ES2350202T3 - ALTERATION OF UNION AFFINITIES BY FCRN OR SERUM SEMIVIDES OF ANTIBODIES THROUGH MUTAGENESIS. - Google Patents

Abstract

An antibody or IgG class fusion protein, which comprises a constant region of the heavy chain or FcRn portion thereof of a human IgG molecule, which comprises glutamic acid or glutamine in amino acid residue 250 and leucine or phenylalanine in the amino acid residue 428 and in which the amino acid residues are numbered by the EU numbering system.

Description

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and protein engineering. In particular, it refers to modified IgG class antibodies that have altered FcRn binding affinities or serum half-lives altered as a result of one or more amino acid modifications in the Fc region thereof.

BACKGROUND OF THE INVENTION

Antibodies are proteins that exhibit binding specificity for a particular antigen. Native (ie, natural or wild) antibodies are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). As shown in Figure 1, each light chain is linked to a heavy chain by a covalent disulfide bond, although the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy chain has, at one end, a variable domain (VH) followed by a series of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain.

Certain portions of the variable domains differ widely in sequence between the antibodies and are responsible for the specificity of binding of each specific antibody to its specific antigen. Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit several effector functions. Depending on the amino acid sequence of the heavy chain constant region, antibodies or immunoglobulins can be assigned to different classes. There are five main classes (isotypes) of immunoglobulins in humans: IgA, IgD, IgE, IgG, and IgM, and several of these can also be divided into subclasses (subtypes), such as IgG1, IgG2, IgG3 and IgG4, as well as IgA1 and IgA2.

A schematic representation of the structure of native IgG is shown in Figure 1, in which the various portions of the native antibody molecule are indicated. The constant region of the heavy chain includes CH1, the hinge region, CH2, and CH3. Papain digestion of the antibodies produces two fragments, Fab and Fc. The Fc fragment consists of CH2, CH3, and part of the hinge region. The crystal structure of the Fc fragment of human IgG1 has been determined (Deisenhofer, Biochemistry 20: 2361-2370 (1981)). In human IgG molecules, the Fc fragment is generated by cleavage with papain from the N-terminal hinge region to Cys 226. Therefore, the Fc region of the human IgG heavy chain is usually defined as the extent from the residue from amino acid at position 226 to end C (numbered according to the Kabat EU index, and co., "Sequences of Proteins of Immunological Interest", 5th ed., National Institutes of Health, Bethesda, MD (1991) ; hereinafter the EU numbering scheme is used).

The Fc region is essential for the effector functions of the antibodies. Effector functions include initiating complement-dependent cytotoxicity (CDC), initiating phagocytosis and antibody-dependent cell-mediated cytotoxicity (AD-CC) and transferring antibodies across cell barriers through transcytosis. In addition, the Fc region is crucial for maintaining the serum half-life of an IgG class antibody (Ward and Ghetie, Ther. Immunol. 2: 77-94 (1995)).

Studies have found that the serum half-life of an IgG antibody is mediated by the binding of Fc to the neonatal Fc receptor (FcRn). FcRn is a heterodimer consisting of a transmembrane α chain and a soluble β chain (β2-microglobulin). The FcRn shares a 22-29% sequence identity with MHC class I molecules and has a non-functional version of the peptide-MHC binding slot (Simister and Mostov, Nature 337: 184-187 (1989)). The α1 and α2 domains of FcRn interact with the CH2 and CH3 domains of the Fc region (Raghavan et al., Immunity 1: 303-315 (1994)).

A model for how FcRn could regulate the serum half-life of an antibody has been proposed. As shown in Figure 2, IgGs are captured by endothelial cells through nonspecific pinocytosis and then enter the acid endosomes. FcRn binds to IgG at an acidic pH (<6.5) in the endosomes and releases IgG at a basic pH (> 7.4) in the bloodstream. Accordingly, FcRn saves IgG from a lysosomal degradation pathway. When serum IgG levels decrease, more FcRn molecules are available for IgG binding so that an increase in the amount of IgG is saved. On the contrary, if serum IgG levels increase, the FcRn becomes saturated and, thus, the proportion of pinecytosed IgG that degrades is increased (Ghetie and Ward, Annu. Rev. Immunol. 18: 739-766 ( 2000)).

Consistent with the previous model, the results of numerous studies support a correlation between the affinity of the FcRn binding and the serum half-life of an antibody (Ghetie and Ward, ibid.). The fact that this correlation has extended to engineered antibodies with greater affinity for FcRn than its wild parental molecules is significant.

Ghetie et al. performed a random mutagenesis at position 252, position 254 and position 256 on a fragment of mouse IgG1 Fc hinge. One mutant showed an affinity three and a half times higher for mouse FcRn and a half-life of approximately 23% or 65% higher in two mouse strains, respectively, compared to those of the wild type (Ghetie et al., Nat. Biotechnol 15: 637-640 (1997)).

Shields et al. they used Alanine scanning mutagenesis to alter residues in the Fc region of a human IgG1 antibody and then evaluated the binding to human FcRn. They found several mutants with a higher binding affinity for human FcRn than the wild type, but did not identify mutations at positions 250, 314 or 428 (Shields et al., J. BioI. Chem. 276: 6591-6604 (2001)) .

Martin et al. proposed mutagenesis in a series of positions in the Fc of human IgG to increase binding to FcRn, including, among many others, positions 250, 314 and 428. However, none of the mutants proposed by Martin et al. it was constructed or analyzed according to its binding to FcRn (Martin et al., Mol. Cell 7: 867-877 (2001)).

Dall'Acqua et al. have described the random mutagenesis and selective detection of phage expression libraries of the human IgG1 Fc-hinge fragment against the mouse FcRn. They reported random mutagenesis from positions 428-436, but did not identify that mutagenesis at position 428 had any effect on mouse FcRn binding affinity and indicated that the wild-type amino acid, methionine, in this position is favorable to a binding effective (Dall'Acqua et al., J. Immunol. 169: 5171-5180 (2002)).

Kim et al. performed mutagenesis in human IgG1 by amino acid substitutions at position 253, position 310 or position 435 of the Fc region. They found that the Fc-hinge mutant fragments have reduced half-lives in mice compared to the Fc-hinge fragment of wild IgG1 and concluded that lIe253, His310 and His435 play a fundamental role in the regulation of serum half-life of IgG (Kim and CO., Eur. J. Immunol. 29: 2819-2825 (1999)).

Hornick et al. showed that a substitution of a single amino acid at position 253 in the Fc region of a chimeric human IgG1 antibody accelerates the clearance in mice and improves the immuno-scintillation of solid tumors (Hornick et al., J. Nucl. Med. 41: 355 -362 (2000)).

U.S. Pat. No. 6,165,745 discloses a method of producing an antibody with a lower biological half-life by introducing a mutation in the segment of DNA encoding the antibody. The mutation includes an amino acid substitution at positions 253, 310, 311, 433 or 434 of the Fc-hinge domain.

U.S. patents Nos. 5,530,101; 5,585.89; 5,693,761; 5,693,762; Y

6,180,370 disclose the humanization of immunoglobulins.

U.S. Pat. No. 6,277,357 B1 discloses a composition comprising a mutant IgG molecule that has a higher serum half-life compared to wild IgG, in which the mutant IgG molecule comprises amino acid substitutions: threonine to leucine at position 252 , threonine to serine at position 254 or threonine to phenylalanine at position 256. A mutant IgG with an amino acid substitution at position 433, 435 or 436 is also disclosed.

U.S. Patent Application Publication US 20020098193 A1 and PCT Publication No. WO 97/34621 disclose mutant IgG molecules that have higher serum half-lives relative to IgG in which the mutant IgG molecule has at least one amino acid substitution in the Fc-hinge region. However, no experimental support is provided for mutations at positions 250, 314 or 428.

U.S. Pat. No. 6,528,624 discloses a variant of an antibody comprising an Fc region of IgG, wherein the variant comprises an amino acid substitution at one or more of the amino acid positions 270, 322, 326, 327, 329, 331, 333, and 334 of the Fc region of human IgG.

PCT Publication No. WO 98/05787 discloses the deletion or substitution of amino acids at positions 310-331 of the BR96 antibody in order to reduce its induced toxicity, but does not disclose amino acid modifications that result in impaired FcRn binding.

PCT Publication No. WO 00/42072 discloses a polypeptide comprising a variant Fc region with impaired FcRn binding affinity, in which the polypeptide comprises an amino acid modification at any one or more of amino acid positions 238, 252 , 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400 , 413, 415, 424, 433, 434, 435, 436, 439, and 447 of the Fc region, in which the numbering of residues in the Fc region is that of the EU index (Kabat et al., Op cit.)

PCT Publication No. WO 02/060919 A2 discloses a modified IgG comprising a constant domain of IgG comprising one or more amino acid modifications with respect to a constant domain of wild IgG, in which the modified IgG has a greater half-life compared to the half-life of an IgG that has the constant domain of wild IgG, and in which the one or more amino acid modifications are in one or more of positions 251, 253, 255, 285-290, 308-314, 385-389 and 428-435. However, examples of mutations at positions 314 or 428 with impaired binding to FcRn are not disclosed.

Martin, W.L. (Doctoral dissertation entitled, "Protein-Protein Recognition: The Neonatal Fc Receptor and Immunoglobulin G," California Institute of Technology (2001)) proposes theoretical mutations at various Fc positions of the rat gamma-2a constant region, including positions 250 to 428, which can increase the binding affinity for fcRn. Martin suggests the possibility of replacing, among others, isoleucine with valine at position 250 or replacing phenylalanine with leucine at position 428. Martin does not suggest any substitution for position 314. Martin does not demonstrate an increase in FcRn binding affinity for any of these proposed mutations.

The publications referred to above have not shown that serum half-life or FcRn binding affinity of an IgG class antibody can be altered by amino acid modifications at position 250, position 314

or position 428 of the Fc region. The present invention has used molecular modeling to select Fc residues near the FcRn contact site that could have an effect on binding, but does not necessarily have to be pH dependent binding. Amino acid modifications were made at positions 250,314 or 428 of the constant region of an IGG class immunoglobulin heavy chain. The serum half-lives or FcRn binding affinities of the antibodies comprising said modifications were altered and, therefore, were different from those of the unmodified antibodies.

SUMMARY OF THE INVENTION

The present invention is based on the identification by the inventors of several mutations in the constant domain of a human IgG molecule that alter (i.e. increase or decrease) the affinity of the IgG molecule for FcRn. The present invention provides modified antibodies that have altered FcRn binding affinity and / or serum half-life relative to that of the corresponding unmodified antibody. The in vivo half-life (i.e., persistence in serum or other tissues of a subject) of antibodies, and other bioactive molecules, is an important clinical parameter that determines the amount and frequency of antibody administration (or any other pharmaceutical molecule). Accordingly, said molecules, including antibodies, with greater (or lesser) half-life have significant pharmaceutical significance.

The present invention relates to a modified fusion antibody or protein that has a greater half-life in vivo by virtue of the presence of a constant modified human IgG domain or an FcRn binding portion thereof (preferably, the Fc or the domain Fc-hinge), in which the constant domain of the IgG, or a fragment thereof, is modified as defined in the appended claims.

In a specific embodiment, the present invention relates to modified IgG class antibodies, whose half-lives in vivo have been extended by changes in amino acid residues as defined in the appended claims.

In preferred embodiments, the constant domain (or a fragment thereof) has a higher affinity for FcRn at pH 6.0 than at pH 7.4. That is, the pH dependence of the FcRn binding affinity mimics the wild pH dependence. In alternative embodiments, the modified antibodies of the present invention may exhibit altered pH dependence profiles with respect to those of the unmodified antibody. Such altered pH dependence profiles may be useful in some therapeutic or diagnostic applications.

In some embodiments, the modifications of the antibody of the present invention will alter FcRn binding and / or serum half-life without altering other effector functions of the antibody, such as ADCC or CDC. In particularly preferred embodiments, the modified antibodies of the invention do not exhibit changes in binding to the Fc-gamma or C1q receptors. In alternative embodiments, the antibody modifications of the present invention may result in an increase (or decrease) in effector functions, as well as an increase in serum half-life. In particularly preferred embodiments, the modified antibodies of the invention may have greater (or lesser) ADCC activities, as well as an increase in serum half-life.

It should be noted that the modifications of the present invention may also alter (i.e. increase or decrease) the bioavailability (e.g., transport to mucosal surfaces or other target tissues) of the modified antibodies (or other molecules).

The present disclosure provides a modified IgG class antibody, in which at least one amino acid of the constant region of the heavy chain selected from the group consisting of amino acid residues 250, 314 and 428 is substituted by an amino acid residue different from the present in the unmodified antibody. Preferably, this substitution alters the binding affinity for FcRn and / or the serum half-life of said modified antibody with respect to the unmodified wild antibody. The present invention further provides a modified antibody that has a higher binding affinity for FcRn and an increase in serum half-life compared to the unmodified antibody, in which amino acid residue 250 of the heavy chain constant region is substituted with glutamic acid or glutamine; and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine or leucine.

The present invention further provides a modified antibody that has a higher binding affinity for FcRn and / or greater serum half-life compared to the unmodified antibody, in which (a) amino acid residue 250 of the constant region of the chain heavy is substituted with glutamic acid and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine; (b) amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine; or (c) amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with leucine.

The present disclosure further provides a modified antibody having a lower binding affinity for fcRn and / or lower serum half-life compared to the unmodified antibody, in which amino acid residue 314 of the heavy chain constant region is substituted. with another amino acid that is different from that present in an unmodified antibody.

The present disclosure further provides a modified antibody that has a lower binding affinity for FcRn and / or lower serum half-life compared to the unmodified antibody, in which amino acid residue 250 of the heavy chain constant region is substituted. with arginine, asparagine, aspartic acid, lysine, phenylalanine, proline, tryptophan or tyrosine, or amino acid residue 428 of the constant region of the heavy chain is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine , glycine, histidine, lysine, proline, serine, threonine, tyrosine or valine.

The present disclosure also provides an antibody having a constant region substantially identical to a constant region of natural IgG class antibody, in which at least one amino acid residue selected from the group consisting of residues 250,314 and 428 is different from that present in the natural IgG class antibody, so that it alters the binding affinity for FcRn and / or the serum half-life of said antibody with respect to the natural antibody. In preferred embodiments, the natural IgG class antibody comprises a constant region of the heavy chain of a molecule of human IgG1, IgG2, IgG2M3, IgG3 or IgG4. Also, in preferred embodiments, amino acid residue 250 of the heavy chain constant region of the antibody having a constant region substantially identical to the natural IgG class antibody is glutamic acid or glutamine; or amino acid residue 428 of the heavy chain constant region is phenylalanine or leucine. In other preferred embodiments, the antibody having a constant region substantially identical to the natural IgG class antibody has a glutamic acid residue at position 250 and the phenylalanine residue at position 428; or amino acid residue 250 is glutamine and amino acid residue 428 is phenylalanine; or amino acid residue 250 is glutamine and amino acid residue 428 is leucine.

In some aspects of the disclosure, the antibody having a constant region substantially identical to a constant region of natural IgG class antibody includes an amino acid residue at position 314 different from that present in the natural antibody, so that affinity is reduced of binding to FcRn and / or the half-life in serum is reduced with respect to the natural antibody. Embodiments include antibodies in which amino acid residue 314 is alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. In a preferred embodiment, amino acid residue 314 is arginine.

In other aspects of the disclosure, the antibody having a constant region substantially identical to a constant region of natural IgG class antibody includes an amino acid residue at position 250 selected from the group consisting of arginine, asparagine, aspartic acid, lysine phenylalanine, proline, tryptophan or tyrosine, so that the affinity of binding to FcRn is reduced and / or the half-life in serum with respect to the natural antibody is reduced. Similarly, the amino acid residue at position 428 may be substituted with an amino acid residue selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, proline, serine , threonine, tyrosine or valine, so that the affinity of binding to FcRn is reduced and / or the serum half-life is reduced with respect to the natural antibody.

The present invention further provides a method of modifying an IgG class antibody or fusion protein as defined in the appended claims.

The present invention further provides a method of producing a modified IgG class antibody with a binding affinity altered by FcRn and / or serum half-life alteration compared to an unmodified antibody, wherein said method comprises:

(a) preparing an expression vector (preferably a replicable expression vector), which comprises a suitable promoter operably linked to DNA encoding at least one constant region of a human immunoglobulin heavy chain or an FcRn binding portion of the same, in which amino acid residue 250 is substituted with glutamic acid

or glutamine and amino acid residue 428 is substituted with phenylalanine or leucine;

(b)

transforming host cells with said vector; Y

(C)

culturing said transformed host cells to produce said modified antibody.

Optionally, said method further comprises: preparing a second expression vector (preferably a replicable expression vector), which comprises a promoter operably linked to DNA encoding a complementary immunoglobulin light chain and further transforming said cell line with said second vector.

The present invention also includes pharmaceutical compositions and methods of prophylaxis and therapy using modified immunoglobulins (including immunoglobulins conjugated to toxins and radionuclides), proteins and other bioactive molecules of the invention that have altered half-lives. Diagnostic procedures are also included using immunoglobulins, proteins and other modified bioactive molecules of the invention that have half-lives altered. In preferred embodiments, the amino acid modifications of the present invention can be used to extend the serum half-life of a therapeutic or diagnostic antibody. For example, the present invention provides a modified IgG class therapeutic or diagnostic antibody with an in vivo elimination half-life of at least about 1.3 times longer than that of the corresponding unmodified antibody. The modified therapeutic or diagnostic antibody of the disclosure, wherein at least one amino acid residue selected from the group consisting of residues 250, 314 or 428 is different from that present in the unmodified antibody. In preferred embodiments, the modified therapeutic or diagnostic antibody has an in vivo elimination half-life of at least about 1.3, 1.5, 1.8, 1.9 or greater than 2.0 times longer than that of the corresponding unmodified antibody.

The present invention also provides a modified therapeutic or diagnostic IgG class antibody with an in vivo clearance of at least about 1.3 times less than that of the corresponding unmodified antibody. The modified therapeutic or diagnostic antibody of the disclosure, wherein at least one amino acid residue selected from the group consisting of residues 250, 314 and 428 is different from that present in the unmodified antibody. In preferred embodiments, the modified therapeutic or diagnostic antibody has an in vivo clearance of at least about 1.3, 1.5, 1.8, 2.0, 2.3, 2.5, 2.8 times or greater than 3.0 times less than that of the corresponding unmodified antibody.

The present invention further provides a modified therapeutic or diagnostic antibody of the IgG class with an area under the concentration-time in vivo curve of at least about 1.3 times greater than that of the corresponding unmodified antibody. The modified therapeutic or diagnostic antibody of the disclosure, wherein at least one amino acid residue selected from the group consisting of residues 250, 314 or 428 is different from that present in the unmodified antibody.

In preferred embodiments, the modified therapeutic or diagnostic antibody has an in vivo elimination half-life of at least about 1.3, 1.5, 1.8, 2.0, 2.3, 2.6, 2.8 times or more than 3.0 times greater than that of the corresponding unmodified antibody. In alternative embodiments, the amino acid modifications of the present invention can also be used to reduce the serum half-life of a therapeutic or diagnostic antibody. Such therapeutic or diagnostic antibodies are well known in the art and are listed in the following description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Illustration of the structure of an IgG molecule

Figure 2. Rescue route of IgG molecules.

Figure 3A Amino acid sequences of OST577-lgG2M3 and OST577-lgGI with positions 250, 314 and 428 of the heavy chain highlighted "OST577-VH" (SEQ ID NO. 1) represents the amino acid sequence of the variable region of the heavy chain of OST577 -lgG2M3 or OST577-lgG1. "lgG2M3-CH" (SEQ ID NO. 2) represents the amino acid sequence of the heavy chain constant region of OST577-lgG2M3. "lgG1-CH" (SEQ ID NO. 3) represents the amino acid sequence of the heavy chain constant region of OST577-lgG1. "OST577-VL" (SEQ ID No. 4) represents the amino acid sequence of the variable region of the light chain of OST577-lgG2M3 or OST577-lgG1 .. "LAMBDA2-CL" (SEQ ID No. 5) represents the amino acid sequence of the light chain constant region of OST577-lgG2M3 or OST577-lgG1.

Figure 3B Amino acid sequences of the constant regions of the modified OST577-lgG2M3 compared to unmodified OST577-lgG2M3 (see Table 1 for SEQ ID NO. Of each amino acid sequence disclosed).

Figure 3C Amino acid sequences of the constant regions of modified OST577-lgGI compared to unmodified OST577-lgGI.

3D figure Amino acid sequences of Hu1 D10-lgG2M3 and Hu1 D10-lgG1 with positions 250, 314 and 428 of the heavy chain highlighted "Hu1 D10-VH" (SEQ ID NO: 6) represents the amino acid sequence of the variable region of the chain heavy of Hu1 D10-lgG2M3 or Hu1 D10-lgG1. "lgG2M3-CH" (SEQ ID NO. 2) represents the amino acid sequence of the heavy chain constant region of Hu1 D10-lgG2M3. "lgG1-CH" (SEQ ID NO. 7) represents the amino acid sequence of the constant region of the heavy chain of Hu1 D10-lgG1. "Hu1 D1O-VL" (SEQ ID NO. 8) represents the amino acid sequence of the variable region of the light chain of Hu1 D10-lgG2M3 or Hu1 D10-lgG1. "KAPPA-CL" (SEQ ID NO. 9) represents the amino acid sequence of the constant region of the light chain of Hu1 D10-lgG2M3 or Hu1 D10-lgG1.

Figure 3E Amino acid sequences of the constant regions of modified Hu1 D10-lgG2M3 compared to unmodified Hu1 D10-lgG2M3.

Figure 3F. Amino acid sequences of the constant regions of modified Hu1 D10-lgG1 compared to unmodified Hu1 D10-lgG1. Figure 4. Illustration of the Superposition-Extension PCR procedure.

Figure 5A Vector restriction map of the heavy chain pVAg2M3-OST577

Figure 5B Vector restriction map of the heavy chain pVAg1.N-OST577

Figure 6. Restriction map of the pVA light chain vector "A2-0ST577

Figure 7A Vector restriction map of the heavy chain pVAg2M3-Hu1 D10

Figure 7B Vector restriction map of the heavy chain pVAg1.N-Hu1 010

Figure 8. Restriction map of the light chain vector pVk-Hu1D10.

Figure 9A Restriction map of human FcRN vector pDL208

Figure 9B Rhesus pDL410 FcRN vector restriction map

Figure 10A SDS-PAGE Analysis of OST577-lgG2M3 Wild Antibodies and Mutants The purified OST577-lgG2M3 mutant antibodies were analyzed by SDS-PAGE under reducing conditions, as described in Example 5.

Figure 10B SDS-PAGE analysis of wild antibodies and mutants OST577-lgG1. Purified wild OST577-lgG1 mutant antibodies were analyzed by SDS-PAGE under reducing conditions, as described in Example 5.

