternatively, only at 3, 5, or 7 days) to measure the vascular response. The assay is quantified by calculating the percentage of the area of ​​new blood vessels relative to the total corneal area.

[0541] The disc is infused with 300 ng of VEGF or PBS as a control and implanted for 7 days. Vascular elongation from the corneal margin to the disc is monitored over time on days 3, 5, and / or 7. One day before disc implantation, the antibody is administered intravenously at a dose of 10 mg / kg to test its anti-angiogenic effect against VEGF-induced angiogenesis in vivo (as this is an intravenous application, serum-stable VEGF / ANG2-0015 (without IHH-AAA mutation) (which differs from VEGF / ANG2-0016 only in the IHH-AAA mutation and possesses the same VEGF and ANG2-binding VH and VL that mediate efficacy) is used as a substitute). Animals in the control group receive excipients. The application volume is 10 mL / kg.

[0542] Example 8 Pharmacokinetic (PK) properties of antibodies with HHY-AAA mutations PK data for human FcRn using transgenic Fc-Rn mice Survival phase: The study included female C57BL / 6J mice (background), which are deficient in mouse FcRn but are hemizygous transgenic for human FcRn (huFcRn, strain 276- / tg).

[0543] Part 1: All mice were given a single intravitreous injection into the right eye using appropriate solutions of IGF-1R 0033, IGF-1R 0035, and IGF-1R 0045 (i.e., 22.2 μg compound / animal IGF-1R 0033, 24.4 μg compound / animal IGF-1R 0035, 32.0 μg compound / animal IGF-1R, and 32.0 μg compound / animal IGF-1R 0045).

[0544] Thirteen mice were divided into two groups of six and seven mice each. Blood samples were collected from group 1 at 2, 24, and 96 hours after administration, and from group 2 at 7, 48, and 168 hours.

[0545] Intravitreal injection into the right eye of mice was performed using a NanoFil Microsyringe system for nanoliter injection (World Precision Instruments, Inc., Berlin, Germany). Mice were anesthetized with 2.5% isoflurane, and the mouse eyeballs were visualized using a Leica MZFL3 microscope (40x magnification, with a ring light from a Leica KL2500 LCD). Subsequently, 2 μL of the compound was injected using a 35-gauge needle.

[0546] To measure the levels of compounds in the serum, blood was collected from each animal via the retrobulbar venous plexus of the opposite eye.

[0547] At least 50 μL of serum sample was obtained from blood after being left at room temperature for 1 hour by centrifugation (9300xg) at 4°C for 3 minutes. The serum sample was frozen immediately after centrifugation and stored at -80°C until analysis. The treated eyeballs were isolated 96 hours after treatment for animals in Group 1 and 168 hours after treatment for animals in Group 2. The samples were stored at -80°C until analysis.

[0548] Part 2: All mice were administered intravenously once via the tail vein using appropriate solutions of IGF-1R 0033, IGF-1R 0035, and IGF-1R 0045 (i.e., 22.2 μg compound / animal IGF-1R 0033, 24.4 μg compound / animal IGF-1R 0035, 32.0 μg compound / animal IGF-1R, and 32.0 μg compound / animal IGF-1R 0045).

[0549] Twelve mice were divided into two groups of six. Blood samples were collected from group 1 at 1, 24, and 96 hours after administration, and from group 2 at 7, 48, and 168 hours. Blood was collected from each animal via the retrobulbar venous plexus to measure the levels of compounds in the serum.

[0550] After leaving blood at room temperature for 1 hour, at least 50 μL of serum sample was obtained by centrifugation at 4°C for 3 minutes (9300xg). The serum sample was frozen immediately after centrifugation and stored frozen at -80°C until analysis.

[0551] Preparation of cell lysis buffer Carefully mix 100 μL of factor 1, 50 μL of factor 2, and 24.73 mL of cell lysis buffer (all from Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011), and add 125 μL of PMSF solution (phenylmethylsulfonyl fluoride 174.4 mg, diluted in 2.0 mL DMSO).

[0552] Preparation of whole eyeball lysate (mouse) Eye lysates were obtained by physicochemical degradation of the entire eyeball from experimental animals. For mechanical disruption, each eyeball was transferred to a 1.5 mL cone-bottomed microvial. After thawing, the eyeballs were washed once with 1 mL of cell washing buffer (Bio-Rad, Bio-Plex Cell Lysis Kit, Cat. No. 171-304011). In the next step, 500 μL of freshly prepared cell lysis buffer was added, and the eyeballs were disrupted using a 1.5 mL tissue disruption pestle (VWR Int., Art. No. 431-0098). The mixture was then frozen and thawed five times and pulverized again. To separate the lysate from the remaining tissue, the samples were centrifuged at 4500 × g for 4 minutes. After centrifugation, the supernatant was collected and stored at -20°C until further analysis in quantitative ELISA.

[0553] Analysis (serum) To quantify antibodies in mouse serum samples, a standard solid-phase serial sandwich immunoassay was performed using biotinylated and digoxigenin-modified monoclonal antibodies as capture and detection antibodies. Serum accounts for approximately 50% of the total blood sample volume.

[0554] More specifically, the antibody concentration in mouse serum samples was determined using a human-IgG(Fab)-specific solid-phase enzyme immunoassay. 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, at room temperature for 1 hour with agitation. After three washes with phosphate-buffered saline-polysorbate 20 (Tween20), serum samples of various dilutions were added, followed by the second incubation at room temperature for 1 hour. After repeating the three washes, the conjugated antibodies were detected by incubation with digoxigenin-conjugated anti-human Fab(CH1) monoclonal antibody M-1.19.31-IgG, and then with horseradish peroxidase (HRP)-conjugated anti-digoxigenin antibody. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Roche Diagnostics GmbH, Mannheim, Germany) was used as the substrate for HRP to form a color reaction product. The absorbance of the obtained reaction product was read at 405 nm (ABTS; reference wavelength: 490 nm).

[0555] All samples, including positive and negative control samples, were repeatedly analyzed and calibrated against the provided antibody standards.

[0556] Analysis (ocular lysate) The concentrations of analytes in mouse eye lysate samples were determined using qualified electrochemiluminescence immunoassay (ECLIA) based on the ELECSYS® instrument platform (Roche Diagnostics GmbH, Mannheim, Germany) under non-GLP conditions.

[0557] The undiluted supernatant (ocular lysate) was incubated with the 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(bispyridyl) 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 9 minutes to allow the previously formed immune complex to bind via biotin-streptavidin interaction. The microparticles were magnetically trapped on an electrode, and a chemiluminescent signal was generated using the co-reactant tripropylamine (TPA). The resulting signal was measured using a photomultiplier tube detector.

[0558] [Table 33]

[0559] [Table 34]

[0560] [Table 35] ...

Claims

1. A polypeptide comprising: The antibody comprises a first polypeptide and a second polypeptide, each of which comprises, from N-terminal to C-terminal, at least a portion of an immunoglobulin hinge region, an immunoglobulin CH2 domain, and an immunoglobulin CH3 domain, each of which comprises one or more cysteine ​​residues. 、 i i) the first and second polypeptides comprise the mutations L251D, L314D and L432D; nothing, Polypeptide.

2. The polypeptide of claim 1, which does not specifically bind to human FcRn but specifically binds to staphylococcal protein A.

3. 3. The polypeptide of any one of claims 1 to 2, which is a homodimeric polypeptide.

4. 3. The polypeptide of any one of claims 1 to 2, which is a heterodimeric polypeptide.

5. 5. The polypeptide of any one of claims 1 to 4, wherein 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 comprises the mutations S354C, T366S, L368A and Y407V and the second polypeptide comprises the mutations Y349C and T366W.

6. 6. The polypeptide of claim 1, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of the human IgG1 subclass.

7. 7. The polypeptide of claim 1, wherein the first polypeptide and the second polypeptide further comprise the mutations L234A and L235A.

8. 6. The polypeptide of claim 1, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of the human IgG2 subclass.

9. 6. The polypeptide of claim 1, wherein the immunoglobulin hinge region, the immunoglobulin CH2 domain and the immunoglobulin CH3 domain are of the human IgG4 subclass.

10. 10. The polypeptide of any one of claims 1 to 5 and 9, wherein the first polypeptide and the second polypeptide further comprise the mutations S228P and L235E.

11. 11. The polypeptide of any one of claims 1 to 10, wherein the first polypeptide and the second polypeptide further comprise the mutation P329G.

12. 11. The polypeptide of any one of claims 1 to 10, which is a bispecific antibody.

13. 13. The polypeptide of any one of claims 1 to 12 for intravitreal application.

14. 13. The polypeptide of any one of claims 1 to 12 for treating ocular vascular diseases.

15. 13. A pharmaceutical formulation comprising a polypeptide according to any one of claims 1 to 12 and optionally a pharmaceutically acceptable carrier.

16. 13. A polypeptide according to any one of claims 1 to 12 for use in the treatment of an eye disease.

17. 13. A polypeptide according to any one of claims 1 to 12 for use in transporting a soluble receptor ligand from the eye into the circulation across the blood-ocular barrier.

18. 13. A polypeptide according to any one of claims 1 to 12 for use in the removal of one or more soluble receptor ligands from the eye.

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