Figure 11A Single-point competitive binding assay of the various mutants of position 250 of OST577-lgG2M3 to human FcRn

Binding of biotinylated OST577-lgG2M3 antibody to human FcRn in NS0 cells transfected in the presence of wild or mutant competing OST577-lgG2M3 antibodies at position 250 in FBB, at Ph 6.0, was detected with streptavidin conjugated RPE and analyzed by flow cytometry, as described in Example 6.

Figure 11B Single-point competitive binding assay of the various mutants of position 314 of OST577-lgG2M3 to human FcRn

Binding of biotinylated OST577-lgG2M3 antibody to human FcRn in NS0 cells transfected in the presence of wild-type or mutant competitor OST577-lgG2M3 antibodies at position 314 in FBB, at Ph 6.0, was detected with streptavidin-conjugated RPE and analyzed by flow cytometry, as described in Example 6.

Figure 11C. Single-point competitive binding assay of the various mutants of position 428 from OST577-lgG2M3 to human FcRn

Binding of the biotinylated OST577-lgG2M3 antibody to human FcRn in NS0 cells transfected in the presence of wild-type or mutant competitor OST577-lgG2M3 antibodies at position 428 in FBB, at Ph 6.0, was detected with streptavidin-conjugated RPE and analyzed by flow cytometry, as described in Example 6.

Figure 12A Competitive binding assay of wild and mutant OST577-lgG2M3 antibodies to human FcRn

Binding of the biotinylated HuEP5C7-lgG2M3 antibody to human FcRn in transfected NS0 cells in the presence of increasing concentrations of wild-type competitor OST577lgG2M3 antibodies at FBB, at pH 6.0, was detected with streptavidin-conjugated RPE and analyzed by flow cytometry, as described in Example 6.

Figure 12B Competitive binding assay of wild and mutant OST577-lgG2M3 antibodies to human FcRn

Binding of the biotinylated OST577-lgG2M3 antibody to the human FcRn in transfected NS0 cells in the presence of increasing concentrations of the wild competitor OST577lgG2M3 antibodies in FBB, at pH 6.0, was detected with streptavidin conjugated RPE and analyzed by flow cytometry, as described in Example 6.

Figure 13. Binding of the antibody to cells transfected with human FcRn against non-transfected cells

Binding of wild or mutant OST577-lgG2M3 antibodies to human FcRn in transfected NS0 cells or non-transfected NS0 cells in FBB, to Ph 6.0, was analyzed by flow cytometry, as described in Example 7.

Figure 14. Competitive binding assay of wild and mutant OST577-lgG2M3 antibodies to human FcRn at 37 ° C

Binding of the biotinylated OST577-lgG2M3 antibody to the human FcRn in transfected NS0 cells in the presence of increasing concentrations of the wild competitor OST577lgG2M3 antibodies in FBB, at pH 6.0, was detected with streptavidin conjugated RPE and analyzed by flow cytometry, as described in Example 7. All incubations were performed at 37 ° C.

Figure 15A PH-dependent binding and release of wild and mutant OST577-lgG2M3 antibodies to human FcRn.

The binding and release of wild or mutant OST577-lgG2M3 antibodies to human FcRn in NS0 cells transfected in FBB, at pH 6.0, 6.5, 7.0, 7.5 or 8.0, was analyzed by cytometry of flow, as described in Example 7.

Figure 15B PH-dependent binding and release of wild and mutant OST577-lgG1 antibodies to human FcRn.

The binding and release of OST577-lgG1 antibodies or mutants to human FcRn in NS0 cells transfected in FBB, at pH 6.0, 6.5, 7.0, 7.5 or 8.0, was analyzed by flow cytometry , as described in Example 7.

Figure 15C PH-dependent binding and release of wild and mutant OST577-lgG1 antibodies to human FcRn.

Binding and release of wild or mutant OST577-lgG1 antibodies to human FcRn in NS0 cells transfected in FBB, at pH 6.0, 6.5, 7.0, 7.5 or 8.0, was analyzed by cytometry of flow, as described in Example 7.

Figure 15D. PH-dependent binding and release of wild and mutant OST577-lgG2M3 antibodies to rhesus FcRn

Binding and release of wild or mutant OST577-lgG2M3 antibodies to rhesus FcRn in NS0 cells transfected in FBB, at pH 6.0, 6.5, 7.0, 7.5 or 8.0, was analyzed by cytometry flow rate, as described in Example 7.

Figure 15E PH-dependent binding and release of wild-type and mutant OST577-lgG1 antibodies to rhesus FcRn

The binding and release of wild-type or mutant OST577-lgG1 antibodies to rhesus FcRn in NS0 cells transfected in FBB, at pH 6.0, 6.5, 7.0, 7.5 or 8.0, was analyzed by cytometry flow rate, as described in Example 7.

Figure 16A. Competitive binding assay of wild and mutant OST577-lgG2M3 antibodies to HBsAg

Binding of wild or mutant OST577-lgG2M3 antibodies to HBsAg was analyzed in an ELISA competition, as described in Example 8.

Figure 16B. Competitive binding assay of wild and mutant OST577-lgG 1 antibodies to HBsAg

Binding of wild or mutant OST577-lgG1 antibodies to HBsAg was analyzed in an ELISA competition, as described in Example 8.

Figure 17A Binding assay of wild and mutant Hu1D10-lgG2M3 antibodies to the HLA-DR β chain allele

Binding of wild or mutant Hu1D10-lgG2M3 antibodies to Raji cells was analyzed in a FACS binding assay, as described in Example 8.

Figure 17B. Binding assay of wild and mutant Hu1D10-lgG1 antibodies to the HLA-DR β chain allele

Binding of wild or mutant Hu1D10-lgG1 antibodies to Raji cells was analyzed in a FACS binding assay, as described in Example 8.

Figure 18A ADCC Assay for Hu1D10-IgG1 and Hu1D10-IgG2M3 Wild and Mutant Antibodies Using PBMC from a 158V / V Donor. The ADCC activity of wild and mutant Hu1D10-IgG1 and Hu1D10-IgG2M3 antibodies in Raji cells was determined using PBMC isolated from a donor carrying FcγRIII 158V / V alleles, as described in Example 8

Figure 18B ADCC Assay for Hu1D10-IgG1 and Hu1D10-IgG2M3 Wild and Mutant Antibodies Using PBMC from a 158F / F Donor. The ADCC activity of wild and mutant Hu1D10-IgG1 and Hu1D10-IgG2M3 antibodies in Raji cells was determined using PBMC isolated from a donor carrying FcγRIII 158F / F alleles, as described in Example 8

Figure 19. Pharmacokinetics of wild OST577-lgG2M3 antibodies and variants in rhesus macaque.

The mean serum concentrations observed and modeled (µg / ml) and the standard deviations of wild OST577-lgG2M3 antibodies and variants administered by infusion at a dose of 1 mg / kg to groups of four rhesus macaques were represented as a function. of time (days after infusion), as described in Example 9.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Modified antibodies with impaired affinity for binding to FcRn and / or serum half-lives

In order for the invention to be more fully understood, several definitions are set forth.

As used herein, the terms "immunoglobulin" and "antibody" refer to proteins composed of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (γ1, γ2, γ3, γ4), delta, epsilon and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. The "light chains" of full-length immunoglobulin (approximately 25 Kd or 214 amino acids) are encoded by a gene from the kappa or lambda variable region at the NH2 end (approximately 110 amino acids) and a gene from the kappa or lamdba constant region in the COOH end. The "heavy chains" of full-length immunoglobulins (approximately 50 Kd or 446 amino acids) are similarly encoded by the heavy chain variable region gene (approximately 116 amino acids) and one of the other genes in the region constant mentioned above, for example gamma (which encodes approximately 330 amino acids).

One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, in which each pair has a light chain and a heavy chain. In each pair, the variable regions of the light and heavy chains are, together, responsible for binding to an antigen, and the constant regions are responsible for the effector functions of the antibody. In addition to tetrameric antibodies, immunoglobulins can exist in various other forms, including, for example, Fv, Fab and (Fab ') 2, as well as bifunctional hybrid antibodies (eg, Lanzavecchia and Scheidegger, Eur. J. Immunol. 17: 105-111 (1987)) and in single chains (eg, Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988), and Bird et al., Science , 242: 423-426 (1988). (See, in general, Hood et al., "Immunology", 2nd ed., Benjamin, New York (1984), and Hunkapiller and Hood, Nature, 323: 15-16 ( 1986)).

The term "genetically altered antibodies" refers to antibodies in which the sequence of amino acid residues has changed from that of a native or wild antibody. Given the relevance of recombinant DNA techniques, the present invention is not limited to the modification of amino acid sequences found in natural antibodies. As described below, previously engineered antibodies can be redesigned in accordance with the present invention in order to obtain the desired characteristics of impaired FcRn binding affinity and / or serum half-life. The possible variants of the modified antibodies useful in the present invention are many and vary from the change of only one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will be made, in general, in order to improve or alter the characteristics, such as complement fixation, interaction with several Fc-gamma receptors and other effector functions. Changes in the variable region will be made in order to improve antigen binding characteristics.

An antibody having a constant region substantially identical to the constant region of the natural IgG class antibody refers to an antibody in which any constant region present is substantially identical, ie at least about 85-90% and, preferably, at minus 95% identical, to the amino acid sequence of the constant region of the natural IgG class antibody.

In many preferred uses of the present invention, including in vivo use of modified antibodies in humans and in in vitro detection assays, it may be preferable to use privatized chimeric or humanized humanized antibodies that have been modified (i.e. mutated) in accordance with the present invention.

The term "chimeric antibody" refers to an antibody in which the constant region comes from an antibody of one species (usually human) and the variable region comes from an antibody of another species (usually from a rodent). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214-221 (1986); Gillies et al., J. Immunol. Methods 125: 191-202 (1989); U.S. patents No. 5,807,715; 4,816,567; Y

4,816,397.

The term "primatized antibody" refers to an antibody comprising variable regions of mono and constant regions of human. Methods for producing primed antibodies are known in the art See, for example, US Pat. No. 5,658,570; 5,681,722; and 5,693,780.

The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human structure, at least one, and preferably all, the complementarity determining regions (CDR) of a non-human antibody and in which any constant region present is substantially identical to a constant region of human immunoglobulin, that is at least about 85-90% and, preferably, at least 95% identical. Thus, all parts of a humanized immunoglobulin, except possibly the CRDs, are substantially identical to the corresponding parts of one or more native immunoglobulin sequences. Often, the structure residues in the human structure regions will be substituted with the corresponding residue of the CDR donor antibody to preferentially alter, binding to the antigen. These structure substitutions are identified by methods well known in the art by, for example, modeling the interactions of the CDR residues and the structure to identify the structure residues important for antigen binding and sequence comparison for identify residues of unusual structures in concrete positions. See, for example, Queen et al., US Pat. No. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91/09967; U.S. Patent No. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (cover the surface) (EP 592,106; EP 519,596; Padlan, Mol. Immunol.,

28: 489-498 (1991); Studnicka et al., Prot. Eng. 7: 805-814 (1994); Roguska et al., Proc. Natl Acad. Sci. 91: 969-973 (1994), and transposition of chains (US Patent No. 5,565,332).

Fully human antibodies may be desirable for therapeutic treatment of human patients. Human antibodies can be made by various methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See US patents. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For a review of this technology to produce human antibodies, see Lonberg and Huszar, Int Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies see, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. patents No. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In addition, companies such as Abgenix, Inc. (Fremont, CA) and Medarex (Princeton, NJ) can commit to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can be generated using a technique called "guided selection."

In this approach a selected non-human monoclonal antibody, for example a mouse antibody, is used to guide the selection of a completely human antibody that recognizes the same epitope (Jespers et al., Biotechnology 12: 899-903 (1988).

As used herein, the term "alter" may refer to "increase" or "reduce". The invention is defined in the appended claims.

The present disclosure provides a modified IgG class antibody, in which at least one amino acid of the constant region of the heavy chain selected from the group consisting of amino acid residues 250, 314 and 428 is substituted by an amino acid residue different from the present in the unmodified antibody. IgG class antibodies include antibodies of IgG1, IgG2, IgG3 and IgG4. The constant region of the last chain of an IgG molecule is indicated in Figure 1. The numbering of the residues in the heavy chain is that of the EU index (Kabat et al., Op. Cit.). The replacement can be carried out in position 250, 314 or 428 alone, or in any combination thereof, such as in positions 250 and 428, or in positions 250 and 314 or in positions 314 and 428, or in the positions 250, 314 and 428, with positions 250 and 428 as the preferred combination.

For each position, the substitute amino acid can be any amino acid residue 5 different from that present in said position of the unmodified antibody.

For position 250, the amino acid residue may be any amino acid residue other than threonine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine , proline, glutamine, arginine, serine, valine, tryptophan or tyrosine.

10 For position 314, the substitute amino acid residue may be any amino acid residue other than leucine, including, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine valine, tryptophan or tyrosine. For position 428, the substitute amino acid residues can be any residue.

15 amino acid other than methionine, including but not limited to alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine valine, tryptophan or tyrosine . The present disclosure provides IgG class antibodies comprising at least one of the amino acid substitutions described above. For example, the present invention provides

20 mutated constant regions of IgG2M3, comprising two of the substitutions mentioned above at position 250, 314 and / or 428. The amino acid sequences are some specific substitutions (ie, mutations) of the constant region provided by the The present invention is disclosed in Table 1 (SEQ ID NO: 10-66) and Figure 3B.

25

Table 1

Amino Acid Substitute

250 314 428

Alanina (A)

T250A; SEQ ID NO. 10 L314A; SEQ ID No. 29 M428A; SEQ ID No. 48

Cysteine (C)

T250C; SEQ ID No. 11 L314C; SEQ ID No. 30 M428C; SEQ ID No. 49

Aspartic acid (D)

T250D; SEQ ID No. 12 L314D; SEQ ID No. 31 M428D; SEQ ID No. 50

Glutamic acid (E)

T250E; SEQ ID NO. 13 L314E; SEQ ID No. 32 M428E; SEQ ID No. 51

Phenylalanine (F)

T250F; SEQ ID No. 14 L314F; SEQ ID No. 33 M428F; SEQ ID No. 52

Glycine (G)

T250G; SEQ ID No. 15 L314G; SEQ ID No. 34 M428G; SEQ ID No. 53

Histidine (H)

T250H; SEQ ID No. 16 L314H; SEQ ID No. 35 M428H; SEQ ID No. 54

Amino Acid Substitute

250 314 428

Isoleucine (I)

T250I; SEQ ID No. 17 L314I; SEQ ID No. 36 M428I; SEQ ID No. 55

Lysine (K)

T250K; SEQ ID No. 18 L314K; SEQ ID No. 37 M428K; SEQ ID No. 56

Leucine (L)

T250L; SEQ ID No. 19 Wild M428L; SEQ ID No. 57

Methionine (M)

T250M; SEQ ID No. 20 L314M; SEQ ID No. 38 Wild

Asparagine (N)

T250N; SEQ ID No. 21 L314N; SEQ ID No. 39 M428N; SEQ ID No. 58

Proline (P)

T250P; SEQ ID No. 22 L314P; SEQ ID No. 40 M428P; SEQ ID No. 59

Glutamine (Q)

T250Q; SEQ ID No. 23 L314Q; SEQ ID No. 41 M428Q; SEQ ID No. 60

Arginine (R)

T250R; SEQ ID No. 24 L314R; SEQ ID No. 42 M428R; SEQ ID No. 61

Serina (S)

T250S; SEQ ID No. 25 L314S; SEQ ID No. 43 M428S; SEQ ID No. 62

Threonine (T)

Wild L314T; SEQ ID No. 44 M428T; SEQ ID No. 63

Valine (V)

T250V; SEQ ID No. 26 L314V; SEQ ID No. 45 M428V; SEQ ID No. 64

Tryptophan (W)

T250W; SEQ ID No. 27 L314W; SEQ ID No. 46 M428W; SEQ ID No. 65

Tyrosine (Y)

T250Y; SEQ ID No. 28 L314Y; SEQ ID No. 47 M428Y; SEQ ID No. 66

In a preferred embodiment, the present invention provides a modified antibody that has a serum half-life or FcRn binding affinity with respect to the unmodified antibody. The present disclosure further provides an antibody

5 modified from class IgG, in which at least one amino acid of the heavy chain constant region, selected from the group consisting of amino acid residues 250, 314 and 428, is substituted with another amino acid that is different from that present in the antibody unmodified, so that it alters the binding affinity for FcRn and / or the serum half-life of the modified antibody compared to the binding affinity and / or serum half-life of

10 said unmodified antibody. The unmodified antibodies of the present invention include natural antibodies of all species. The term "natural antibodies" refers to all antibodies produced by a host animal. Non-limiting examples of natural antibodies of the present invention include antibodies derived from humans, chicken, goat and rodents.

15 (e.g., rats, mice, hamsters and rabbits), including genetically engineered transgenic rodents to produce human antibodies (see, for example, Lonberg and Co., WO93 / 12227; US Pat. No. 5,545,806; and Kucherlapati, et al., WO 91/10741; U.S. Patent No. 6,150,584). ).

The unmodified antibodies of the present invention also include recombinant antibodies that have the same amino acid sequences as a natural antibody or genetically altered antibodies that have amino acid sequences modified in

Comparison with natural antibodies. They can be manufactured in any expression system, including prokaryotic and eukaryotic expression systems, or using phage display methods (see, eg, Dower et al., WO 91/17271 and McCafferty et al., WP92 / 01 047; U.S. Patent No. 5,969,108.

The unmodified antibodies of the present invention also include chimeric, primatized, humanized and human antibodies (see discussion above). Accordingly, a modified antibody of the present invention can be produced by replacing an amino acid residue at position 250, 314 or 428 in a humanized, primatized or chimeric antibody, in which in humanized, primatized or chimeric antibody it has been previously derived to from a native antibody.

Preferably, the chimeric antibodies comprise variable regions derived from rodents and constant regions derived from humans, so that the chimeric antibodies have longer half-lives and are less immunogenic when administered to a human subject. Normally, humanized antibodies comprise at least one CDR of donor antibodies (eg, murine or chicken antibodies) and human heavy and / or light chain structures. Occasionally, some amino acid residues in human structures will be replaced by residues in equivalent positions of donor antibodies to ensure proper binding of humanized antibodies to their antigens. Detailed guides to antibody humanization are disclosed in US Pat. No. 5,530,101; 5,585,089; 5,693,761; 5,693,762; Y

6,180,370.

The unmodified antibodies of the present invention may include genetically altered antibodies that are functionally equivalent to the corresponding natural antibodies. Unmodified antibodies that are genetically altered to provide better stability and / or therapeutic efficacy are preferred. Examples of altered antibodies include those with conservative substitutions of amino acid residues and one or more amino acid deletions or additions that do not significantly alter the usefulness of antigen binding. Substitutions may vary from changing or modifying one or more amino acid residues to complete the redesign of a region with the condition that the binding or functional utility be maintained. The antibodies of the present invention can be posttranslationally altered (eg, acetylation and phosphorylation) or can be synthetically altered (eg, the fixation of a marker group).

The unmodified antibodies of the present invention may include antibodies that have binding affinities enhanced by their antigens through genetic alterations of the variable regions (see, for example, U.S. Patent No. 6,350,861). ). In an alternative embodiment, the modified IgGs of the present invention that have longer half-lives than wild (or unmodified) IgGs may also include IgGs whose bioactive sites, such as antigen binding sites, binding sites to Fcreceptor or complement binding sites, are modified by genetic engineering to increase or reduce these activities compared to wild antibodies,

The unmodified and modified antibodies of the present invention may be of any of the recognized isotypes, but all four isotypes of IgG are preferred, with IgG1 and IgG2 being especially preferred. Antibodies with and other IgG2 mutants described in US Pat. No. 5,834,597. In a preferred aspect, the unmodified and modified antibodies in the present invention comprise constant regions of the human IgG heavy chain.

The present invention can be applied to any antibody comprising constant regions of the IgG class heavy chain, preferably IgG1, IgG2, IgG2M3, IgG3 and Egg4. The variable regions of the heavy chain of said antibodies can be derived from any selected antibody. Examples of antibodies disclosed herein include OST577-lgG1 and OST577-lgG2M3, comprising the variable regions of the heavy chain and the light chain of the human hepatitis B virus antibody OST577 (Ehrlich et al., Hum. Antibodies Hybridomas 3: 2-7 (1992)), the constant region of the human lambda-2 light chain and the constant region of the human IgG1 and IgG2M3 heavy chain, respectively. Also disclosed herein are Hu1D10-lgG1 and Hu1D10-IgG2M3, which comprise the heavy and light chain variable regions of the Hu1D10 anti-allele antibody of the humanized HLA-DR β chain (Kostelny et al., L. J. Cancer 93: 556-565 (2001)), the constant region of the human kappa light chain and the constant regions of the heavy chain mentioned above from human IgG1 and IgG2M3, respectively.

Other examples of the present invention disclosed herein include mutants of the human antibodies IgG1, IgG2M3 (a genetically altered variant of human IgG2), IgG3 and IgG4, which illustrate the alteration of the serum half-life of an IgQ class antibody. The constant region of the IgG2M3 heavy chain derives from that of IgG2 by replacing residues 234 and 237 of the constant region of the IgG2 heavy chain with alanine. The generation of the IgG2M3 heavy chain constant region is disclosed in US Pat. No. 5,834,597.

Generally, the modified antibodies of the present invention include any immunoglobulin molecule that binds (preferably immunospecifically, that is, non-specific binding competes, determined by immunoassays well known in the art to analyze specific antibody-antigen binding) to an antigen and It contains an FcRn binding fragment. Such antibodies include, among others, polyclonal, monoclonal, bispecific, multispecific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, disulfide-linked Fv fragments and fragments containing a VL domain or VH, or even a complementarity determining region (CDR) that specifically binds to an antigen, in certain cases, engineered to contain or condense to an FcRn binding domain.

The modified IgG molecules of the invention can include the IgG subclasses of any given animal. For example, in humans, the IgG class includes IgG1, IgG2, IgG3, and IgG4; and the mouse IgG class includes IgG1, IgG2a, IgG2b, and IgG3; and the rat IgG class includes IgG1, IgG2a, IgG2b, IgG2c, and IgG3. It is known that certain IgG subclasses, for example, rat IgG2b and IgG2c, have higher clearance rates than, for example, IgG1 (Medesan et al., Eur. J. lmmunol., 28: 2092-2100 (1998) ). Therefore, when other subclasses of IgG1 other than IgG1 are used, it may be advantageous to replace one or more of the residues, particularly in the CH2 and CH3 domains, which differ from the sequence of IgG1 with those of IgG1, so that it is increased the in vivo half-life of the other types of IgG.

The immunoglobulins of the present invention can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, rodent, donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from libraries of human immunoglobulins or transgenic animals for one or more human immunoglobulins, as described below. and, for example, in US Pat. No. 5,939,598 to Kucherlapati et al.

In addition, the modified antibodies of the present invention may be monospecific, bispecific, triespecific or of greater multispecificity antibodies. Multispecific antibodies can be specific for different epitopes of a polypeptide or they can be specific for heterologous epitopes, such as a heterologous polypeptides or solid support material. See, for example, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. lmmunol. 147: 60-69 (1991); U.S. patents No. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. lmmunol. 148: 1547-1553 (1992).

Modified antibodies of the invention include derivatives that are otherwise modified, that is, by covalent binding of any type of molecule to the antibody so that covalent binding does not prevent antibody binding to the antigen and / or generate a anti-idiotypic response. For example, although not as a limitation, antibody derivatives include antibodies that have been modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivation with known protecting / blocking groups, proteolytic cleavage, ligand binding or other cellular protein, etc. Any of the numerous chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

Monoclonal antibodies useful with the present invention can be prepared using a wide variety of techniques known in the art, including the use of phage, hybridoma and recombinant expression technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known and taught in the art, for example in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York (1988); Hammerling et al., In: "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, New Yolk (1981), pp. 563

681.

The term "monoclonal antibody" as used herein, is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including a eukaryotic, prokaryotic or phage clone, and not the method by which it is produced.

Procedures for producing and selecting specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing said antigen. Once a response is detected, for example specific antibodies are detected by the antigen in the mouse serum, the spleen is removed from the mouse and the splenocytes are isolated. Splenocytes are then condensed by well known techniques with suitable myeloma cells. The hybridomas are selected and cloned by limit dilution. Then, the hybridoma clones are analyzed by methods known in the art for cells capable of secreting antibodies capable of binding to the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating positive hybridoma clones into the mice intraperitoneally.

Antibody fragments that recognize specific epitopes can also be useful with the present invention and can be generated by well known techniques. For example, Fab and F (ab ’) 2 fragments can be produced, by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab’) 2 fragments). F (ab ’) 2 fragments contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various expression procedures known in the art, including phage expression. In phage display procedures, functional antibody domains are expressed on the surface of phage particles that carry the polynucleotide sequences that encode them. In a specific embodiment, said phage can be used to express antigen-binding domains, such as Fab and Fv or Fv stabilized by disulfide, expressed from a repertoire or combinatorial library of antibodies (e.g., human or murine). Phages that express an antigen binding domain that binds to the antigen of interest can be selected or identified with the antigen, for example using labeled antigen or antigen bound or captured by a solid surface or sphere. The phages used in these procedures are usually filamentous phages, including phages fd and M13. The antigen binding domains are expressed in the form of a protein recombinantly condensed to the protein of gene III or phage gene VIII. Alternatively, the modified FcRn binding portion of the immunoglobulins of the present invention can also be expressed in a phage display system. Examples of phage display procedures that can be used to make immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettieborough et al., Eur. J. Immunol. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT publication WO 92/01047; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. patents Nos. 5,698,426; 5,223,409; 5,403. 484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the previous references, after phage selection, regions encoding the antibody can be isolated from the phage and used to generate whole antibodies, including human antibodies, or any other desired fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeasts and bacteria, for example as described in detail below. For example, techniques can also be used to recombinantly produce Fab, Fab 'and F (ab ’) 2 fragments using methods known in the art such as those described in PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12: 864-869 (1992); Sawai et al., Amer. J. Reprod. Immunol 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988). Examples of techniques that can be used to produce Fv and single chain antibodies include those described in US Pat. No. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88 (1991); Shu et al., Proc. Natl Acad. Sci. 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988).

In specific embodiments, the modified antibodies have therapeutic and / or prophylactic uses in vivo. Examples of therapeutic and prophylactic antibodies that can be modified in this way include, among others, SYNAGIS® (palivizumab Medimmune, MD) which is a humanized monoclonal antibody against respiratory syncytial virus (RSV) for the treatment of patients with RSV infection; HERCEPTIN® (Trastuzumab) (Genentech, CA), which is a humanized anti-HBR2 monoclonal antibody for the treatment of patients with metastatic breast cancer; RBMICADE® (infliximab) (Centocor, PA), which is an anti-TNF-α chimeric monoclonal antibody for the treatment of patients with Crohn's disease; REPRO® (abciximab) (Centocor) which is an anti-glycoprotein lIb / IlIa receptor on platelets for the prevention of clot formation; ZBNAPAX® (daclizumab) (Roche Pharmaceuticals, Switzerland), which is a humanized anti-CD25 immunosuppressive monoclonal antibody for the prevention of acute renal allograft rejection. Other examples are humanized anti-CD18 F (ab ') 2 (Genentech); CDP860 which is a humanized anti-CD18 F (ab ') 2 (Celltech, UK); PR0542 which is an anti-gp120 HIV antibody condensed with CD4 (Progenics / Genzyme Transgenics); OSTAVIRTM, which is a human hepatitis B virus antibody (Protein Design Labs / Novartis); PROTOVIRTM, which is a humanized anti-CMV IgG1 antibody (Protein Design Labs / Novartis); IC14 which is an anti-CD14 antibody (ICOS); AVASTINTM (bevacizumab), which is a humanized anti-VEGF IgG1 antibody (Genentech); ERBITUXTM (cetuximab) which is a chimeric anti-EGFR IgG antibody (ImClone Systems); VITAXINTM, which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution / Medimmune); Campath-1H / LDP-03 (alemtuzumab), which is a humanized anti-CD52 IgG1 antibody (Leukosite); ZAMYLTM, which is a humanized anti-CD33 IgG antibody (Protein Design Labs / Kanebo); RITUXANTM (rituximab, which is a chimeric anti-CD20 IgG1 antibody (IDEC Pharmaceuticals / Genentech, Roche / Zenyaku); LYMPHOCIDETM, which is a humanized anti-CD22 IgG antibody (Immunomedics); REMITOGENTM, which is an anti-HLA-DR antibody humanized (Protein Design Labs); ABXIL8 which is a human anti-IL8 antibody (Abgenix); RAPTIVATM (efalizumab) which is a humanized IgG1 antibody (Genetech / Xoma); ICM3, which is a humanized anti-ICAM3 antibody (ICOS); IDEC-114, which is a primed anti-CD80 antibody (IDEC Pharmaceuticals / Mitsubishi); IDEC-131, which is a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151, which is a primed anti-CD4 antibody ( IDEC); IDEC-152, which is a primitive anti-CD23 antibody (IDEC / Seikagaku); NUIVONTM (visilizumab), which is a humanized anti-CD3 IgG (Protein Design Labs); SG1.1, which is an anti- antibody humanized complement factor 5 (C5) (Alexion Pharmaceuticals); HUMIRATM (adalimumab), which is a human anti-TNF-α antibody (CAT / BASF); CDP870, which is a humanized antiTNF-α Fab fragment (Celltech); IDEC-151, which is a primitive anti-CD4 IgG1 antibody (IDEC Pharmaceuticals / Smith-Kline Beecham); MDX-CD4, which is a human anti-CD4 IgG antibody (Medarex / Eisai / Genmab); CDP571, which is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02, which is a humanized anti-α4β7 antibody (LoukoSite / Genentech); OrthoClone OKT4A, which is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVATM, which is a humanized anti-CD40L IgG antibody (Biogen); ANTBGRENTM (natalizumab), which is a humanized anti-VLA-4 IgG antibody (Elan); MDX33, which is a human anti-CD64 antibody (FcγR) (MedarexiCenteon); SCH55700, which is a humanized anti-IL-5 IgG4 antibody (Celltech / Schering); SB-240563 and SB-240683 which are humanized anti-IL-5 and IL-4 antibodies, respectively (SmithKline Beecham); rhuMabE25, which is a humanized anti-IgE IgG1 antibody (Genentech / Novards / Tanox Biosystems); IDEC-152, which is a primed anti-CD23 antibody (IDEC Phatmaceuticals); SIMULECTM (basiliximab), which is a chimeric anti-CD25 IgG1 antibody (Novartis Pharmaceuticals); LDPD1, which is a humanized anti-β2-integrin IgG antibody (Leukosite); CAT-152, which is a human anti-TGF-β2 antibody (Cambridge Antibody Technology); and Corsevin M, which is a chimeric anti-Factor VII antibody (Centocor). The present invention allows the modification of these and other therapeutic antibodies to increase the half-life in vivo, allowing the administration of less effective dosages and / or less frequent dosing of the therapeutic antibody. Such modification to increase half-life in vivo may also be useful for improving diagnostic immunoglobulins. For example, increasing the serum half-life of a diagnostic antibody may allow the administration of lower doses to achieve sufficient diagnostic sensitivity. Alternatively, the decrease in serum half-life may be advantageous in applications where rapid clearance of a diagnostic antibody is desired.

The amino acid sequence of OST577-lgG2M3 is disclosed herein, including the amino acid sequence of its heavy chain variable region (SEQ ID No. 1) (OST577-VH) and the constant region (SEQ ID No. 2) ( lgG2M3-CH) with positions 250, 314 and 428 highlighted, and the amino acid sequence of the variable region of its light chain (SEQ ID No. 4) (OST577-VL) and the constant region (SEQ ID No. 5) (LAMBDA2 -CL) (Figure 3A).

The amino acid sequence of the constant region of the heavy chain of OST577-lgG1 (SE ID No. 3) is disclosed herein, with positions 250, 314 and 428 highlighted (Figure 3C).

The present invention further provides a modified antibody that has a higher binding affinity for FcRn and / or an increase in serum half-life compared to the unmodified antibody, in which amino acid residues 250 and 428 of the constant region of the Human heavy chain are substituted, namely, amino acid residue 250 of the constant region of the heavy chain is substituted with glutamic acid or glutamine and amino acid residue 428 of the constant region of the heavy chain is substituted with phenylalanine or leucine.

In one example, said unmodified antibody comprises the heavy chain constant region of a molecule of IgG1, or IgG2, or IgG2M3, including, among others, OST577lgG2M3 or OST577-lgG1. IgG1, IgG2 and IgG2M3 have a threonine residue at position 250 and a methionine residue at position 428. The threonine residue at position 250 is substituted with glutamic acid (T250E) or glutamine (T250Q) and the methionine residue at position 428 it is substituted with phenylalanine (M428F) or leucine (M428L). Figure 3B discloses the amino acid sequence of the constant region of the modified IgG2M3 heavy chain having the amino acid substitution of T250E (SEQ ID No. 13), T250Q (SEQ ID No. 23), M428F; SEQ ID No. 52), or M428L (SEQ ID No. 57). Figure 3C discloses the amino acid sequence of the heavy chain constant region of the modified IgG1 having the amino acid substitution of T250D (SEQ ID NO: 67) T250E; SEQ ID No. 68), T250Q (SEQ ID No. 69), M428F; SEQ ID No. 70), or M428L (SEQ ID No. 71).

The present invention provides a modified antibody that has a higher binding affinity for FcRn and / or a higher serum half-life compared to the unmodified antibody. The amino acid modification can be any one of the following substitutions:

one)

amino acid residue 250 of the heavy chain constant region is

replaced

with glutamic acid and amino acid residue 428 of the region

Heavy chain constant is substituted with phenylalanine.

2)

amino acid residue 250 of the heavy chain constant region is

substituted with glutamine and amino acid residue 428 of the constant region of

the heavy chain is substituted with phenylalanine.

3)

amino acid residue 250 of the heavy chain constant region is

substituted with glutamine and amino acid residue 428 of the constant region of

The heavy chain is substituted with leucine.

The amino acid sequence of the constant region of the modified IgG2M3 heavy chain having the double amino acid substitution of T250E / M428F (SEQ ID No. 72), T250Q / M428F (SEQ ID No. 73), or T250Q / M428L (SEQ ID No. 74) is disclosed in Figure 3B.

The amino acid sequence of the constant region of the modified IgG1 heavy chain having the double amino acid substitution of T250E / M428F (SEQ ID No. 75), or T250Q / M428L (SEQ ID No. 76) is disclosed in Figure 3C.

The antibodies modified with the double amino acid substitutions described in positions 250 and 428 show extremely high binding affinities for FcRn compared to those of the unmodified antibodies.

In a preferred embodiment of the present invention, the FcRn binding affinity and / or the serum half-life of the modified antibody are increased by at least about 30%, 50%, 80%, 2 times, 3 times, 4 times , 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times.

The present disclosure provides a modified antibody that has a lower binding affinity for fcRn and / or lower serum half-life compared to the unmodified antibody, in which amino acid residue 314 of the heavy chain constant region is substituted with another amino acid that is different from that present in an unmodified antibody. Modified antibodies that have an amino acid substitution at position 314 have been shown to show reduced binding affinity, suggesting that position 314 should be modified if a reduction in serum half-life of an antibody is desired. Preferably, amino acid residue 314 of the heavy chain constant region is substituted with alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. More preferably, the amino acid substitution is from leucine to alanine or arginine at position 314. As shown in the Examples, the FcRn binding affinity of the modified OST577-lgG2M3 comprising a substitution of leucine to arginine is reduced by 11 % with respect to the unmodified OST577-lgG2M3.

As shown in Figure 3B, L314A represents the amino acid sequence of the heavy chain constant region of the modified IgG2M3, which has the amino acid substitution of leucine for Alanine at position 314 (SEQ ID NO: 29). L314A represents the amino acid sequence of the heavy chain constant region of the modified IgG2M3, which has the amino acid substitution of leucine for arginine at position 314 (SEQ ID NO: 42).

The present disclosure provides a modified antibody that has a lower binding affinity for FcRn and / or lower serum half-life compared to the unmodified antibody, in which (1) amino acid residue 250 of the heavy chain constant region is substituted with arginine, asparagine, aspartic acid, lysine, phenylalanine, proline, tryptophan or tyrosine, or (2) amino acid residue 428 of the constant region of the heavy chain is substituted with alanine, arginine, asparagine, aspartic acid, cysteine , glutamic acid, glutamine, glycine, histidine, lysine, proline, serine, threonine, tyrosine or valine. Preferably, amino acid residue 250 of the heavy chain constant region is substituted with aspartic acid or amino acid residue 428 of the heavy chain constant region is substituted with glycine. Said amino acid substitution can dramatically reduce the serum half-life of an antibody. As shown in the Examples, the FcRn binding affinity of modified OST577-lgG2M3 having such amino acid substitutions is reduced by approximately 5-7% with respect to unmodified OST577-lgG2M3.

As shown in Figure 3B, T250D represents the amino acid sequence of the heavy chain constant region of the modified IgG2M3, which has the substitution of threonine amino acid to aspartic acid at position 250 (SEQ ID NO: 12). M428 represents the amino acid sequence of the heavy chain constant region of the modified IgG2M3, which has the amino acid substitution of methionine to glycine at position 428 (SEQ ID NO: 53).

In a particular aspect of the present disclosure, the binding affinity for FcRn and / or the serum half-life of said modified antibody is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

The present invention includes the heavy chain constant regions, the Fc regions, or the CH2-CH3 regions of the modified IgG antibodies described herein, preferably, of the modified IgG1, IgG2 or IgG2M3 antibodies having amino acid substitutions described herein.

The present disclosure also includes a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 10-76. In a preferred embodiment, these polypeptides are mutated constant regions of IgG1, IgG2 or IgG2M3. The heavy chain constant regions of the modified antibodies of the present invention can be linked to the heavy chain variable region of any selected antibody to generate the desired hybrid heavy chain. Examples of the selected antibodies include, among others, antibodies against IL-2, IL-4, IL-10, IL-12, HSV, CD3, CD33, CMV and IFN-γ. In addition, the variable regions may be those of natural antibodies of any species such as a human being, a primate or a rodent. Alternatively, they may be those of genetically altered antibodies, including but not limited to humanized antibodies, antibodies that have greater binding affinities to their antigen through genetic modification or fully human antibodies. Such a hybrid heavy chain may be linked to a variety of light chains to produce the desired antibody. The light chains can be lambda or kappa-light chains. Since the serum half-life of an antibody is determined primarily in the constant region of the heavy chain, a desired serum half-life of an antibody produced through the substitutions of amino acid in the constant region of the heavy chain described herein.

II. Production of modified antibodies with impaired affinity for binding to FcRn and / or serum half-lives

The present invention provides methods of producing proteins, particularly antibodies with impaired binding affinity for FcRn and / or serum half-lives. Preferably, the present invention provides methods for modifying a given antibody of class IgG in one or more positions disclosed herein. This can be achieved chemically or by site-directed mutagenesis and recombinant production using any known production method.

The present disclosure provides a method of modifying an IgG class antibody comprising replacing at least one amino acid of the constant region of the heavy chain selected from the group consisting of amino acid residues 250, 314 and 428 with an amino acid that is different from the present in an unmodified antibody, so that it causes alteration of the binding affinity for FcRn and / or the serum half-life of said unmodified antibody.

The replacement can be carried out in position 250, 314 or 428 alone or in any combination thereof, such as in positions 250 and 428.

In the process according to the invention, to increase the binding affinity for FcRn and / or increase the serum half-life of an antibody, amino acid residue 250 of the heavy chain constant region is substituted with glutamic acid or glutamine, and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine or leucine. Alternatively, amino acid residue 250 of the heavy chain constant region is substituted with glutamic acid and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine; or amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine; or amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with leucine. These antibodies that have these double mutations show exceptionally high FcRn binding affinities.

To produce a modified antibody having a lower binding affinity for fcRn and / or lower serum half-life compared to the unmodified antibody, in which amino acid residue 314 of the heavy chain constant region is substituted with another amino acid which is different from the present in an unmodified antibody. Preferably, amino acid residue 314 of the heavy chain constant region is substituted with alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. More preferably, amino acid residue 314 of the heavy chain constant region is substituted with alanine or arginine.

To produce a modified antibody that has a lower binding affinity for FcRn and lower serum half-life compared to the unmodified antibody, amino acid residue 250 of the heavy chain constant region is substituted with arginine, asparagine, aspartic acid, lysine, phenylalanine, proline, tryptophan or tyrosine, or amino acid residue 428 of the heavy chain constant region is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, proline , serine, threonine, tyrosine or valine. More preferably, amino acid residue 250 is substituted with aspartic acid or amino acid 428 is substituted with glycine.

The amino acid substitutions described herein are achieved by recombinant DNA technology. In one embodiment, site-directed mutagenesis can be used to introduce amino acid substitutions into the DNA encoding an unmodified antibody. DNAs resulting from modified antibodies are released into host cells and modified antibodies are produced in this way. The desired alteration of the FcRn binding affinity of the modified antibodies can be selected using phage display technology or any other suitable method known in the art and shaped by measuring binding affinity.

Preferably, a method of producing modified IgG class antibody with impaired binding affinity for FcRN and serum half-life alteration compared to an unmodified antibody:

(to)

preparing a replicable expression vector comprising a suitable promoter operably linked to a DNA that encodes at least one constant region of an immunoglobulin heavy chain and in which at least one amino acid from the constant region of the heavy chain selected from the group consisting of amino acid residues 250, 314 and 428 are substituted with an amino acid that is different from that present in an unmodified antibody, so that it causes an alteration in serum half-life;

(b)

transforming host cells with said vector; Y

(C)

culturing said transformed host cells to produce said modified antibody.

Said method additionally and optionally comprises, after step (a) preparing a second replicable expression vector comprising a promoter operably linked to a DNA encoding a complementary immunoglobulin light chain and into which said cell line is further transformed. with said vector.

To generate the DNA in step (a), amino acid substitutions can be introduced by mutagenesis, including but not limited to site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985) ), PCR mutagenesis (Higuchi, in "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego (1990) pp. 177183), and cassette mutagenesis (Wells et al., Gene 34: 315-323 (1985)). Preferably, site-directed mutagenesis is performed by the extension overlap PCR procedure, which is disclosed in the Examples (Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification", Stockton Press, New York (1989), p. 61-70).

The PCR technique with overlap-extension (Higuchi, ibid.) Can be used to introduce any desired mutation (portions) into a target sequence (the starting DNA). For example, as shown in Figure 4, the first PCR cycle in the overlap-extension procedure involves amplifying the target sequence with an external primer (Primer 1) and an internal primer for mutagenesis (primer 3) and, separately , with a second external primer (Primer 4) and an internal primer (primer 2), which gives two PCR segments (segments A and B). The internal mutagenesis primer (primer 3) is designed to contain mismatches with the target sequence specifying the desired mutation (s). In the second PCR cycle, the products of the first PCR cycle (segments A and B) are amplified by PCR using the two external primers (primers 1 and 4). The resulting full length PCR segment (segment C) is digested with restriction enzymes and the resulting restriction fragment is cloned into a suitable vector.

As the first stage of mutagenesis, the starting DNA is operably cloned into a vector for mutagenesis. The primers are designed to reflect the desired amino acid substitution (see more details in the Examples). In one example, vectors used for in vitro mutagenesis can be used to direct protein expression. Thus, the DNA resulting from the overlap-extension PCR can be cloned back into the vector for mutagenesis so that an expression vector comprising the DNA with the desired mutation is created. An example of the vector for mutagenesis comprising the starting DNA includes, among others, pVAg2M3-OST577.

For example, mutations at position 250 were made by amplification of the region surrounding the PinAI-BamHI fragment of pVAg2M3-OST577 (see the restriction map of Figure 5A) in a two-stage procedure using the overlapping procedure. extension described above, then digesting the resulting PCR segment with PinAl and BamHI and cloning the resulting restriction fragment into pVAg2M3OST577. Similarly, mutations at position 314 or 428 were performed by amplification of the region surrounding the Pmll-BamHI fragment by overlapping PCR extension, then digesting the resulting PCR segment with Pmll-and BamHI and cloning the resulting restriction fragments in pVAg2M3-OST577.

The starting DNA may be a DNA encoding an unmodified whole antibody, an entire immunoglobulin heavy chain of an unmodified antibody, the constant region of a heavy chain or part of the heavy chain constant region of an unmodified antibody , as long as it includes the amino acid residue to be modified.

If the DNA encoding the entire unmodified antibody is used as the starting DNA for mutagenesis, all modified antibody can be produced by performing steps (a), (b) and (c) of the procedure described herein. The stage between the stage

(a) and step (b) of said procedure to generate the complementary light chain would not be necessary.

If the starting DNA for DNA mutagenesis encoding the entire heavy chain of an unmodified antibody, the mutagenesis will result in a vector comprising the DNA encoding the entire modified heavy chain. In order to produce an entire modified antibody, the step between steps (a) and (b) of the process disclosed herein is performed. That is, another replicable expression vector comprising a promoter operably linked to a DNA encoding the complementary immunoglobulin light chain is co-transfected into the same host cells. As a result, both the complementary light chain and the modified heavy chain are expressed in the same host cells and properly assembled to give rise to the entire modified antibody. An example of said expression vector comprising a DNA encoding an immunoglobulin light chain includes, among others, pVAλ2-0ST577.

If the starting DNA for mutagenesis is a DNA that encodes part of the heavy chain constant region, such as the CH2-CH3 segment or an Fc domain, the resulting DNA encoding such modified partial heavy chain is first connected in the frame with the rest of the unmodified heavy chain, so that the DNA encoding an entire heavy chain is generated with the modification described herein in Step (a).

An entire modified antibody is then produced by co-transfection of the host cells with the vector comprising the DNA encoding the complementary light chain and the vector comprising the DNA encoding said modified heavy chain. The connection of the DNA encoding the modified partial heavy chain and the remaining unmodified heavy chain can be achieved using standard molecular cloning techniques known in the art of molecular biology, such as digestions and restriction junctions (Sambrook and Russell, " Molecular Cloning: A Laboratory, Manual ", 3rd edition, Cold Spring Harbor Laboratory Press, New York (2001)).

Light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector (s) that guarantee the expression of immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer and a transcription termination sequence (see, Queen and coI., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989); W0O90 / 07861; Co et al., J. Immunol. 148: 1149-1154 (1992); "Antibody Engineering: A Practical Guide", Borrebaeck, Ed., Freeman, New York (1997)).

Host cells are transformed using techniques known in the art, such as liposomes, calcium phosphate, electroporation etc. (Sambrook and Russell, op. Cit.). Preferably, the host cells are transiently transfected using the liposome method.

The host cells used to produce the modified antibody of the present invention can be cultured in various means known in the art.

The modified antibodies described herein can be produced intracellularly, in the periplasmic space or secreted directly in the medium. Preferably, the modified antibodies in the present invention are secreted into the culture medium. Host cell culture media producing modified antibodies are collected and cell debris precipitated by centrifugation. Supernatants are collected and subjected to protein expression assays (see more details in the Examples).

The expression of a modified antibody is confirmed by Gel electrophoresis using protein analysis in SDS-PAGE under reducing or non-reducing conditions or any other technique known in the art. ELIS can also be used to detect the expression of a modified antibody and the amount of said antibody.

Modified antibodies should retain adequate antigen binding compared to unmodified antibodies. Accordingly, the appropriate antibody-antigen binding is analyzed by methods known in the art of immunology, such as ELISA.

Additional experiments can be performed to confirm that the modified antibodies have structural properties similar to those of the unmodified antibodies. These experiments include, among others, SDS-PAGE, SEC, ELISA and protein A binding assay. Protein A binding assay is preferred, since protein A binds to the same region of the CH2-CH3 binding that the FcRn, although the union implies different residues.

Modified antibodies prepared from host cells can be purified using techniques known in the art, including but not limited to gel filtration and column chromatography (eg, affinity chromatography by protein A, cation exchange chromatography , anion exchange chromatography and gel filtration). The minimum acceptable purity of the antibody for use in pharmaceutical formulation will be 90%, 95% being preferred, 98% more preferred and 99% or more preferred.

The binding affinities of the antibodies produced for FcRn can be detected by performing a competitive binding assay at pH 6.0, the optimal condition for binding to FcRn. Binding affinities can be analyzed by immobilizing FcRn on a solid substrate such as Sepharose® beads. Alternatively, binding affinities can be evaluated using an ELISA. Preferably, the present invention investigates binding affinities by performing a competitive binding assay in a cell-based system. A series of dilutions of a modified antibody produced and the unmodified antibody are compared according to the binding to the FcRn expressed in a cell line, preferably an NS0 cell line. Experimental procedures for conducting a competitive binding assay are described in detail in the Examples.

Experiments in the present invention show that similar binding affinity results can be achieved with purified antibodies or culture supernatants of antibody producing cells. Accordingly, supernatants can be used directly to analyze the FcRn binding affinities of the antibodies produced in order to confirm that the desired alteration of binding affinities has been achieved. After such confirmation, the antibody produced is subjected to more complex purification procedures.

Direct binding assays should also be performed to confirm that the modified antibodies bind to the FcRn in a pH dependent manner. In particular, the binding affinity of FcRn modified antibodies is analyzed at pH 6.0 and at pH 8.0 (see more details in the Examples). In general, the binding affinity at pH 6.0 should be higher than that obtained at pH 8.0.

Biological stability (or serum half-life) can be measured by various means in vitro or in vivo, by, for example, the use of a radiolabeled protein and measuring serum radioactivity levels as a function of time, or by analysis of the levels of intact antibody (of known specificity) present in the serum using ELISA as a function of time, in which a particularly preferred measure of the increase in biological stability is evidenced by an increase in serum half-life and decrease in clearance rates.

The present disclosure provides polynucleotide molecules encoding modified antibodies or polynucleotide molecules encoding a modified partial or entire heavy chain of modified antibodies, such as constant regions, Fc regions, or CH2-CH3 regions, with the mutations described in This memory.

The present disclosure provides vectors comprising polynucleotide molecules that encode modified antibodies or polynucleotide molecules that encode partial or whole modified heavy chains of modified antibodies, such as constant regions, Fc regions, or CH2-CH3 regions, with the mutations (substitutions) described herein.

The present disclosure includes a host cell containing said vectors comprising said nucleic acid molecules, as described herein. Suitable host cells for the expression of the modified antibodies described herein are derived from prokaryotic organisms, such as Escherichia coli, or eukaryotic multicellular organisms, including yeasts, plants, insects and mammals.

E. coli is a prokaryotic host particularly useful for cloning and / or expression of the AND sequences of the present invention. Other suitable microbial hosts for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various species of Pseudomonas. In these prokaryotic hosts, expression vectors can also be made that typically contain expression control sequences compatible with the host cell (eg, an origin of replication). In addition, there may be any number of various well known promoters, such as the lactose promoter system, or tryptophan promoter system (trp), a beta-lactamase promoter system or a lambda phage promoter system. Typically, promoters control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, to initiate and complete transcription and translation.

Other microbes, such as yeasts, can also be used for expression. Saccharomyces is a preferred host, and the vectors have expression control sequences such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.

Plants and plant cell cultures can be used for the expression of the DNA sequence of the invention (Larrick and Fry, Hum. Antibodies Hybridomas 2: 172-189 (1991); Benvenuto et al., Plant Mol. BioI. 17: 865-874 (1991); During et al., Plant Mol. BioI. 15: 281-293 (1990); Hiatt et al., Nature 342: 76-78 (1989)). Preferred plant guests include, for example: Arabidopsis, Nicotiana tabacum, Nicotiana rustica and Solanum tuberosum. A preferred expression cassette for expressing polynucleotide sequences encoding the modified antibodies of the invention is plasmid pMOG18, in which the inserted polynucleotide sequence encoding the modified antibody is operably linked to the CaMV 35S promoter with a duplicated enhancer; pMOG18 is used according to the method of Sijmons et al., BiolTechnology 8: 217-221 (1990). Alternatively, a preferred embodiment for the expression of modified antibodies in plants follows the methods of Hiatt and coI., Ant., With the substitution of the polynucleotide sequences encoding the modified antibodies of the invention for immunoglobulin sequences used by Hiatt et al., Ant. Agrobacterium tumifaciens T-DNA-based vectors can also be used to express the AND sequences of the invention; preferably, said vectors include a marker gene encoding resistance to spectinomycin or another selectable marker.

Insect cell cultures can also be used to produce the modified antibodies of the invention, normally using a baculovirus-based expression system. Modified antibodies can be produced by expression of the polynucleotide sequences encoding the modified antibodies according to the methods of Putlitz et al., Bio / Technology 8: 651-654 (1990)

In addition to microorganisms and plants, mammalian cell cultures can also be used to express and produce the polypeptides of the present invention (see, Winnacker, "From Genes to Clones", VCH Publishers, New York (1987)). In reality, mammalian cells are preferred, since in the art numerous suitable host cell lines capable of secreting intact immunoglobulins have been developed in include CHO cell lines, several COS cell lines, HeLa cells, preferably myeloma cell lines etc. ,, or transformed B cells or hybridomas. Expression vectors for these cells may include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen and co., Immunol. Rev. 89: 49-68 (1986)) and necessary sites to process information, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription termination sequences. Preferred expression control sequences are promoters derived from the immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus and the like. In general, a selectable marker, such as a neo expression cassette, is included in the expression vector.

The present disclosure provides a method for manufacturing an agent with impaired affinity of FcRn binding and / or serum half-life by conjugation or, otherwise, binding of said agent to an identified moiety that has an increase or reduction in serum half-life through its interaction with FcRn. Said moiety includes, among others, a modified IgG or a modified partial or complete heavy chain comprising the amino acid substitutions described herein. Such agents would include, among others, antibodies, antibody fragments, hormones, receptor ligands, immunotoxins, therapeutic drugs of any type, T-cell receptor binding antigens and any other agent that can bind to the residues with greater half-life in serum of the present invention. To create a fusion protein with impaired stability in vivo, DNA fragments encoding said proteins can be operatively incorporated into a recombinant vector, within the framework of the constant region of a modified antibody, in the anterior or posterior direction, in a position so that the vector is capable of expressing a fusion protein comprising said protein operably linked to the constant region. Techniques for manipulating DNA segments in this way by, for example, genetic engineering using restriction endonucleases, will be known to those skilled in the art in light of the present disclosure and references such as Sambrook and Russell, ant . The above procedure has been proposed for use in the generation of a series of therapeutic compounds with better biological stability. Such compounds include, for example, interleukin 2. insulin, interleukin 4 and interferon gamma, or even T-cell receptors. The use of the recombinant Fc domains of the present invention is also contemplated also in the stabilization of a wide range of drugs, that would probably ease the need to repeat the administration. However, the present methods are not limited solely to the production of proteins for human administration and can be used to produce large amounts of any protein with greater stability, so that they can be used in, for example, immunization protocols, in treatment of animals by veterinarians or in rodent therapy models in vivo.

III. Uses of modified antibodies with impaired FcRn binding affinity and / or serum half-lives

The present disclosure provides a composition comprising the modified antibodies described herein and a pharmaceutically acceptable carrier. Compositions for parenteral administration typically comprise a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers can be used, for example, water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate material. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, chloride calcium, sodium lactate. The concentration of the antibodies in these formulations can vary widely, that is from less than about 0.01%, usually at least about 0.1%, to 5% by weight, and are mainly selected on the basis of fluid volumes and viscosities according to the particular mode of administration selected.

A typical composition for intravenous infusion can be prepared to contain 250 ml of sterile Ringer's solution and 10 mg to 100 mg of the antibody (see, "Remington's Pharmaceutical Science", 15th ed., Mack Publishing Company, Easton, PA ( 1980)).

The modified antibodies and the fusion protein of the present invention can be used for various non-therapeutic purposes. They can be used as an affinity purification agent. They may also be useful in diagnostic assays, such as the detection of the expression of an antigen of interest in specific cells, tissues or serum. For diagnostic applications, antibodies will normally be labeled with a detectable moiety, including radioisotopes, fluorescent markers and various enzyme substrate markers. Antibodies can also be used in any known assay procedure, such as competitive binding assays, direct and indirect sandwich assays and immunoprecipitation assays. Antibodies can also be used for diagnostic tests in vivo. In general, the antibodies are labeled with a radionuclide so that the antigen or the cell that expresses it can be localized using immunocentelleo.

Also kit can be supplied for use with the modified antibodies in the protection against, or the detection of, a cellular activity or the presence of a selected cell surface receptor or the diagnosis of disease. Therefore, the subject composition of the present invention can be provided, usually in lyophilized form in a container, either alone or together with additional antibodies specific to the desired cell type. Modified antibodies, which may be conjugated to a label or toxin, or unconjugated, are included in the kit with buffers such as Tris, phosphate, carbonate etc., stabilizers, biocides, inert proteins, for example serum albumin, or the like, and a group of instructions for use. In general, these materials will be present in less than about 5% by weight based on the amount of active antibody and will normally be present in a total amount of at least about 0.001% by weight based, again, on the antibody concentration.

Frequently, it will be desirable to include an inert expander or excipient to dilute the active ingredients, in which the excipient may be present in about 1 to 99% by weight of the total composition. When a second antibody capable of binding to the modified antibody is used in an assay, it will normally be present in a separate vial. Normally, the second antibody is conjugated to a label and is formulated analogously to the antibody combinations described above.

Modified antibodies have several therapeutic applications. The modified antibodies can be used to treat a patient who suffers, or is predisposed to suffer, a disease or disorder, which could benefit from the administration of the modified antibodies. Conditions that can be treated with antibodies include cancer, inflammatory conditions, such as asthma, autoimmune diseases, and viral infections etc.

The types of cancer that can be treated with the antibodies described herein include, but are not limited to, breast cancer, squamous cell cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, cancer of pancreas, glioblastoma, cervical cancer, ovarian cancer, urinary bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer , liver carcinoma and various types of head and neck cancer.

Autoimmune diseases include, among others, Addison's disease, autoimmune diseases of the ear, autoimmune diseases of the eye, such as uveitis, autoimmune hepatitis, Crohn's disease, diabetes (type I), epididymits, glomerulonephritis, Graves' disease, syndrome of Guillain-Barre, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis and ulcerative colitis.

The antibodies modified with lower serum half-lives of the present invention can be used in the treatment of diseases or disorders in which destruction or removal of tissue or foreign microorganisms is desired. For example, the antibody can be used to treat cancer, inflammatory diseases, infections and other conditions in which tissue removal is desired. Generally, the antibody would be useful in that faster biological clearance times would result in a reduction in the immunogenicity of any antibody administered. Other applications would include antibody-based imaging regimens, antibody-based drug elimination.

or creation of immunotoxins with a shorter life.

The antibodies modified with higher serum half-lives may be an anti-tissue factor (TF) antibody, anti-IgE antibody and an anti-integrin antibody. The desired mechanism of action may be to block ligand-receptor binding pairs. The antibodies modified with higher serum half-lives may also be agonist antibodies. Antibodies can also be used as therapeutic agents such as vaccines. The dosage and frequency of immunization of said vaccines will be reduced due to the extent of serum half-lives of the antibodies.

The compositions comprising the present antibodies are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal administration and, if desired, for immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are suitably administered by pulse infusion, particularly with decreasing doses of antibodies. Compositions containing the present antibodies or a cocktail thereof can be administered for prophylactic and / or therapeutic treatments. In therapeutic application, the compositions are administered to a patient already affected by the specific disease, in an amount sufficient to cure, or at least partially stop, the condition and its complications. An adequate amount to achieve this is defined as a "therapeutically effective dose." The amounts effective for this use will depend on the severity of the condition and the general condition of the patient's own immune system but, in general, vary from about 0.01 to about 100 mg of dose modified antibody, the doses being from 1 to 10 mg per most used patient.

In prophylactic applications, compositions containing the modified antibodies or a cocktail thereof are administered to a patient who is not yet suffering from the disease to enhance the patient's resistance. This amount is defined as a "prophylactically effective dose." In this use, the precise amounts depend, again, on the patient's state of exit and the general level of immunity, but in general they vary from 0.1 to 100 mg per dose, especially doses from 1 to 10 mg per patient.

Single or multiple administrations of the compositions can be carried out, from which the levels and dose pattern are selected by the treating physician. In any case, the pharmaceutical formulations will provide an amount of the mutant antibodies of the present invention sufficient to treat the patient effectively.

The following examples are offered by way of illustration and not as a limitation.

EXAMPLES

Example 1

This example describes the antibody expression vectors used in the present invention.

The components of the heavy chain expression plasmid pVAg2M3-OST577, a derivative of the M3 variant of pVg2.D.Tt (Cole et al., J. Immunol. 159: 3613-3621 (1997)) are the following. As shown in Figure 5A, starting clockwise from the EcoRI site, the heavy chain unit begins with the immediate early promoter (IE) of the human cytomegalovirus (hCMV) and the Boshart enhancer and coI. , Cell 41: 521-530 (1985)) as an EcoRI-XbaI fragment. Behind the hCMV region is the

OST577 VH region as an XbaI fragment, including the signal sequence, the J segment and the donor's splicing sequence. The VH region is followed by a modified genomic DNA fragment that contains the constant region of the gamma-2M3 heavy chain

human (Cole et al., op. cit.) as Xbal-BamHI fragment, including exons CH1, hinge (H), CH2, and CH3 with intermediate introns, preceding part of the intron to CH1, and a polyadenylation signal ( polyA) for mRNA processing after CH3, followed by the transcription terminator of the human complement gene C2 (Ashfield et al., EM BO J. 10: 41974207 (1991)) as a BamHI-EcoRI fragment. The heavy chain unit is followed by a ge encoding a mutant form of dihydrofolate reductase (dhfr), together with regulatory elements (enhancer, promoter, splicing signals and POlyA signal) of simian virus 40 (SV40 ) necessary for transcription. This region, which was taken as the BamHI-EcoRI fragment of plasmid pVg1 (Co and coI., Op. Cit.), Was modified by converting the BamHI site into an EcoRI site. Against the clockwise inside this unit from the original EcoRI site, there is first part of the plasmid pBR322 (Sutcliffe, Cold Spring Harbor Symp. Quant. BioI. 43: 77-90 (1979)) that comprises the origin of bacterial replication and the ampicillin resistance gene for selection in E.coli, except that the origin of bacterial replication was replaced by the corresponding segment of pUC 18 (Yanisch-Perron et al. Gene 33: 103-119 (1985) ) to increase the number of copies of the vector in bacterial hosts. Below is a segment of SV40 (Reddy et al., Science 200: 494-502 (1978)) that contains the SV40 enhancer and early promoter to ensure the strong onset of transcription. This segment is followed by the dosing sequence of the E.coli dhfr gene (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499 (1983)). After the dhfr gene, there is an SV40 segment that contains the small intron of the t antigen, which is believed to increase mRNA levels, and then the plasmid contains another SV40 segment that contains a polyA signal to terminate the mRNA transcript.

The components of the heavy chain expression plasmid pVAg1.N-OST577, a derivative of pVg1 (Co et al., Op. Cit.), Are as follows. As shown in Figure 5B, clockwise from the EcoRI site, the heavy chain unit begins with the same EcoRI-Xbal fragment that contains the hCMV IE promoter and the one used in the vector pVAg2M3-OST577, followed by the VH region of OST577 as a fragment

XbaI The VH region is followed by the genomic DNA fragment that contains the constant region of the human gamma-1 heavy chain (Ellison et al., Nucleic Acids Res 10: 4071-4079 (1982)) as an Xbal-BamHI fragment, including exons CH1, hinge (H), CH2, and CH3 with the intermediate introns, preceding part of the intron to CH1, and a polyA signal for mRNA processing after CH3. To facilitate subsequent manipulations of the non-coding regions, overlap-extension PCR mutagenesis (Higuchi, op. Cit.) Was used to create an NheI site in the intron between the hinge and CH2 exons. The heavy chain unit is followed by the same BamHI-EcoRI restriction fragment encoding the dhfr, together with regulatory elements and a portion of plasmid pBR322, which was used in the vector pVAg2M3-OST577.

The components of the light chain expression plasmid pVAλ2-OST577, a derivative of pVk (Co et al., Op. Cit.), Are as follows. As shown in Figure 6, starting clockwise from the EcoRI site, the light chain unit begins with the same EcoRI-Xbal fragment that contains the hCMV IE promoter and enhancer that were used in the heavy chain vectors, followed by the VL region of OST577 as an XbaI fragment, including a signal sequence, J segment and a donor splice sequence. The VL region is followed by a genomic DNA fragment that contains the constant region of the human lambda-1 light chain (Hieter et al., Nature 294: 536-540 (1981)) as an Xbal-Sau3AI fragment that was modified by PCR to encode the constant region of the human lambda-2 light chain (Hieter and coI., Ibid.), Including the intron of the human lambda 1 light chain, the exon of the constant region of the lambda-light chain 2 human (Cλ2) and a portion of the 3 'untranslated region of the human lambda-2 light chain and a polyA signal for mRNA processing of the human lambda-1 light chain. Following the light chain gene is a gene that encodes the xanthine guanine phosphoribosyl transferase (gpt), along with SV40 regulatory elements necessary for transcription. The function of this region, which was taken as a BamHI-EcoRI fragment of plasmid pSV2-gpt (Mulligan and Berg, op. Cit.), Is to provide a selectable drug resistance marker after transfection of the plasmid into mammalian cells. Against the clockwise inside this unit from the original EcoRI site, there is first part of plasmid pBR322 (Sutcliffe, op. Cit.) That comprises the origin of bacterial replication and the ampicillin resistance gene for selection in E.coli, except that the origin of bacterial replication was replaced by the corresponding segment of pUC 18 (Yanisch-Perron op. cit.) to increase the number of copies of the vector in bacterial hosts. Below is a segment of SV40 (Reddy and coI., Op. Cit.) Containing the enhancer and the SV40 early promoter to ensure the strong onset of transcription. This segment is followed by the coding sequence of the E. coli gpt gene (Richardson et al., Nucleic Acids Res. 11: 8809-8816 (1983)). After the gpt gene, there is an SV40 segment that contains the small intron of the t antigen, which is believed to increase mRNA levels and, then, the plasmid contains another SV40 segment that contains a polyA signal to terminate the mRNA transcript.

The components of the pVAg2M3-Hu1D10 heavy chain expression plasmid (see Figure 7A) are identical to those of pVAg2M3-OST577 from which it was derived, except that the VH region of OST577 was replaced with the VH region of Hu1D10 of the plasmid pHu1D10. lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.). The components of the heavy chain expression plasmid pVAg1.N-Hu1D10 (see Figure 7B) are identical to those of pVAgI.N-OST577 from which it was derived, except that the VH region of OST577 was replaced with the VH region of Hu1D10 of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al.

The components of the light chain expression plasmid pVk-Hu1D10, a derivative of pVk (Co et al., Op. Cit.), Are as follows. As shown in Figure 8, clockwise from the EcoRI site, the light chain unit begins with the same EcoRI-Xbal fragment that contains the hCMV IE promoter and enhancer that was used in the heavy chain vectors, followed by the VL region of Hu1D10 (Kostelny et al. (2001), op. cit.) as an Xbal fragment, including the signal sequence, the J segment and the donor's splicing sequence. The VL region is followed by a genomic DNA fragment that contains the constant region of the human kappa light chain (Hieter et al., Cell 22: 197-207 (1980)) as an Xbal-BamHI fragment, including the exon of the constant region of the human kappa light chain (Ck), preceding part of the intron to Ck, and a polyA signal for mRNA processing after Ck. The light chain unit is followed by the same BamHI-EcoRI fragment encoding gpt, together with regulatory elements necessary for transcription and a portion of plasmid pBR322, which was used in the pVk vector.

Example 2

This example describes the plasmids used in the present invention.

OST577 light and heavy chain cDNAs were cloned in their entirety by PCR in a trioma cell line expressing the human monoclonal anti-HBV OST577 antibody (Ehrlich et al., Op. Cit.). The variable regions of the heavy and light chains were converted by PCR into miniexons, faltered by XbaI sites at both ends, comprising the signal sequences, V, (D), and J segments, donor splicing sequences and a portion of the corresponding introns, as indicated in Co and co., supra. The expression vector pVAg2M3-OST577 (see Figure 5A), a derivative of the M3 variant of pVg2.D.Tt (Cole and coI., Op. Cit.) Was constructed, replacing the XbaI fragment containing the OKT3 miniexon -V with the OST577-V miniexon, then the Pcil fragment-

H H.

Fspl containing the origin of bacterial replication was replaced with the corresponding Pcil-Fspl fragment of pUC18 (Yanisch-Perron et al., Op. Cit.) To increase the number of copies of the vector in the bacterial hosts. The expression vector pVAg1.N-OST577 (see Figure 5B), a derivative of pVg1 (Co et aI., Op. Cit.) Was constructed, inserting an XbaI fragment containing the OST577-VH miniexon into the single Xbal site of pVg1, modifying the intron hinge-CH2 by overlap-extension PCR (Higuchi, op. cit.) to create a unique NheI site, and replacing the Hindlll-Xhol fragment that contains the origin of bacterial replication with the corresponding Hindlll fragment -Xhol of pVAg2M3OST577 to increase the number of copies of the vector in bacterial hosts.

The expression vector pVλ2-0ST577, a derivative of pVk (Co et aI., Op. Cit.), Was constructed by first replacing the Xbal-BamHI fragment of pVk containing the genomic constant region of human kappa with an Xbal-Bglll product of the PCR containing the genomic constant region of human lambda-1. The coding region and a 3 ’untranslated portion were replaced with the corresponding OST577 light chain cDNA fragment by PCR, which essentially gives an exon of the human lambda-2 genomic constant region. Finally, the OST577-VL miniexon was inserted into the XBaI site of the vector. The expression vector pVλ2-0ST577 (see Figure 6), a derivative of pVλ2-0ST577 was constructed by first replacing the Sapl-Fspl fragment containing the bacterial replication origin with the corresponding Sapl-Fspl fragment of pVAg2M3-OST577 to increase the number of copies of the vector in bacterial hosts.

The pVAg2M3-Hu1D10 expression vector (see Figure 7A) was constructed by replacing the Xhol-Xbal fragment of plasmid pVAg2M3-OST577, which contains the hCMV promoter and enhancer (Boshart et aI., Op. Cit.) And the region VH of OST577 with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. Cit.) Containing the hCMV promoter and enhancer and the Hu1D10 VH region. The pVAg1.N-Hu1D10 expression vector (see Figure 7B) was constructed by replacing the Xhol-Xbal fragment of plasmid pvAg1.N-OST577, which contains the hCMV promoter and enhancer (Boshart et al., Op. Cit. ) and the VH region of OST577 with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the region Hu1D10 VH.

The pVk-Hu1D10 expression vector (see Figure 8) was constructed by replacing the Xhol-Xbal fragment of plasmid pVk (Co and coI., Op. Cit.), Which contains the hCMV promoter and enhancer (Boshart and coI. , op. cit.) with the Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the VL region of Hu1D10.

The base expression vector pDL172, a derivative of pVkrg (Cole et al., Op. Cit.), Was constructed by replacing the Xbal-Sphl fragment containing the genomic constant region of human kappa with an Xbal-Sphl fragment composed of a fragment Xbal-Nhel containing the N-terminal portion of the signal sequence of the M195 heavy chain (Co et a.,

op. cit.), a 0.7 kb Nhel-Agel fragment, a synthetic Agel-Eagl fragment encoding a human c-myc decapeptide, flanked by linker peptides, which is recognized by mouse monoclonal antibody 9E10 (Evan and coI. , Mol. Cell. BioI. 5: 3610-3616 (1985)), followed by the GPI binding signal of the human acceleration factor (Caras et al., Nature 325: 545-549 (1987)), and an Eagl fragment -Sphl containing the polyA signal of the human gamma 1 immunoglobulin gene (Ellison et al., Op. Cit.).

The human beta-2 (β2m) and extracellular microglobulin domains of the FcRn human neonatal Fc receptor alpha chain gene were cloned by PCR from a cDNA library prepared with peripheral blood mononuclear cells. The human FcRn alpha chain gene was modified by PCR to add a flanking NeheI site and the C-terminal portion of the M195 heavy chain signal sequence (Co et aI., Op. Cit.) At end 5 'and a flanking AgeI site at the 3' end and was used to replace the Nhel-Agelf fragment of pDL172, which results in the expression vector pDL172 + HuFcRn. The human β2m gene was modified by PCR to add flanking Xbal and Sall sites at the 5 'and 3' ends, respectively, and to remove an internal EcoRI site. The resulting Xbal-Sall fragment was subcloned into an intermediate vector, flanked at its 5 'end by an EcoRI-Xbal fragment containing the IE promoter and the hCMV enhancer (Boshart and coI., Op. Cit.), And, in its 3 'end, by the Sall-BamHI fragment containing the polyadenylation signal of the murine gamma-2a immunoglobulin gene (Kostelny et al. (1992), op. cit.), followed by a BamHI-EcoRI fragment containing the transcription terminator of the human complement C2 gene (Ashfield et al., op. cit.). The resulting EcoRI-EcoRI fragment containing a functional human transcriptional unit was cloned into the unique EcoRI site of pDL172 + HuFcRn, which results in the expression vector pDL172 + HuFcRn + Huβ2m, hereinafter referred to as pDL208 (see Figure 9A ). The rhesus and extracellular β2m domains of the rhesus FcRn alpha chain were cloned by PCR from a cDNA library prepared from rhesus peripheral blood mononuclear cells. The rhesus β2m gene was modified by PCR to add flanking Xbal and Sall sites at the 5 'and 3' ends, respectively, and to remove an internal EcoRI site. The resulting Xbal-Sall fragment was subcloned into an intermediate vector, flanked at its 5 'end by an EcoRI-Xbal fragment containing the IE promoter and the hCMV enhancer (Boshart and coI., Op. Cit.), And, in its 3 'end, by the Sall-BamHI fragment containing the polyadenylation signal of the murine gamma-2a immunoglobulin gene (Kostelny et al. (1992), op. cit.), followed by a BamHI-EcoRI fragment containing the transcription terminator of the human complement C2 gene (Ashfield et al., op. cit.). The resulting EcoRI-EcoRI fragment containing a functional transcriptional unit of β2m of rhesus was used to replace the EcoRI-EcoRI fragment (containing the transcriptional unit of human β2m) of pDL172 + HuFcRn + Huβ2m, resulting in pDL172 + HuFcRn + Rhβ2m. The rhesus FcRn alpha chain gene was modified by PCR to add a flanking NheI site and the C-terminal portion of the M195 heavy chain signal sequence (Co et aI., Op. Cit.) At the end 5 'and a flanking ageI site at the 3' end and was used to replace the Nhel-Agel fragment (which contains the human FcRn alpha chain gene) of pDL172 + HuFcRn + Rhβ2m, which results in the vector of expression pDL172 + RhFcRn + Rhβ2m, hereinafter referred to as Pdl410 (see Figure 9B).

Example 3

This example describes the mutagenesis of the Fc region of the human γ2M3 heavy chain gene

Molecular modeling:

An initial model of the human Fc / FcRn complex was generated based on a low resolution crystal structure of the rat Fc / FcRn complex (Burmeister et al., Nature 372: 379-383 (1994); RCSB protein databank code 1FRT ). First, the rat β2m of the complex was replaced by superposition of human β2m taken from a high resolution crystal structure of the human histocompatibility HLA-A1 antigen (Saper et al. J. Mol. BioI. 219: 277- 319 (1991); RCSB code 3HLA) in the same orientation as that of the rat in the complex. Next, the rat FcRn alpha chain was replaced with superposition of the human alpha chain taken from a high resolution crystal structure of the human FcRn West and Bjorkman, Biochemistry 29: 9698-9708 (2000); RCSB code 1EXU) in the same orientation as the rat alpha chain in the complex. Then, the rat residues in the Fc of the complex were replaced with the corresponding Fc residues of the human IgG1 (Kabat et al., Op. Cit.) And energy minimization calculations were performed using the SEGMOD and ENCAD programs (Levitt , J. Mol. BioI. 226: 507-533 (1992); Levitt, J. Mol. BioI. 168: 595-620 (1983)) to produce an Fc / FcRn complex model of human IgG1. Finally, the Fc residues of the IgG1 of the model were replaced with the corresponding Fc residues of the IgG2M3 Cole et aI., Op. cit.) and the energy minimization calculations were repeated to produce an Fc / FcRn complex model of human IgG2M3, hereinafter referred to as model 1.

A second model of the human IgG2M3 Fc / FcRn complex was generated as described above, based on a model of the rat Fc / FcRn complex (Weng et al., J. Mol. BioI. 282: 217-225 (1998); RCSB code 2FRT), hereinafter referred to as model 2.

A third model was generated as described above, based on a high resolution crystal structure of a heterodimeric rat fc / FcRn complex (Martin et al., Mol. Cell 7: 867-877 ( 2001); RCSB code 1/1 A), hereinafter referred to as model 3.

Mutagenesis:

The polymerase chain reaction (PCR) procedure with overlap-extension (Higuchi, op. Cit.) Was used to generate random amino acid substitutions at positions 250, 314 and 428 of the OST577-lgG2M3 heavy chain ( numbered according to the EU index of Kabat and coI., op. cit.). To generate random mutants at position 250, mutagenesis primers JY24 (5 'GAC CTC AGG GGT CCG GGA GAT CAT GAG MNN GTC GG -3') (SEQ ID No. 77) and JY25 (5 '-CTC ATG ATC) were used TCC CGG ACCCCT GAGGTC-3 ') (SECIDN ° 78), in M = AoC, and N = A, C, GoT. The first overlap-extension PCR cycle used the external primer msc g2-1 (5' -CCA GCT CTG TCC CAC ACC G -3 ') (SEQ ID No. 79) and JY24 for the left fragment and the external primer kco8 (5'-GCC AGG ATC CGA CCC ACT -3') (SEQ ID No. 80) and JY25 for the right fragment. The PCR reaction was performed using the Expand ™ High Fidelity PCR System (Roche Diagnostics Corporation, Indianapolis, IN), following the manufacturer's recommendations, by incubation at 94 ° C for 5 minutes, followed by 25 cycles of 94 ° C for 5 seconds , 55 ° C for 5 seconds and 72 ° C for 60 seconds, followed by incubation at 72 ° C for 7 minutes in a GeneAmp® PCR System 9600 (Applied BiosystemS®, Foster City, CA). The PCR products were passed through a low melting agarose gel, cleaved from the gel and melted at 70 ° C. The second PCR cycle to combine the left and right fragments was performed as described above, using the external primers msc g2-1 and kco8 for 35 cycles. The final PCR products were passed through a low melting agarose gel and the DNA fragments of the expected size were cleaved and purified using the QIAEXTM II Gel Extraction Kit (QIAGEN®, Valencia, CA). The purified fragments were digested with PinAl and BamHI, gel purified as described above and cloned between the corresponding sites in pVAg2M3-OST577.

To generate the T2501 and T250L mutants, the mutagenesis primers KH4 (5'-GAC CTC AGG GGT CCG GGA GAT CAT GAG AAK GTC GG -3 ') (SEQ ID No. 81) and KH3 (5'-CTC ATG ATC) were used TCC CGG ACC CCT GAG GTC -3 ') (SEQ ID No. 82), in which K = G

or T. To generate the T250C and T250G mutants, mutagenesis primers KH5 (5'-GAC CTC AGG GGT CCG GGA GAT CAT GAG GCM GTC GG -3 ') (SEQ ID No. 83) and KH3, in which M = A or C. To generate mutants T250N and T250Q, mutants KH6 (5'-GAC CTC AGG GGT CCG GGA GAT CAT GAG NTK GTC GG -3 ') (SEQ ID No. 84) and KH3 were used in those that K = G or T, and N = A, C, GoT. The first PCR cycle used the external primer msc g2-1 and KH4, KH5 or KH6 for the left fragment and the external primer MGD-1 (5'-GCC AGG ATC CGA CCC ACT -3 ') (SEQ ID No. 85) and KH3 for the right fragment. PCR reactions were performed using the Expand ™ High Fidelity PCR System (Roche Diagnostics Corporation) by incubation at 94 ° C for 5 minutes, followed by 25 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 60 seconds, followed by incubation at 72 ° C for 7 minutes. The PCR products were passed through a low melting agarose gel, cleaved from the gel and melted at 70 ° C. The second PCR cycle to combine the left and right fragments was performed as described above, using the external primers msc g2-1 and MGD-1, by incubation at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 60 seconds, followed by incubation at 72 ° C for 7 minutes. The final PCR products were passed through a low melting agarose gel and the DNA fragments of the expected size were cleaved and purified using the QIAquick ™ Gel Extraction Kit (QIAGEN®). The purified fragments were digested with PinAl and BamHI, gel purified as described above and cloned between the corresponding sites in pVAg2M3OST577.

To generate random mutants at position 314, the mutagenesis primers kc078 (5 '-ACC GTG CAC CAG GAC TGG NNK AAC GGC AAG GAG -3') (SEQ ID No. 86) and kc079 (5'-CCA GTC CTG) were used GTG CAC AAC GG -3 ') (SEQ ID No. 87), in which K = G

or T, and N = A, C, G or T. The first PCR cycle used the external primer ks g2-5 (5 '-CTC CCG GAC CCC TGA GGT C -3') (SEQ ID No. 88) and kc079 for the left fragment and the external primer kco8 and kc078 for the right fragment. All subsequent steps were performed as described above for random mutagenesis at position 250, except that the second PCR cycle used external primers ks g2-5 and kco8, and the final PCR fragments were digested with Pmll and BamHI and were cloned between the corresponding sites in pVAg2M3-OST577.

To generate the L3141 mutant, the mutagenesis primers MGD-10 (5'ACC GTG CAC CAG GAC TGG ATC AAC GGC AAG GA -3 ') (SEQ ID No. 89) and kc079 were used. To generate the L314Y mutant, the mutagenesis primers MGD-11 (5'-ACC GTG CAC CAG GAC TGG TAT AAC GGC AAG GA -3 ') (SEQ ID No. 90) and kc079 were used. To generate the L314H mutant, mutagenesis primers MGD-12 (5'-ACC GTG CAC CAG GAC TGG CAC AAC GGC AAG GA -3 ') (SEQ ID No. 91) and kc079 were used. To generate the L314M mutant, mutagenesis primers MGD-13 (5'-ACC GTG CAC CAG GAC TGG ATG AAC GGC AAG GA -3 ') (SEQ ID No. 92) and kc079 were used. To generate the L314N mutant, the mutagenesis primers MGD-14 (5'-ACC GTG CAC CAG GAC TGG AAT AAC GGC AAG GA -3 ') (SEQ ID No. 93) and kc079 were used. The first PCR cycle used the external primer jt240 (5'-GGA CAC CTC TCC TCC C -3 ') (SEQ ID No. 94) and kc079 for the left fragment and the external primer kc041 (5'-CTA GTG GnTGT CC - 3 ') (SEQ ID No. 95) and MGD-10, MGD-11, MGD-12, MGD-13 or MGD14 for the right fragment. PCR reactions were performed using the Expand ™ High Fidelity PCR System (Roche Diagnostics Corporation) by incubation at 94 ° C for 5 minutes, followed by 25 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 60 seconds, followed by incubation at 72 ° C for 7 minutes. The PCR products were passed through a low melting agarose gel, cleaved from the gel and melted at 70 ° C. The second PCR cycle to combine the left and right fragments was performed as described above, using the external primers jt240 and kc041, by incubation at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 60 seconds, followed by incubation at 72 ° C for 7 minutes. The final PCR products were passed through a low melting agarose gel and the DNA fragments of the expected size were cleaved and purified using the QIAquick ™ Gel Extraction Kit (QIAGEN®). The purified fragments were subcloned into pCR®4Blunt-TOPO® (Invitrogen ™, Carlsbad, CA), then digested with Pmll and BamHI, gel purified as described above and cloned between the corresponding sites in pVAg2M3 -OST577.

To generate random mutants at position 428, mutagenesis primers JY22 (5'-GAA CGT CTC ATG CTC CGT GNN KCA TGA GGC TCT G -3 ') (SEQ ID No. 96) and JY23 (5'-CAC GGA) were used GCA TGA GM GAC C-3 ') (SEQ ID No. 97), in which K = G or T, and N = A, C, G or T. The first PCR cycle used the external primer ks g2-5 and JY23 for the left fragment and the external primer kco8 and JY22 for the right fragment. All subsequent steps were performed as described above for random mutagenesis at position 314.

To generate random mutants at position 428, the mutagenesis primers MGD-2 (5'-GTG TAG TGG TGC AGA GCC TCA TGM NNC ACG GAG CAT GAG AAG -3 ') were subsequently used (SEQ ID No. 98) and KH1 ( 5'-CAT GAG GCT CTG CAC AAC CAC TAC AC -3 ') (SEQ ID No. 99), in which M = A or C, and N = A, C, G or T. The first PCR cycle used the external primer msc g2-1 and MGD-2 for the left fragment and the external primer MGD-1 and KH1 for the right fragment. The PCR reaction was performed using the Expand ™ High Fidelity PCR System (Roche Diagnostics Corporation) by incubation at 94 ° C for 5 minutes, followed by 25 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 90 seconds, followed by incubation at 72 ° C for 7 minutes. The PCR products were passed through a low melting agarose gel, cleaved from the gel and melted at 70 ° C. The second PCR cycle to combine the left and right fragments was performed as described above, using the external primers msc g2-1 and MGD-1, by incubation at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 5 seconds, 60 ° C for 5 seconds and 72 ° C for 75 seconds, followed by incubation at 72 ° C for 7 minutes. The final PCR products were passed through a low melting agarose gel and the DNA fragments of the expected size were cleaved and purified using the QIAquick ™ Gel Extraction Kit (QIAGEN®). The purified fragments were digested with PinAl and BamHI, gel purified as described above and cloned between the corresponding sites in pVAg2M3-OST577.

To generate the M428E mutant, the mutagenesis primers MGD-8 (5'GTG TAG TGG TGC AGA GCC TCA TGT TCC ACG GAG CAT GAG AAG -3 ') (SEQ ID No. 100) and KH1 were used. The first PCR cycle used the external primer msc g2-1 and MGD-8 for the left fragment and the external primer MGD-1 and KH1 for the right fragment. All subsequent steps were performed as described above for random mutagenesis at position 428.

The double mutant T250E / M428F was generated by replacing the Pmll-BamHI fragment in plasmid pVAg2M3-OST577 containing the T250E mutation with the corresponding Pmll-BamHI fragment of plasmid pVAg2M3-OST577 containing the M428F mutation. The double mutants T250Q / M428F and T250Q / M428L were generated by replacing the Pmll-BamHI fragment in plasmid pVAg2M3-OST577 containing the T250Q mutation with the corresponding Pmll-BamHI fragment of plasmids pVAg2M3-OST577 containing M4 mutations and M4428 respectively.

Several amino acid substitutions were also created at positions 250 and 428 of the Hu1D10-lgG2M3 heavy chain. To generate the M428L mutant, the Xhol-Xbal fragment of plasmid pVAg2M3-OST577 (M428L), which contains the hCMV promoter and enhancer (Boshart et al., Op. Cit.) And the VH region of OST577 was replaced with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the Hu1D10 VH region. To generate the T250Q / M428L mutant, the Xhol-Xbal fragment of plasmid pVAg2M3-OST577 (T250Q / M428L), which contains the hCMV promoter and enhancer (Boshart et aI., Op. Cit.) And the VH region of OST577 it was replaced with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the Hu1D10 VH region.

Plasmid DNA was prepared using the QIAprepTM Spin Miniprep Kit (QIAGEN®) and nucleotide substitutions were identified by sequencing. Large-scale plasmid DNA preparations were made using the EndoFree® Plasmid Maxi Kit (QIAGEN®). The coding regions of the OST577-lgG2M3 expression plasmids were verified by nucleotide sequencing.

Results:

In order to assimilate human IgG mutants with greater or lesser affinity for the neonatal Fc receptor (FcRn), which would be expected to have serum half-lives altered, random amino acid substitutions were generated at positions 250, 314 and 428 of the γ2M3 heavy chain (numbered according to the Kabat EU index and coI., op. cit.). These three positions were chosen on the basis of ordering modeling of a complex of the human IgG2M3 Fc and the human FcRn (see models 1, 2 and 3, which have been described above in the Example), which was deduced from of the X-ray crystal structure of the rat Fc / FcRn complex (Burmeister et al., op. cit.). Although wild amino acids at positions 250, 314 and 428 are located near the Fc / FcRn interface, these residues do not appear to contribute directly to the pH-dependent interaction between Fc and FcRn. Therefore, amino acid substitutions at these positions can increase (or decrease) the affinity of Fc for FcRn while maintaining the pH-dependent binding. Among the simple mutants generated by PCR mutagenesis, 19 mutants were observed that converted the wild T in 250 to AC, 0, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, oY; 19 mutants that converted the wild L at position 314 into A, C, 0, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; 19 mutants that converted the wild M at position 428 into A, C, 0, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y (see Table 1). Some of the single mutants with the highest FcRn binding were combined to generate several double mutants, including T250E / M428F, T250Q / M428F, and 1750Q / M428L.

Example 4

This example describes the mutagenesis of the Fc region of the human γ1 heavy chain gene

Mutagenesis:

The overlap-extension PCR procedure (Higuchi, op. Cit.) Was used to generate random amino acid substitutions at positions 250, 314 and 428 of the OST577-lgG1 heavy chain (numbered according to the EU index of Kabat and coI., Op. Cit.). To generate the T250E mutant, primers for mutagenesis JX076 (5 '-AAC CCA AGG ACG AAC TCA TGA TCT CCC G -3') (SEQ ID No. 101) and JX077 (5 '-GGA GAT CAT GAG TTC GTC CTT were used GGG TG -3 ') (SEQ ID No. 102). The first overlap-extension PCR cycle used the external primer JX080 (5 '-CCT CAG CTC GGA CAC cn CTC -3') (SEQ ID No. 103) and JX077 for the left fragment and the external primer NT244 (5 '- GCC TCC CTC ATG CCA CTC A-3 ') (SEQ ID No. 104) and JX076 for the right fragment. The PCR reaction was performed using the Expand ™ High Fidelity PCR System (Roche Diagnostics Corporation) PCR system, following the manufacturer's recommendations, by incubation at 94 ° C for 5 minutes, followed by 35 cycles of 94 ° C for 20 seconds, 55 ° C for 20 seconds and 72 ° C for 90 seconds, followed by incubation at 72 ° C for 7 minutes in a GeneAmp® PCR System 9600 (Applied BiosystemS®). The PCR products were passed through a low melting agarose gel, cleaved from the gel and melted at 70 ° C. The second PCR cycle to combine the left and right fragments was performed as described above, using external primers JX080 and NT244 for 35 cycles. The final PCR products were passed through a low melting agarose gel and the DNA fragments of the expected size were cleaved and purified using the QIAquick ™ II Gel Extraction Kit (QIAGEN®). The purified fragments were digested with Nhel and Eagl, gel purified as described above and cloned between the corresponding sites in pVAg1.N-OST577.

To generate the T250D mutant, primers for mutagenesis JX087 (5 'AAC CCA AGG ACG ACC TCA TGA TCT CCC G -3') (SEQ ID No. 105) and JX088 (5 '-GGA GAT CAT GAG GTC GTC cn GGG were used TG -3 ') (SEQ ID No. 106). The first PCR cycle used the external primer JX080 and JX088 for the left fragment and the external primer NT244 and JX087 for the right fragment.

All subsequent steps were performed as described above.

To generate the M428F mutant, primers for mutagenesis JX078 (5'CTC ATG CTC CGT GnCCATGAGGC TCT GC -3) (SEQ ID No. 107) and JX079 (5 '-AGA GCC TCA TGG AAC ACG GAG CAT GAG -3' were used ) (SEQ ID No. 108). The first PCR cycle used the external primer JX080 and JX079 for the left fragment and the external primer NT244 and JX078 for the right fragment. All subsequent steps were performed as described above.

To generate the M428L mutant, primers for mutagenesis JXM428L1 (5 '-CTCATG CTC CGTGnGCATGA GGC TCT GC -3') (SEQ ID No. 109) and JXM428L2 (5 '-AGA GCC TCA TGC AAC ACG GAG CAT GAG -3 were used ') (SEQ ID No. 110). The first PCR cycle used the external primer JX080 and JXM428L2 for the left fragment and the external primer NT244 and JXM428L1 for the right fragment. All subsequent steps were performed as described above.

To generate the T250Q mutant, the primers for mutagenesis JXT250Q1 (5 '-AAC CCA AGG ACC AAC TCA TGA TCT CCC G -3' ') (SEQ ID No. 111) and JXT250Q2 (5' GGA GAT CAT GAG TTG GTC CTT) were used GGG TTT TG -3 ') (SEQ ID No. 112). The first PCR cycle used the external primer JX080 and JXT250Q2 for the left fragment and the external primer NT244 and JXT250Q1 for the right fragment. All subsequent steps were performed as described above.

To generate the double mutant T250E / M428F, primers for mutagenesis JX076 and JX077 were used to create the T250E mutation in a template containing the M428F mutation. The first PCR cycle used the external primer JX080 and JX077 for the left fragment and the external primer NT244 and JX076 for the right fragment. All subsequent steps were performed as described above.

To generate the double mutant T250Q / M428L the primers for mutagenesis JXT250Q1 and JXT250Q2 were used to create the mutation T250Q and the primers for mutagenesis JXM428L1 and JXM428L2 to create the mutation M428L. The first PCR cycle used the external primer JX080 and JXT250Q2 to create the left fragment, JXT250Q1 and JXM428L2 to create the middle fragment and the external primer NT244 and JXM428L1 to create the right fragment. All subsequent steps were performed as described above.

Several amino acid substitutions were also created at positions 250 and 428 of the Hu1D10-IgG1 heavy chain. To generate the M428L mutant, the Xhol-Xbal fragment of plasmid pVAg1.N-OST577 (M428L), which contains the hCMV promoter and enhancer (Boshart and coI., Op. Cit.) And the VH region of OST577 was replaced with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the Hu1D10 VH region. To generate the T250Q / M428L mutant, the Xhol-Xbal fragment of plasmid pVAg1.N-OST577 (T250Q / M428L), which contains the hCMV promoter and enhancer (Boshart and coI., Op. Cit.) And the VH region of OST577 was replaced with the corresponding Xhol-Xbal fragment of plasmid pHu1D10.lgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing the hCMV promoter and enhancer and the Hu1D10 VH region.

Plasmid DNA was prepared using the QIAprepTM Spin Miniprep Kit (QIAGEN®) and nucleotide substitutions were confirmed by sequencing. Large-scale plasmid DNA preparations were made using the EndoFreeTM Plasmid Maxi Kit (QIAGEN®). The coding regions of the OST577-lgG1 expression plasmids were verified by nucleotide sequencing.

Results:

In order to identify human IgG mutants with affinity alteration for the neonatal Fc receptor (FcRn), which would be expected to have serum half-lives altered, several amino acid substitutions were generated at positions 250 and 428 of the heavy chain of γ1 (numbered according to the EU index of Kabat and coI., op. cit.). These two positions were chosen based on the identification of mutations in these positions in the γ2M3 heavy chain that resulted in an increase or decrease in FcRn binding. Although wild amino acids at positions 250 and 428 are located near the Fc / FcRn interface, these residues do not appear to contribute directly to the pH-dependent interaction between Fc and FcRn. Therefore, amino acid substitutions at these positions can increase (or decrease) the affinity of Fc for FcRn while maintaining the pH-dependent binding. Both single and double mutants that exhibited increased binding in the context of the human γ2M3 heavy chain were evaluated in the human γ1 heavy chain, including the single mutants T250E, T250Q, M428F, and M428L, and the double mutants T250E / M428F and T250Q / M428L A single mutant that exhibited decreased binding in the context of the human γ2M3 heavy chain (T250D) was also evaluated in the human γ1 heavy chain.

Example 5

This example describes the characterization of IgG2M3 and IgG1 mutant antibodies

Cell culture:

The 293H human kidney cell line (Life TechnologieS®, Rockville, MD) was maintained in DMEM (BioWhittaker ™, Walkersville, MD) containing 10% fetal bovine serum (FBS) (HyClone®, Logan, UT), 0 , 1 Mm of non-essential amino acids MEM (Invitrogen ™) and 2 mM Lglutamine (Invitrogen ™), hereinafter referred to as medium 293, at 37 ° C in an incubator at 7.5% CO2. For expression and purification of monoclonal antibodies after transient transfection, 293-H cells were incubated in DMEM containing 10% FBS with low levels of IgG (HyClone®), 0.1 mM non-essential amino acid MEME and L-glutamine 2 mm, hereinafter referred to as medium 293 poor in IgG. The mouse myeloma cell line Sp2 / 0 (American Type Culture Collection, Manassus, VA) was maintained in DMEM with 10% FBS and 2 mM L-glutamine. For the purification of the monoclonal antibodies after stable transfection, the Sp2 / 0 cells were adapted to grow in Hybridoma-SFM (Life Technologies®).

Transient Transfections:

293-H cells were transiently co-transfected with the appropriate light chain plasmid and the appropriate wild plasmid or one of several mutated heavy chain plasmids, which contain a single or double substitution at position 250, 314 or

428. For small-scale transient transfections, approximately 1 x 10 6 cells per transfection were seeded in a 6-well plate in 3 ml of 293 medium and grown overnight until confluence. The next day, 2 µg of the light chain plasmid and 2 µg of the mutated wild or heavy chain plasmid were combined with 0.25 ml of HSFM. In a separate tube, 10 µl of Lipofectamine ™ 2000 reagent (Invitrogen ™) and 0.25 ml of HSFM were combined and incubated for 5 minutes at room temperature. The 0.25 ml mixture of Lipofectamine ™ 2000-HSFM was gently mixed with the 0.25 ml mixture of ADn-HSFM and incubated at room temperature for 20 minutes. The medium covering the 293-H cells was aspirated and replaced with 293 medium poor in IgG, then, dropwise, the lipofectamine-DNA complexes were added dropwise, mixed gently by rotating movements and the cells were incubated for 5 -7 days at 37 ° C in a 7.5% CO2 incubator before collecting supernatants.

For large-scale transient transfections, approximately 7 x 10 6 cells per transfection were seeded in a T-75 flask in 25 ml of 293 medium and grown overnight until confluence. The next day, 12 µg of the light chain plasmid and 12 µg of the mutated wild or heavy chain plasmid were combined with 1.5 ml of HSFM. In a separate tube, 60 µl of Lipofectamine ™ 2000 reagent and 1.5 ml of HSFM were combined and incubated for 5 minutes at room temperature. The 1.5 ml mixture of Lipofectamine ™ 2000HSFM was gently mixed with the 1.5 ml mixture of HSFM-DNA and incubated at room temperature for 20 minutes. The medium covering the 293-H cells was aspirated and replaced with 293 medium poor in IgG, then, dropwise, the lipofectamine-DNA complexes were added dropwise, mixed gently by rotating movements and the cells were incubated for 5 -7 days at 37 ° C in a 7.5% CO2 incubator before collecting supernatants.

Antibody concentration:

Supernatants from small-scale transfections were collected by centrifugation at ∼ 1,200 rpm for 5 minutes and filtered in sterility using MilleX®-GV 0.22 µm microfilters (Millipore® Corporation, Bedford, MA). Samples were concentrated approximately 6 times to 0.5 ml using Vivaspin® 6 ml concentrators (50,000 MWCO) (Vivascience® AG, Hannover, Germany) by centrifugation at 3,000 rpm. The concentrated protein samples were resuspended in 5 ml of PBS, Ph 6.0 and concentrated to a volume of 0.5 ml as described above. The ELISA procedure described below was used to measure the antibody concentration in each sample.

Stable Transfections:

Sp2 / 0 cells were stably transfected with the appropriate light chain plasmid and the appropriate wild plasmid or one of several mutated heavy chain plasmids, containing a single or double substitution at position 250 or 428.

Approximately 1 x 107 cells were washed with 10 ml of PBS and resuspended in 1 ml of PBS. Approximately 25-30 µg of the light chain plasmid and 50-60 µg of the heavy chain plasmid were linearized with Fspl and added to the cells. Cells and DNA were mixed gently and transferred to a Gene Pulser Cuvette (Bio-Rad® Laboratories, Hercules, CA) on ice. The cells were electroporated using a Gene Pulser II (Bio-Rad® Laboratories) set at 0.360 kV. 25 µF, and returned to the ice for 10-20 minutes. The cells were diluted in 40 ml of DMEM, 10% FBS, 2 mM L-glutamine and seeded in four 96-well plates at 100 µl / well. After 48 hours, 100 µl / well of 2 x selection medium with mycophenolic acid (MPA) (DMEM, 10% FBS, 1X HT Media Supplement Hybri-MaX® (Sigma®, St. Louis, MO), 300 were added µg / ml of xanthine Sigma®), 2 µg / ml of mycophenolic acid (Life Technologies®) and 2 mM L-glutamine) Supernatants from wells that apparently contained single colonies were subjected to ELISA after 10-14 days. Clones producing more antibodies were chosen for expansion and adaptation to HSFM (Life Technologies®). The adapted clones were expanded to rotating bottles in 450 ml of HSFM, gasified with 5% CO2 in air, supplemented after 2 days with 50 ml of feed medium 2 without proteins (PFFM-2) (Sauer et a., Biotechnol. Bioeng. 67: 585-597 (2000)), and grown to exhaustion.

Some of the most antibody-producing clones were also adapted to basal medium 1 without protein (PFBM-1) (Protein Design Labs ™, Inc.), expanded to 10 l rotating flasks, supplemented after 2 days with a volume of 1/10 of PFFM-2 and grown until depletion.

ELISA:

To quantify the amount of OST577 or Hu1D10 antibody present in the culture supernatants, an ELISA was performed. 4 Immulon ™ plates (DYNEX® Technologies, Inc., Chantilly, VA) were coated overnight at 4 ° C with 1.0 µg / ml of goat F (ab`) 2 anti-human IgG gamma chain antibody ( BioSource International, Camarillo, CA) or goat anti-human IgG Fcγ fragment specific antibody or AffiniPure ™ (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in 100 µl / well of 0.2M carbonate-bicarbonate buffer, pH 9.4. The next day, the plates were washed with ELISA wash buffer (EWB) (PBS, 0.1% Tween 20) and blocked with 300 µl / well of SuperBlocik® blocking buffer in TBS (Pierce Chemical Company, Rockford , IL) for 20-30 minutes at room temperature. The plates were washed with EWB and properly diluted test samples were added to each well. Purified OST577 or Hu1D10 antibodies, as appropriate, were serially diluted twice in 100 µl / well of ELISA buffer (EB) (PBS, 1% bovine serum albumin, 0.1% Tween 20) starting at 0, 2 µg / ml EB and were used as standards. The culture supernatants were initially diluted to 1:10 in 100 µl / well of EB, then serially diluted twice in 100 µl / well of EB. Plates were incubated for 1-2 hours at room temperature, then washed with EWB and 100 µl / well of HRP-conjugated goat lambda light chain antibody ((BioSource International, or Southern Biotechnology Associates) was added , Inc., Birmingham, AL) or the HRP-conjugated antibody of the goat anti-human kappa light chain (Southern Biotechnology Associates, Inc.), as appropriate, at 1.0 µg / ml in EB. for 1 hour at room temperature, the plates were washed with EWB, followed by the addition of 100 µl / well of ABTS substrate peroxidase / peroxidase solution B (Kirkegaard & Perry Laboratories, Gaithersburg, MD) The reaction was stopped with 100 µl / well of 2% oxalic acid and absorbance at 415 nm was measured using a VERSAmax ™ microtiter plate reader (Molecular Devices Corporation®, Sunnyvale, CA).

Antibody Purification:

Supernatants from transient transfection cultures were collected by centrifugation and filtered in sterility. The pH of the filtered supernatants was adjusted by adding a 1/50 volume of 1M sodium citrate, Ph 7.0. The supernatants were passed through a 1 ml HiTrap® HP protein A column (Amersham Biosciences ™ Corporation, Piscataway, NJ) which was pre-equilibrated with 20 mM sodium citrate, 150 mM NaCl, pH 7.0. The column was washed with the same buffer and the bound antibody eluted with 20 mM sodium citrate, pH 3.5. After neutralizing by adding a volume of 1/50 of 1.5M sodium citrate, pH 6.5, the combined antibody fractions were passed through a 5 ml HiTrap® Desalting column (Amersham Biosciences ™ Corporation) that was prepared equilibrated with 20 mM sodium citrate, 120 mM NaCl, pH 6.0. The flow rate was collected and the fractions with OD280> 0.1 were combined and concentrated to ∼ 0.5-1.0 mg / ml using 2 ml concentrators Vivaspin® (50,000 dalton MWCO) (Vivascience® AG). The samples were then filtered in sterility using 0.2 µm microflexers Millex®-GV (Millipore® Corporation) Concentrations of the purified antibodies were determined by UV spectroscopy measuring absorbance at 280 nm (1 mg / ml = 1.4 A280).

For small-scale purification of antibodies from stable transfections, culture supernatants were collected by centrifugation and filtered in sterility. The supernatants were passed through a 5 ml POROS® 50A protein column (Applied Biosystems®) pre-equilibrated with PBS, pH 7.4-The column was washed with the same buffer and the bound antibody eluted with 0.1 glycine mM, 0.1M NaCl pH 3.0. After neutralizing by adding a 1/20 volume of 1M Tris base, the combined fractions were exchanged with buffer in PBS, Ph, 7.4, using a PD-10 desalination column (Amersham Biosciences ™ Corporation) or by dialysis . The samples were then filtered in sterility using 0.2 µm microflexers Millex®-GV (Millipore® Corporation) Concentrations of the purified antibodies were determined by UV spectroscopy measuring absorbance at 280 nm (1 mg / ml = 1.4 A280).

For large-scale purification of antibodies from stable transfections, those collected from cell cultures were clarified by terminal filtration using Sartorius® filtration capsules (Sartorius® AG, Goettingen, Germany). The clarified collection was concentrated from about 10 to 750 ml using a Pellicon® 2 cassette (30,000 dalton MWCO) (Millipore® Corporation), then purified by protein A affinity chromatography on a rProtein A Sepharose FF column (Amersham Biosciences ™ Corporation ) using the citrate buffer system described above. The protein A eluate was concentrated in an Amicon® cell shaking apparatus using a YM30 membrane (Millipore® Corporation), then the sample was exchanged with buffer in 20 mN sodium citrate, 120 mM NaCl, pH 6.0, using a Superdex ™ 200 column (Amersham Biosciences ™ Corporation). The pH and osmolality were measured and the concentrations of the purified antibodies were determined by UV spectroscopy by measuring the absorbance at 280 nm (1 mg / ml = 1.4 A280).

SDS-PAGE:

Samples of five µg of purified antibodies were passed under reducing and non-reducing conditions on NuPAGE® Novex 4-12% Bis-Tris gels (Invitrogen ™) and stained using the SimplyBlue ™ SafeStain Kit (Invitrogen ™) kit following manufacturer's recommendations .

Results:

The mutants of Fc IgG2M3 and Fc of IgG1 were expressed as anti-HBV antibodies, which comprise the variable regions of the light and heavy chain of OST577 (Ehrlich et aI., Op. Cit.), The constant region of the light chain of the human lambda-2 (Hieter et al. (1981), op. cit.), and the constant regions of the human gamma.2 mutant heavy chain 3 (lgG2M3) (Cole et aL, op. cit.) and IgG1 (Ellison et aI., Op. Cit.), Respectively. The IgG2M3 variant contains two amino acid substitutions in the CH2 region (V234A and G237A), and shows an extremely low residual binding to human Fcγ receptors (Cole et aI., Op. Cit.). Fc mutants of IgG2M3 and Fc of IgG1 were also expressed as anti-allele antibodies of the HLA-DR β chain, comprising the variable regions of the light and heavy chain of Hu1D10 (Kostelny et al. (2001 ), op. cit.), the constant region of the light chain of the human kappa (Hieter et al. (1980), op. cit.), and the constant regions of the heavy chain of the human IgG2M3 mutant (lgG2M3) (Cole et ai, op. cit.) and IgG1 (Ellison et aI., op. cit.), respectively. As described above, the appropriate wild-type or mutant heavy chain expression vector was transiently co-transfected with the appropriate light chain expression vector in 293-H cells for expression of the OST577 or monoclonal antibodies. Hu1D10. ELIS analysis of the culture supernatants collected 5-7 days after transient transfection showed antibody expression levels that were normally 5-50 µg / ml in 25 ml of the supernatant. The OST577 or Hu1D10 antibodies were purified by affinity chromatography with protein a for a final yield of approximately 100-1000 µg. Stable expression of OST577 or Hu1D10 antibodies in Sp2 / 0 cells typically resulted in expression levels of 5-50 µg / ml determined by ELISA. Yields of approximately 50-80% of the antibody present in the culture supernatants were obtained by small-scale protein A affinity chromatography.

The purified antibodies were characterized by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. SDS-PAGE analysis under non-reducing conditions indicated that the purified antibodies had a molecular weight of approximately 150-160 kD (data not shown); the analysis under reducing conditions indicated that the purified antibodies were composed of a heavy chain with a molecular weight of approximately 50 kD and a light chain with a molecular weight of approximately 25 kD (see Figures 10A and 10B). SDS-PAGE analysis of antibodies purified from stable transfectants with Sp2 / 0 gave results similar to those observed with antibodies purified from transient transfections with 293-H.

Example 6

This example describes the competitive binding analysis of the IgG2M3 and IgG1 mutant antibodies

Cell culture:

The NS0 mouse myeloma cell line (European Collection of Animal Cell Cultures, Salisbury, Wiltshire, UK) was maintained in DMEM with 10% FBS. NS0 transfectants expressing recombinant FcRn, nest to human GPI or rhesus on the surface were maintained in selection medium with mycophenolic acid (MPA) (DMEM, 10% FBS, 1X HT Media Supplement Hybri-MaX® (Sigma®), 250 µg / ml of xanthine Sigma®), 1 µg / ml of mycophenolic acid (Life Technologies®) and 2 mM L-glutamine or selection medium x 2MPA.

Human FcRn cell line:

NS0 cells were stably transfected with pDLl208. Approximately 1x107 cells were washed once and resuspended in 1 ml of normal MDEM, transferred to a Gene Pulser ™ Cuvette (Bio-Rad® Laboratories) and incubated on ice for 10 minutes. 40 µg of plasmid pDL208 was linearized with Fspl and gently mixed with the cells on ice, then the cells were electroporated by double clicking using a Gene Pulser ™ II (Bio-Rad® Laboratories) set at 1.5 kV, 3 µF and returned to ice for 10 minutes. The cells were diluted in 20 ml of DMEM, 10% FBS, and seeded in two 96-well plates at 100 µl / well. After 48 hours the medium was replaced by means of MPA selection. NS0 transfectants resistant to mycophenolic acid from wells that apparently contained single colonies were expanded in MPA selection medium and selected after approximately 3 weeks by FACSTM. Approximately 1.5 x 105 cells / assay were incubated in 100 µl of FACS staining buffer (FSB) (PBS, 1% FBS, 0.1 NaN3) containing 10 µg / ml of anti-β2 microglobulin anti-human antibody biotinylated mouse (Chromaprobe, Inc., Aptos, CA) for 1 hour on ice. The cells were washed once with 4 ml of FSB, then incubated in 25 µl of FSB containing 20 µg / ml of streptavidin-FITC conjugate (Southern Biotechnology Associates, Inc.) for 30 minutes on dark ice. The cells were washed once with 4 ml of FSB and resuspended in 1% formaldehyde. Samples were analyzed for antibody binding to human β2m using a FACScan flow cytometer (BD® Biosciences, San Jose, CA). Several clones with the highest apparent staining were subcloned using a FACStar cell sorter (BD® Biosciences), expanded in DMEM, 10% FBS, 2 mM L-glutamine and re-analyzed using the FACSTM as described herein. foregoing. A subclone, called NSO HuFcRn (memb), clone 7-3, was used in subsequent binding assays.

Rhesus FcRn cell line:

NS0 cells were stably transfected with pDL410. Approximately 6x107 cells were transfected by electroporation as described above. Transfectants were identified by FACSTM as described above with 100 ng staining / assay of a mouse anti-human FcRn alpha chain antibody (Protein Design Labs ™, Inc.) that undergoes cross-chain reaction. rhesus FcRn alpha and is detected with goat mouse anti-kappa anti-kappa conjugated antibody (Southern Biotechnology Associates, Inc.). A cell line designated NS0 RhFcRn, clone R-3, was used in subsequent binding assays.

Single point competitive union test:

The concentrated OST577-lgG2M3 supernatants were analyzed in a single-point competitive binding assay for human FcRn binding in the NS0 HuFcRn cell line (memb), clone 7-3. Approximately 2 x 105 cells / assay were washed once in FACS binding buffer (FBB) (PBS containing 0.5% FSB, 0.1% NaN3), pH 8.0, once in FBB, Ph 6.0 and were resuspended in 120 µl of pre-mixed biotinylated OST577-lgG2M3 antibody (8.3 µg / ml) and the supernatant was concentrated (containing 8.3 µg / ml of the competing antibody) in FBB, Ph 6, 0. The cells were incubated for 1 hour on ice, washed twice in FBB, Ph 6.0, and resuspended in 25 µl of streptavidin-RPE conjugate ((BioSource International) diluted to 2.5 µg / ml in FBB, pH 6 0. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB, Ph 6.0, and resuspended in 1% formaldehyde.The samples were analyzed to determine the binding of the antibody to the FcRn by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences) .The average fluorescence in the channel (MCF) of each mutant was compared with that of the wild antibody and represented with Excel (Microsoft® Corporation, Redmond, WA).

Competitive Union Trials:

A series of dilutions of each purified OST577-lgG2M3 antibody were presented against the biotinylated HuEP5C7-IgG2M3 antibody (He et al., J. Immunol. 160: 1029-1035 (1998)) to determine FcRn binding in the NS0 cell line HuFcRn (memb,) clone 7-3. For the initial detection experiments, approximately 2 x 105 cells / assay were washed once in FSB, pH 6.0, and resuspended in 100 µl of the premixed biotinylated HuEP5C7lgG2M3 antibody (10 µg / ml) and the competitor antibody OST577-lgG2M3 (serial dilutions per two from 208 µg / ml to 0.102 µg / ml) in FSB, pH 6.0. The cells were incubated with the antibody mixture for 1 hour on ice, washed twice in FSB, pH 6.0, and resuspended in 25 µl of streptavidin-RPE conjugate ((BioSource International) diluted to 2.5 µg / ML in FSB, pH 6.0 After incubation for 30 minutes on dark ice, the cells were washed twice in FSB, pH 6.0, and resuspended in 1% formaldehyde.The samples were analyzed for binding. of the antibody to FcRn by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences) .The average channel fluorescence (MCF) was plotted against the competitor concentration and IC50 values were calculated using GraphPad Prism® (GraphPad ™ Software, Inc., San Diego, GA) For consistency, the IC50 values shown in the Tables are based on the competitor's final concentrations.

The following competitive binding experiments were performed as described above, except that the cells were washed once in FBB, Ph 8.0, and once in FBB, Ph 6.0, then resuspended in 100 µl of pre-mixed biotinylated HuEP5G7-lgG2M3 antibody (10 100 µg / ml) and the competing antibody OST577-lgG2M3 (serial dilutions by two of 208 µg / ml to 0.102 µg / ml) in FBB, pH 6.0. All subsequent incubations and washes were performed using FBB, Ph 6.0, as described above. A group of experiments was performed on 120 µl of premixed biotinylated OST577-lgG2M3 antibody (8.3 µg / ml) and the competing antibody OST577-lgG2M3 (serial dilutions by two from 208 µg / ml to 0.102 µg / ml) in FBB , pH 6.0, as described above. Another group of experiments was performed on 200 µl of premixed biotinylated OST577-lgG1 antibody (5.0 µg / ml) and the competing antibody OST577-lgG1 (serial dilutions by two starting from 125 µg / ml or serial dilutions by three starting from 250 µg / ml) in FBB, pH 6.0, as described above. A further group of experiments was performed on 200 µl of pre-mixed biotinylated OST577-lgG1 antibody (5.0 µg / ml of the competitor OST577-lgG1 antibody (serial dilutions by three starting from 750 µg / ml) in FBB, pH 6 , 0, as described above.

A series of dilutions of each purified OST577-lgG2M3 antibody were presented against biotinylated OST577-lgG2M3 antibody to determine rhesus FcRn binding in the NS0 RhFcRn cell line, clone R-3. In a group of experiments, approximately 2 x

5

10 cells / assay were washed once in FBB, Ph 8.0, and once in FBB, Ph 6.0, then resuspended in 120 µl of pre-mixed biotinylated OST577-lgG2M3 antibody (8.3 µg / ml of competitor antibody OST577-lgG2M3 (serial dilutions by two starting from 208 µg / ml to 0.102 µg / ml) in FBB, pH 6.0 The cells were incubated with the antibody mixture for 1 hour on ice, washed twice in FBB, pH 6.0, and resuspended in 25 µl of streptavidin-RPE conjugate ((BioSource International) diluted to 2.5 µg / ml in FBB, pH 6.0. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB, Ph 6.0, and resuspended in 1% formaldehyde.The samples were analyzed to determine the binding of the antibody to the FcRn by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences). More group experiments were performed on 200 µl of pre-mixed biotinylated OST577-lgG1 antibody (5.0 µg / ml of competing antibody idor OST577-lgG1 (serial dilutions by three starting from 500 µg / ml) in FBB, pH 6.0, as described above.

Results:

The relative binding of wild OST577-lgG2M3 or OST577-lgG1 antibodies and their various mutants to CRN was determined using a transfected NS0 cell line that stably expresses human FcRn on its surface. As described above, concentrated supernatants were analyzed to determine human FcRn binding according to a single-point competitive binding assay and purified antibodies were analyzed to determine FcRn binding in binding assays. competitive Increasing concentrations of unlabeled competing antibodies were incubated with cells in the presence of a sub-concentration of IgG2M3 or IgG1 antibody labeled in FSB or FBB, pH 6.0.

The results of typical experiments with concentrated supernatants of OST577lgG2M3 are shown in Figures 11A, 11B, and 11C. As Figure 11A is shown, some of the mutants at position 250 (eg T250E, T250Q) were stronger competitors than wild ones, suggesting that these mutants have greater binding to human FcRn compared to the antibody. wild. Other mutants in this position (e.g., T250D, T250F, T250K, T250N, T250P, T250R, T250W, T250Y) were weaker competitors than the wild, suggesting that these mutants have less binding to human FcRn compared to the wild antibody. As shown in Figure 11B, none of the mutants at position 314 were a stronger competitor than the wild, suggesting that none of these mutants have greater binding to FcRn compared to the wild antibody. Most mutants in this position (eg L314A, L314G, L314D, L314E, L314F, L314G, L314H, L314K, L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314V, L314V, L314V, L314V, L314V, L314V weaker competitors than the wild, suggesting that these mutants have less binding to human FcRn compared to the wild antibody. As shown in Figure 11C, some of the mutants at position 428 (eg M428F, M428L) were stronger competitors than the wild, suggesting that these mutants have greater binding to FcRn compared to the wild antibody. Other mutants in this position

(e.g., M428A, M428G, M428D, M428E, M428G, M428H, M428K, M428N, M428P, M428Q, M428R, M428S, M428T, M428V, M428Y) were weaker competitors than the wild, which suggests that these mutants they have less binding to human FcRn compared to the wild antibody. [

Table 2 summarizes the IC50 values (the amount of competing antibody needed to inhibit binding of the labeled antibody to FcRn by 50%) of the purified wild OST577lgG2M3 antibody and some of its mutants for binding to human FcRn. Relative binding values were calculated in the form of the ratio between the IC50 value of the wild OST577-lgG2M3 antibody and that of each of the mutants. At amino acid position 314, none of the purified mutants showed an increase in human FcRn binding to the wild antibody. In fact, the four mutants purified at position 314 showed less binding than the wild antibody. However, at amino acid position 250, one of the mutants (T250E) showed approximately 3 times better binding to the FcRn than the wild antibody. Several mutants at position 250 showed a slightly smaller binding to the wild antibody and a mutant ( T250D) showed a substantially reduced binding to human FcRn relative to the wild antibody. At amino acid position 428, one of the mutants (M428F) also showed approximately 3 times better binding to human FcRn than the wild antibody, while another mutant (M428G) showed substantially reduced binding to human FcRn relative to the wild antibody.

Since two amino acid substitutions were identified in two different positions, namely T250E, T250Q, M428F and M428L, each with an increase in human FcRn binding, the double mutants T250E / M428F, T250Q / M428F, and T250Q were constructed / M428L, transiently transfected into 293-H cells, purified and binding to human FcRn was analyzed. As described above, increasing concentrations of unlabeled competing antibodies were incubated with cells expressing the FcRn in the presence of a sub-concentration of HuEP5C7-lgG2M3 or OST577IgG2M3 antibody labeled in FBB, pH 6.0.

As shown in Figure 12A, the double mutant (T250E / M428F) showed better binding to human FcRn than any of the single mutants (T250E or M428F) and showed approximately 15 times better binding to human FcRn than the wild antibody. As shown in Figure 12B, the double mutant (T250Q / M428L) showed better binding to the human FcRn than either single mutant (T250Q or M428L) or the double mutant (T250Q / M428F) and showed an approximately 28-fold better binding to human FcRn than the wild antibody. As summarized in Table 3, in a series of experiments, the IC50 for the wild OST577-lgG2M3 antibody is ∼9 µg / ml, while the IC50 for each of the single mutants (T250E and M428F) is ∼ 3 µg / ml and the IC50 for the double mutant (T250E / M428F) is less than 1 µg / ml. As summarized in Table 4, in another group of experiments, the IC50 for the wild OST577-lgG2M3 antibody is ∼ 12 µg / ml, while the IC50 for each of the single mutants (T250Q and M428L) and for the mutant double (T250Q / M428F) is ∼ 2.4 µg / ml and the IC50 for the double mutant (T250Q / M428L) is less than 1 µg / ml.

5 Wild OST577-lgG1, single mutants T250E, T250Q, M428F, M428L and double mutants T250E / M428F and T250Q / M428L were also created. As summarized in Table 5, in a group of experiments, the IC50 for the wild OST577-lgG1 antibody is ∼14 µg / ml, while the IC50 for each of the single mutants (T250E and M428F) is ∼ 3- 5 µg / ml and the IC50 for the double mutant (T250E / M428F) is less than 1 µg / ml. As summarized

10 in Table 6, in another group of experiments, the IC50 for the wild OST577-lgG1 antibody is ∼ 10 µg / ml, while the IC50 for the single mutant (T250Q) is ∼ 3 µg / ml and the IC50 for the single mutant (M428L) and double mutant (T250Q / M428L) is less than 1 µg / ml.

The binding of OST577-lgG2M3 and some of its mutants to the rhesus FcRn was analyzed in competitive binding experiments. As summarized in Table 7, the IC50 for the wild OST577-lgG2M3 antibody is ∼ 15 µg / ml, while the IC50 for each of the single mutants (T250Q and M428L) and for the double mutant (T250Q / M428F ) is ∼ 2-4 µg / ml, and the IC50 for the double mutant T250Q / M428L) is less than ∼ 1 µg / ml. The binding of OST577-lgG1 and some of its mutants to the rhesus FcRn was also analyzed in competitive binding experiments. As summarized in Table 8, the IC50 for the OST577-lgG1 antibody is ∼

20 9µg / ml, while the IC for the single mutant (T250Q) IS ∼ 3 µg / ml and the IC50 for the single mutant (M428L) and for the double mutant (T250Q / M428L) is less than 1 µg / m.

TABLE 2

Name a (lgG2M3)

n b IC50 (µg / m I) C Relative union d

Wild

6 10.7 ± 4.6 1.0

L314W

2 20.2 ± 2.0 0.53

L314Q

2 32.0 ± 2.3 0.33

L314A

2 68.7 ± 2.1 0.16

L314R

2 102 ± 4 0.11

T250E

one 3.82 2.8

T250S

2 12.0 ± 0.9 0.89

T250A

2 13.3 ± 0.5 0.80

T250V

3 19.0 ± 1.8 0.56

T250D

one > 210 <0.050

M428F

2 3.40 ± 0.53 3.1

M428G

one 147 0.072

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b n indicates the number of independent trials. c IC50 values ((± SD) are expressed in µg / ml (based on the competitor's final concentrations) and were calculated in competitive binding assays against biotinylated HuEP5C7-IgG2M3 in FSB, pH 6.0, as described in Example 6. d The relative binding to human FcRn was calculated in the form of the ratio between the IC50 value of the wild OST577-lgG2M3 antibody and that of each of the mutants.

TABLE 3

Name a (lgG2M3)

n b IC50 (µg / m I) C Relative union d

Wild

3 9.40 ± 2.87 1.0

T250E

3 2.71 ± 1.16 3.5

M428F

3 2.97 ± 0.30 3.2

T250E / M428F

2 0.639 ± 0.094 fifteen

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b n indicates the number of independent trials. c IC50 values ((± SD) are expressed in µg / ml (based on the competitor's final concentrations) and were calculated in competitive binding assays against biotinylated HuEP5C7-IgG2M3 in FBB, pH 6.0, as described in Example 6. d The relative binding to human FcRn was calculated in the form of the ratio between the IC50 value of the wild OST577-lgG2M3 antibody and that of each of the mutants.

TABLE 4

Name a (lgG2M3)

n b IC50 (µg / m I) C Relative union d

Wild

3 11.9 ± 2.5 1.0

T250Q

3 4.20 ± 1.02 2.8

M428L

3 1.79 ± 0.69 6.7

T250Q / M428F

3 1.50 ± 0.31 8.0

T250Q / M428L

3 0.430 ± 0.084 28

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the

position according to the EU index (Kabat et aI., op. cit.) and the second letter indicates the mutant amino acid b n indicates the number of independent trials. c IC50 values ((± S.D.) are expressed in µg / ml (based on concentrations final competitor) and were calculated in competitive binding assays against biotinylated OST577lgG2M3 in FBB, pH 6.0, as described in Example 6. d The relative binding to human FcRn was calculated in the form of the ratio between the value IC50 of the wild-type OST577-lgG2M3 antibody and that of each of the mutants.

TABLE 5

Name a (lgG1)

n b IC50 (µg / m I) C Relative union d

Wild

6 13.9 ± 4.2 1.0

T250E

3 5.26 ± 0.87 2.6

M428F

5 3.44 ± 1.22 4.0

T250E / M428F

4 0.990 ± 0.786 14

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b n indicates the number of independent trials. c IC50 values ((± SD) are expressed in µg / ml (based on the competitor's final concentrations) and were calculated in competitive binding assays against biotinylated OST577lgG1 in FBB, pH 6.0, as described in Example 6. d The relative binding to human FcRn was calculated in the form of the ratio between the IC50 value of the wild OST577-lgG1 antibody and that of each of the mutants.

TABLE 6

Name a (lgG1)

n b IC50 (µg / m I) C Relative union d

Wild

5 10.3 ± 2.8 1.0

T250Q

5 3.14 ± 0.86 3.3

M428L

5 0.896 ± 0.304 eleven

T250Q / M428L

5 0.351 ± 0.144 29

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et aI., op. cit.) and the second letter indicates the mutant amino acid b n indicates the number of independent trials.

c IC50 values ((± SD) are expressed in µg / ml (based on the competitor's final concentrations) and were calculated in competitive binding assays against biotinylated OST577lgG1 in FBB, pH 6.0, as described in Example 6. d The relative binding to human FcRn was calculated in the form of the ratio between the IC50 value of the wild OST577-lgG1 antibody and that of each of the mutants.

TABLE 7

Name a (lgG2M3)

n b IC50 (µg / m I) C Relative union d

Wild

3 14.8 ± 2.7 1.0

T250Q

3 4.05 ± 0.24 3.6

M428L

3 1.92 ± 0.46 7.7

1750Q / M428F

3 1.77 ± 0.60 8.4

T250Q / M428L

3 0.554 ± 0.052 27

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b n indicates the number of independent trials. c IC50 values ((± SD) are expressed in µg / ml (based on the competitor's final concentrations) and were calculated in competitive binding assays against biotinylated OST577lgG2M3 in FBB, pH 6.0, as described in Example 6. d The relative binding to FcRn rhesus was calculated in the form of the ratio between the IC50 value of the wild OST577-lgG2M3 antibody and that of each of the mutants.

TABLE 8

Name a (lgG1)

n b IC50 (µg / m I) C Relative union d

Wild

3 8.86 ± 0.52 1.0

T250Q

3 2.97 ± 0.59 3.0

M428L

3 0.629 ± 0.060 14

T250Q / M428L

3 0.236 ± 0.013 37

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b n indicates the number of independent trials. c IC50 values ((± S.D.) are expressed in µg / ml (based on concentrations

final competitor) and were calculated in competitive binding assays against biotinylated OST577lgG1 in FBB, pH 6.0, as described in Example 6. d The rhesus FcRn binding was calculated in the form of the ratio between the value IC50 of the wild OST577-lgG1 antibody and that of each of the mutants.

Example 7

5 This example describes the confirmation of the properties of the IgG2M3 and IgG1 mutants in FcRn binding

Direct binding test:

10 The purified OST577-lgG2M3 antibodies were analyzed for binding to human FcRn in the NS0 HuFcRn cell line (memb), clone 7-3, or to non-NS0 cells

5

transfected in FBB at pH 6.0. Approximately 2 x 10 cells / assay were washed once in FBB, pH 8.0, and once in FBB, pH 6.0, then resuspended in 100 µl of antibody at a concentration of 11 µg / ml in FBB, a pH 6.0. The cells were incubated with the antibody for 1 hour on ice, washed twice in FBB, Ph 6.0, and resuspended in 25 µl of goat anti-human IgG conjugated RPE antibody (Southern Biotechnology Associates, Inc.) diluted to 5 µg / ml in FBB, pH 6.0. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB, Ph 6.0, and resuspended in 1% formaldehyde. Samples were analyzed to determine binding

20 of the antibody to FcRn by FACSTM using a FACScan flow cytometer (BD® Biosciences). The mean channel fluorescence (MCF) was represented using Excel (Microsoft® Corporation).

Competitive union test at 37º C

A series of dilutions of each purified OST577-lgG2M3 antibody was presented against the biotinylated OST577-lgG2M3 antibody to determine binding to human FcRn in

5

the NS0 HuFcRn cell line (meb), clone 7-3. Approximately 2 x 10 cells / assay were washed once in FBB, pH 8.0, and once in FBB, pH 6.0, then resuspended in 100 30 µl of pre-mixed biotinylated OST577-lgG2M3 antibody (10 µg / ml of the competitor antibody OST577-lgG2M3 (serial dilutions by two starting from 208 µg / ml to 0.102 µg / ml) in

FBB, pH 6.0. The cells were incubated with the antibody mixture for 1 hour at 37 ° C, washed twice in FBB, pH 6.0, and resuspended in 25 ml of streptavidin-RPE conjugate ((BioSource International) diluted to 2.5 µg / ml in FBB, pH 6.0 After incubation for 30 minutes in the dark, the cells were washed twice in FBB, Ph 6.0, and resuspended in 1% formaldehyde.The samples were analyzed to determine antibody binding to the FcRn by FACSTM using a FACScan flow cytometer (BD® Biosciences) .The mean channel fluorescence (MCF) was plotted against the competitor concentration and the IC50 values were calculated using GraphPad Prism® (GraphPad ™ Software).

PH dependent release and binding assay:

The purified mutant OST577-lgG2M3 and OST577-lgGI antibodies were compared with the respective wild antibodies according to human FcRn binding and then released at various pH values in single-release and single-binding assays.

5

point using the NS0 HuFcRn cell line (meb), clone 7-3. Approximately 2 x 10 cells / assay were washed once in FBB, pH 8.0, and once in FBB, pHh 6.0, then resuspended in 100 µl of purified antibody (10 µg / ml) in FBB, pH 6 , 0-. The cells were incubated with the antibody for 1 hour on ice, washed twice in FBB, Ph 6.0, 6.5, 7.0, 7.5, or 8.0, and resuspended in 25 µl of antibody FITC conjugated to goat antihuman IgG (Southern Biotechnology Associates, Inc.) diluted to 1.25 in FBB at the appropriate pH. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB at the appropriate pH, and resuspended in 1% formaldehyde. Samples were analyzed for antibody binding to FcRn by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences). The mean channel fluorescence (MCF) was represented using Excel (Microsoft® Corporation).

The purified mutant OST577-lgG2M3 and OST577-lgGI antibodies were compared with the respective wild antibodies according to rhesus FcRn binding and then released at various pH values in single-point release and binding assays using the line NS0 RhFcRn cell, clone R-3, as described above.

Results:

The binding properties of the wild OST577-lgG2M3 or OST577-lgG1 antibodies and their various mutants to human FcRn was confirmed using a transfected NS0 cell line that stably expresses human FcRn on its surface. To confirm that the binding of the mutant antibodies was specific to human FcRn on the transfected NS0 cell line, instead of being produced through some other receptor or by an unknown mechanism, the antibodies were analyzed for non-transfected NS0 cell binding. against a transfected NS0 cell line that stably expresses FcRn. As described above, the cells were incubated with a sub-concentration of the antibody in FBB, pH 6.0, and the binding was analyzed by FACSTM. As shown in Figure 13, the results indicated that there was no apparent binding to the parental Ns0 cell line, suggesting that antibodies specifically bind to cells transfected by a human FcRn.

To confirm that each of the mutants generated in the present invention have behaved in a physiologically relevant way, the effects of temperature and pH on human FcRn binding were investigated more closely. Since the initial competitive binding assays were performed at 4 ° C, the experiments were repeated at the most physiologically relevant temperature of 37 ° C to show that the mutants were still active at this temperature. As described above, increasing concentrations of unlabeled competing antibodies were incubated with cells expressing human FcRn in the presence of a subsaturating concentration of labeled OST577-lgG2M3 antibody at 37 ° C in FBB, pH 6.0. As shown in Figure 14, the results indicated that the antibodies maintained their relative pattern of human FcRn binding at 37 ° C.

It is known that the binding of IgG to FcRn depends on the pH. IgG binds strongly to FcRn at pH 6.0, but weakly at Ph 8.0. In order to engineer the mutant antibodies with higher serum half-lives, it is desirable to increase the binding to FcRn at pH 6.0, while maintaining the pH-dependent release of FcRn at pH 8.0. To confirm that the pH-dependent binding, the antibodies were analyzed to determine binding to a transfected NS0 cell line that stably expresses human FcRn and then is released at varying pH values from pH 6.0 to H 8 , 0. As described above, the cells were incubated with a sub-concentration of the antibody in FBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and the binding was analyzed by FACSTM. As shown in Figure 15A, the results indicated that the modified OST577-lgG2M3 antibodies that have mutations T250E, T250Q, M428F, M428L, T250E / M428F, T250Q / M428F, or T250Q / M428L all showed a strong binding to human FcRn at pH 6.0, with decreased binding as the pH values increased to 8.0. As shown in Figure 15B, the results indicated that the modified OST577-lgG1 antibodies having the T250E, M428F, or T250E / M428F mutations all showed a strong binding to human FcRn at pH 6.0, with decreased binding to as the pH values increased to 8.0. The OST577-IgG1 antibody that has the T250D mutation showed a weaker binding (compared to the wild) to human FcRn at pH 6.0, with decreased binding as the pH values increased to 8.0. As shown in Figure 15C, the results indicated that the modified OST577-lgG1 antibodies having the T250Q, M428L, or T250Q / M428L mutations all showed a strong binding to human FcRn at pH 6.0, with decreased binding to as the pH values increased to 8.0. These results indicated that the binding of antibodies to human FcRn was, in fact, pH dependent.

Similarly, the antibodies were analyzed for binding to a transfected NS0 cell line that stably expresses rhesus FcRn and is then released at pH values ranging from pH 6.0 to pH 8.0. As shown in Figure 15D, the results indicated that the modified OST577-lgG2M3 antibodies that have mutations T250E, T250Q, M428F, M428L, T250E / M428F, T250Q / M428F, or T250Q / M428L all showed a strong binding to the FcRn of rhesus at pH 6.0, with decreased binding as the pH values increased to 8.0. As shown in Figure 15E, the results indicated that the modified OST577-lgG1 antibodies having the T250Q, M428L, or T250Q / M428L mutations all showed a strong binding to the rhesus FcRn at pH 6.0, with decreased binding as the pH values increased to 8.0. These results indicated that the binding of antibodies to rhesus FcRn was also dependent on Ph.

Example 8

Cell culture:

The Raji cell line of human Burkitt lymphoma (American Type Culture Collection) was maintained in RPMI 1640 medium with L-glutamine (BioWhittaker ™) containing 10% FBS (HyClone®) and 1% penicillin-streptomycin (Life TechnologieS ®).

This example describes the confirmation of additional properties of the IgG2M3 and IgG1 mutants

Antigen binding assays:

The antigen binding activity of the mutant and wild OST577 antibodies was confirmed by a competitive binding ELISA. 2 Immulon ™ plates (DYNEX® Technologies) were coated overnight at 4 ° C with 1.0 µg / ml recombinant hepatitis B surface antigen antibody (HBsAg) (Advanced ImmunoChemical, Inc., Long Beach, CA). The next day, the plates were washed with EWB and blocked with 300 µl / well of SuperBlocik® blocking buffer in TBS (Pierce Chemical Company) for 30 minutes at room temperature. Plates were washed with EWB and pre-mixed biotinylated OST577-lgG2M3 antibody (0.25 µg / ml) and competitor OST577-lgG2M3 antibody (serial dilutions by two from 33 µg / ml to 0.033 µg / ml) were added to each well or the premixed biotinylated OST577-lgG1 antibody (0.25 µg / ml) and the competing OST577-lgG1 antibody (serial dilutions by two of 67 µg / ml at 0.067 µg / ml) in 100 µl of EB. The plates were incubated for 1 hour at room temperature, then washed with EWB and 100 µl / well of streptavidin-HRP conjugate (Pierce Chemical Company) was added at 1 µg / ml in EB. After incubating for 30 minutes at room temperature, the plates were washed with EWB, followed by the addition of 100 µl / well of ABTS substrate peroxidase / peroxidase solution B (Kirkegaard & Perry Laboratories). The reaction was stopped with 100 µl / well of 2% oxalic acid and the absorbance at 415 nm was measured using a VERSAmax ™ microtiter plate reader (Molecular Devices Corporation®).

The antigen binding activity of the Hu1D10-lgG2M3 wild-type and mutant antibodies was confirmed in a FACSTM binding assay using Raji cells, which express an allele of the HLA-DR β chain that is recognized by Hu1D10 (Kostelny et al. (2001), op.

5

cit.). Approximately 2.5 x 10 cells / assay were washed once with FBB, pH 7.4, and resuspended in 140 µl of the Hu1D10-lgG2M3 antibody (serial dilutions by three of 60 µg / ml to 0.027 µg / ml) in FBB, pH 7.4. The cells were incubated with the antibody for 1 hour on ice, washed twice in FBB, pH 7.4, and resuspended in 25 µl of the RPE conjugated antibody of F (ab`) 2 goat anti-human kappa (Southern Biotechnology Associates, Inc.) diluted to 10 µg / ml in FBB, pH 7.4. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB, Ph 704, and resuspended in 1% formaldehyde. Samples were analyzed for antibody binding to the HLA-DR β chain allele by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences).

Similarly, the antigen binding activity of the Hu1D10-lgG1 wild-type and mutant antibodies was confirmed by binding assay in FACSTM using Raji cells.

5

Approximately 2.0 x 10 cells / assay were washed once with FBB, pH 7.4, and resuspended in 100 µl of Hu1D10-lgG1 antibody (serial dilutions by two from 25 µg / ml to 12.5 µg / ml , then serial dilutions by three from 12.5 µg / ml to 0.0020 µg / ml) in FBB, pH 7.4. A serial dilution of the HuFd79-lgG1 antibody (Co et aI., Proc. Natl. Acad. Sci. 88: 2869-2873 (1991)) was prepared as described above and used as a negative control. The cells were incubated with the antibody for 1 hour on ice, washed twice in FBB, pH 7.4, and resuspended in 25 µl of the antibody conjugated to FITC anti F (ab`) 2 of human goat IgG (Southern Biotechnology Associates, Inc.) diluted to 20 µg / ml in FBB, pH 7.4. After incubation for 30 minutes on dark ice, the cells were washed twice in FBB, pH 7.4, and resuspended in 1% formaldehyde. Samples were analyzed for antibody binding to the HLADR β chain allele by FACSTM using a FACSCalibur flow cytometer (BD® Biosciences).

ADCC test:

The antibody-dependent cell-mediated cytotoxicity (ADCC) activity of wild and mutant Hu1D10 antibodies was confirmed by measuring the release of lactate dehydrogenase (LDH) using peripheral blood mononuclear cells (PBMC) as effectors and Rajo cells as targets following a procedure. published (Shields et aI., op. cit.). PBMCs were prepared from whole blood using a Ficoll-Paque® gradient

6

Plus (Amersham Biosciences ™ Corporation) and were resuspended in a density of 8 x 10 cells / ml in assay medium (RPMI1640, 1% BSA). Raji cells were washed three times in assay medium and resuspended at a density of 0.4 x 106 cells / ml in assay medium. Wild and mutant Hu1D10 antibodies were diluted to 4 µg / ml, 0.25 µg / ml and 0.016 µg / ml in assay medium. Raji cells (50 µl / well) and Hu1D10 antibody (50 µl / well), ie 200 ng / assay, 12.5 ng / assay or 0.8 ng / assay) were combined in the wells of a plate U-bottom 96-well Falcon BD® Biosciences) and incubated for 30 minutes at room temperature. PBMC (100 µl / well, that is

40: 1 effector / target ratio) were added to the opsonized cells and incubated for 4 hours at 37 ° C in a CO2 incubator. Antibody-independent cell-mediated cytotoxicity (AICC) was measured by incubating effector and target cells in the absence of antibody. Spontaneous release was measured by incubating the target cells (SRdiana) or effector cells (SRefectors) in the absence of antibody. Maximum release (MR) was measured by adding 2% Triton X-100 to the target cells. The plates were gently centrifuged and the supernatants (100 µl / well) were transferred to a flat bottom 96-well plate of Falcon type. LDH activity was measured by incubating the supernatants with 100 µl / well of the LDH reaction mixture of the Cytotoxicity Detection Kit (Roche Diagnostics Corporation) for 30 minutes at room temperature. The reaction was stopped with 50 µl / well of 1N HCl and the absorbance at 490 nm was measured using a VERSAmax ™ microtiter plate reader (Molecular Devices Corporation®). The percentage of cytotoxicity was calculated as (release LDHsample-SRefector - SRdiana) / MR target-SRdiana) x 100

Results:

The antigen binding properties of the wild-type OST577 and Hu1D10 antibodies and mutants to their respective antigens were confirmed using the appropriate binding assays. As described above, the binding of OST577 antibodies to HBsAg was determined in a competitive ELISA. As shown in Figure 16A, the binding of wild-type and mutant OST577-IgG2M3 antibodies to HBsAg was essentially identical. Similarly, as shown in Figure 16B, the binding of wild-type and mutant OST577-IgG1 antibodies to HBsAg was essentially identical.

As described above, the binding of Hu1D10 antibodies to an HLA-DR β chain was determined in a FACS binding assay. As shown in Figure 17A, the binding of wild-type and mutant Hu1D10-lgG2M3 antibodies to the HLA-DR β chain allele was essentially identical. Similarly, as shown in Figure 17B, the binding of wild Hu1D10-lgG1 and mutant antibodies to the HLA-DR β chain allele was essentially identical. These results indicate that, as expected, the mutations described in positions 250 and 428 do not affect antigen binding.

The ADCC activity of the Hu1D10 wild and mutant antibodies was confirmed in an LDH release assay using PBMC as effectors and Raji cells as targets. As shown in Figure 18A, using a donor carrying the homozygous V / V alleles of FcγIII, the ADCC activity of the double mutant (T250Q / M428L) of the Hu1 D10-lgG1 antibody was very similar to that of the wild antibody, while ADCC activity of the single mutant (M428L) of the Hu1D10-lgG1 antibody decreased slightly compared to the wild antibody. As expected, the wild and mutant Hu1D10-lgG2M3 antibodies lacked ADCC activity. Similarly, as shown in Figure 18B, using a donor carrying the homozygous F / F alleles of FcγIII, the ADCC activity of the double mutant (T250Q / M428L) of the Hu1 D10-lgG1 antibody was very similar to that of the antibody wild, while the ADCC activity of the single mutant (M428L) of the Hu1D10-lgG1 antibody decreased somewhat compared to the wild antibody, the wild and mutant Hu1D10-lgG2M3 antibodies lacked ADCC activity. These results indicate that the T250Q / M428L mutation described in the present invention does not affect the ADCC activity of the IgG1 form of the antibody, while the M428l mutation slightly reduces the ADCC activity of the IGg1 form. The mutations described in positions 250 and 428 do not affect the ADCC activity of the IgG2M3 form of the antibody.

Example 9

This example describes the in vitro and in vivo assays of serum half-life.

It is expected that human IgG antibodies with greater (or lesser) affinity for FcRn in vitro have a longer (or shorter) serum half-life in vivo, respectively. The affinity of human IgG mutants for FcRn can be measured in vitro by various procedures, such as surface plasmon resonance (SPR) using soluble FcRn conjugated to a suitable biosensor chip or performed a competitive binding experiment using FcRn expressed on the surface of Transfected cells. The FcRn used in in vitro affinity experiments can be of murine, rhesus or human origin. The serum half-life of human IgG mutants with the desired properties can be measured in vivo by injection into suitable experimental animals (eg, mice or monkeys)

or humans of a dose of antibody in the range of 0.1-10 mg of antibody per kg of body weight, then extracting serum samples at various time intervals to cover the expected serum half-life of the antibody and analyzing the samples to determine the presence of intact IgG by a suitable technique such as an ELISA.

Pharmacokinetics study in rhesus:

At the California National Primate Research Center (CNPRC) at the University of California, Davis conducted a non-LPG pharmacokinetic study entitled “Pharmacokinetic Comparison of Three OST577 Variants.” Twelve male rhesus macaques were randomized by weight and assigned to one of three study groups The four animals comprising each study group each received an intravenous dose of wild OST577 or one of two variants at 1 mg / kg administered for fifteen minutes OST577 antibodies were wild OST577-lgG2M3 , a variant of OST577-lgG2M3 containing the single mutation M428L and a variant of OST577-lgG2M3 containing the double mutation T250Q / M428L. The three antibodies were expressed by transfection of the Sp2 / 0 cells and purified as described in Example 5

Blood samples were taken before the dose on day 0 and at 1 and 4 hours after the dose, and at 7, 14, 21, 28, 42 and 56 days. At each time point 4 ml of blood was taken from the saphenous vein, the serum was prepared and 2 aliquots were frozen and kept at 20 ° C until use. For serum chemistry and hematology determinations blood samples were taken 16 days before the study, before the dose on day 0 and at the end of the study on day 56.

ELISA:

The concentrations of the OST577-IgG2M3 antibodies in rhesus serum samples were determined by ELISA using a qualified assay. Combined normal rhesus serum (PNRS) was obtained from CNPRC. The same batch of PNRS was used to prepare calibrators, positive serum controls and for pre-dilution of rhesus serum samples. The calibrators were prepared by standard dilution of OST577lgG2M3 in PNRS at 3000, 1500, 750, 375, 187.5, 93.75, 46.88, 23.44, and 0 ng / ml, equilibrated for 2 hours at room temperature and frozen in aliquots at -20 ° C. Positive serum controls were prepared by contaminating the PNRS with OST577-lgG2M3 at 0.2 µg / ml for the control of low positive serum, 0.4 µg / ml for the control of serum positive medium and 0.8 µg / ml for the High positive serum control, equilibrated for 2 hours at room temperature and frozen in aliquots at -20 ° C. Pre-dose serum samples for each animal were used as negative controls.

2 Immulon ™ plates (DYNEX® Technologies, Inc.) were coated overnight at 28 ° C with 100 µl / well of a mouse anti-OST577-lgG1 monoclonal antibody (OST577-'γ1) anti-id, Protein Design Labs ™, Inc.) at 1.0 µg / ml in PBS. The next day, the plates were washed three times with 300 µl / well of PBST / Tween (phosphate buffered saline, 0.1% Tween 20), tapped on a paper towel and blocked with 300 µl / well of SuperBlock® blocking buffer in PBS (Pierce Chemical Company) for 60 ± 5 minutes at room temperature. Calibrators, positive and negative serum controls, and serum samples were thawed and brought to room temperature before using. Calibrators, positive and negative serum controls, and serum samples were diluted to 1: 10 in SuperBlock® blocking buffer in PBS. Serum samples were adequately pre-diluted (1:10 to 1:80) in PNRS, then diluted to 1:10 in SuperBlock® blocking buffer in PBS. The plates were washed three times with 300 µl / well of PBS / TWEEN and patted on a paper towel. Diluted calibrators, positive and negative serum controls, and serum samples were added at 100 µl / well in duplicate wells and incubated for 60 ± 5 minutes at room temperature. The plates were washed three times with 300 ml / well of PBS / TWEEN and patted on a paper towel. Goat human lambda anti-light chain HRP conjugated antibody (Southern Biotechnology Associates, Inc.) was prepared by dilution of 1: 1000 in PBSBSA / Tween (phosphate buffered saline, 0.5% bovine serum albumin, 0 , 1% Tween 20), were added at 100 µl / well and incubated for 60 ± 5 minutes at room temperature. The plates were washed three times with 300 µl / well of PBS / TWEEN and tapped on a towel. of paper. ABTS Peroxidase Substrate / Peroxidase Solution B (Kirkegaard & Perry Laboratories) was added at 100 µl / well and incubated for 7 ± 1 minute. Development was stopped by the addition of substrate stop solution (2% oxalic acid) at 100 µl / well. The absorbance at 415 nm was measured within 30 minutes after the addition of the substrate stop solution using a VERSAmax ™ microtiter plate reader (Molecular Devices Corporation®).

A calibration curve was prepared using the mean absorbance values obtained from the calibrators and the adjustment of the data to a four-parameter logistic regression curve using SOFTmaX® PRO, version 4.0 (Molecular Devices Corporation®). The average absorbance value for the control of negative serum (ie decor, mean of the pre-dose sample for each animal) was subtracted from each absorbance value obtained for the calibrators. Positive serum control concentrations were determined after subtracting the mean absorbance value obtained for negative serum control from each absorbance value obtained for positive serum controls. The concentrations corresponding to the resulting average absorbance values were obtained by interpolation from the calibration curve. The concentrations of the serum samples were determined by subtracting the mean absorbance value from the negative serum control from the absorbance value of each sample, averaging the resulting absorbance values, deriving the concentration corresponding to the mean values of the absorbance by interpolation from the calibration curve and multiplying the resulting concentration by the pre-dilution factor, if it exists, to reach the final concentration of each sample.

The estimated quantitative interval of the trial was 0.10-0.90. The test was considered adequate when the following two conditions were met: (1) the average backcalculated concentration of the three calibrators in the quantitative range was within 20% of their nominal value; and (2) the calculated average results of four of six positive serum controls were within 30% of their nominal value, and at least one average result of each concentration level was within 30% of their nominal value. Data from the plates that did not meet the above criteria were rejected. Data from individual serum samples were rejected when any of the following three conditions were met: (1) the absorbance values in duplicate wells differed from each other by more than 40%; (2) the average concentration calculated was below the lower qualification limit (LLOQ) of the test (0.10 µg / ml); (3) the calculated average concentration was above the upper limit of quantification (ULOQ) in the assay (0.90 µg / ml)

Results:

The serum antibody concentration data was adjusted with a two-compartment model using WinNonlin® Enterprise Edition, version 3.2 (Pharsight® Corporation, Mountain View, CA). The model assumes a first order distribution and a first order elimination rate and adjusts the data well. The model data (simulated on the basis of each geometric mean of each group of primary pharmacokinetic parameters), as well as the mean concentration of serum antibodies observed and the standard deviation for each group of four animals were represented as a function of the time (days after infusion) using GraphPad Prism®, version 3.02 (GraphPad ™ Software, Inc.). As shown in Figure 19, the data indicate that the mean serum antibody concentrations of the M428L and T250Q / M428L variants of OST577-lgG2M3 were maintained at higher levels than with wild OST577-lgG2M3 at all time points.

Several pharmacokinetic parameters were calculated from the data using WinNonlin® Enterprise Edition, version 3.2 (Pharsight® Corporation). Statistical analysis of the pharmacokinetic parameters was calculated using GraphPad Prism®, version 3.02 (GraphPad ™ Software, Inc.) As shown in Table 9, the maximum mean serum concentration of the antibody (Cmax) was very similar between the three groups. test, indicating that the administered antibodies were distributed in the circulation in a similar way. Therefore, the higher antibody concentrations of the IgG2M3 mutant antibodies following the distribution phase are attributable to their greater persistence in the serum. The analysis of the mean clearance (CL) indicated that this was the case. The mean Vl, the volume of serum antibody removed per unit time was approximately 1.8 times lower for the M428L variant (0.0811 ± 0.0384 ml / h / kg; p = 0.057), and approximately 2.8 - smaller times for the T250Q / M428L variant (0.0514 ± 0.0075 ml / h / kg; p = 0.029) compared to the wild OST577-lgG2M3 (0.144 ± 0.047 ml / h / kg) (Table 9), indicating a significant decrease in clearance of OST577-lgG2M3 M428L and the T250Q / M428L variants of the circulation of rhesus monkeys compared to wild antibodies.

The FC profiles of the OST577-lgG2M3 variants were further analyzed by calculating other parameters (Table 9) Since the AUC (area under the curve) is inversely proportional to the CL, it follows that the average AUC, the area under the concentration curve time extrapolated from time zero to infinity was approximately 2 times longer than the M428L variant (15,200 ± 8,700 hr * µg / ml; p = 0.057) and approximately 2.6 times longer for the T250Q / M428L variant (19,800 ± 2,900 hr * µg / ml; p = 0.029) compared to wild OST577lgG2M3 (7,710 ± 3,110 hr * f, µg / ml) (Table 9), indicating a significant increase in the total exposure of the M428L and T250Q / M428L variants of OST577 -lgG2M3 compared to the wild antibody.

Finally, the mean elimination half-life (ß-phase) was approximately 1.8 times

5 greater for the M428L variant (642 ± 205 h), and approximately 1.9 times greater for the T250Q / M428L variant (652 ± 28 hr, p = 0.029) compared to wild OST577-lgG2M3 (351 ± 121 hr) (Table 9 ). The elimination half-life for wild OST577-lgG2M3 in this study is similar to that of OST577-lgG 1 (324 ± 85 h) in a previous FC study with rhesus monkeys (Ehrlich et a., Op. Cit.).

10 TABLE 9

Half-life of

Namea Cmáxb CLc AUCd

elimination

(lgG2M3) (µg / ml) (ml / h / kg) (h *, µg / ml)

(h)

Wild 36.7 ± 12.8 0.144 ± 0.047 7710 ± 3110 351 ± 121

0.0811 * ± 15200 * ± M428L 0.0384 8700

36.5 ± 20.1 642 ± 205

39.9 ± 6.8 19800 * ± 2900 652 * ± 28

0.0514 * ± T250Q / M428L 0.0075

aFor each mutant, the first letter indicates the wild amino acid, the number indicates the position according to the EU index (Kabat et al., op. cit.) and the second letter indicates the mutant amino acid. b The Cmax (± SD) values are expressed and calculated from the FC data using WinNonlin as described in Example 9. c The CL (± SD) values are expressed in ml / h / kg and are calculated from the FC data using WinNonlin as described in Example 9. d AUC values (± SD) are expressed in h * µg / ml and calculated from the FC data using WinNonlin as described in Example 9. e Elimination half-life (SD) values are expressed in h and calculated from FC data using WinNonlin as described in Example 9. * Indicates a significant difference (p <0.060) between the wild group and each mutant group. Mann-Whitney tests were performed using GraphPad Prism as described in Example 9.

Example 10

This example describes the application of several binding assays described in Examples 6 and 7 to the IgG3 and IgG4 antibody mutants.

The relative binding of the wild OST577-lgG3 or OST577-lgG4 antibodies and the effect of their various mutants on FcRn binding is determined using a transfected NS0 cell line that stably expresses human or rhesus FcRn on its surface. The cell culture and the generation of the human FcRn and rhesus cell line are performed as described in Example 6. The purified OST577-lgG3 or OST577-lgG4 antibodies are analyzed to determine FcRn binding in competitive binding assays. Following the protocols described in Example 6. As described for IgG2M3 and IgG1 in Example 6 using the above binding assays, the effect of mutations in the Fc region on IgG3 or IgG4 on binding affinity can be observed. FcRn of these antibodies.

Similarly, the protocols of the "Direct binding assay" "Competitive binding assay at 37 ° C" and the "pH dependent release and binding assay" described in Example 7 are carried out with mutants of IgG1, IgG3 and IgG4 to confirm the effect of mutants on FcRn binding.

Example 11

This example describes the application of in vivo and in vitro serum half-life assays described in example 9 to the mutants of the IgG 1, IgG3 and IgG4 antibodies.

The protocols of the "Rhesus Pharmacokinetic Study" as described in Example 9 are carried out in mutants of IgG1, IgG3 and IgG4 to confirm the effect of mutations in serum half-life in vivo and the various pharmacokinetic parameters.

Claims (25)

one.

An antibody or IgG class fusion protein, which comprises a constant region of the heavy chain or FcRn portion thereof of a human IgG molecule, which comprises glutamic acid or glutamine in amino acid residue 250 and leucine or phenylalanine in the amino acid residue 428 and in which the amino acid residues are numbered by the EU numbering system.

2.

The antibody or fusion protein according to claim 1, wherein the human IgG molecule is a molecule of IgG1, IgG2, IgG2M3, IgG3 or human IgG4

3.

The antibody or fusion protein according to claim 1, wherein the human IgG molecule is a human IgG1 or IgG2M3 molecule.

Four.

The antibody or fusion protein according to claim 1, wherein the amino acid residue 250 of the heavy chain constant region is glutamine.

5.

The antibody or fusion protein according to claim 1, wherein the amino acid residue 428 of the heavy chain constant region is leucine.

6. The antibody or fusion protein according to claim 1, wherein a) said amino acid residue 250 of the heavy chain constant region is glutamic acid and amino acid residue 428 of the heavy chain constant region is phenylalanine. b) said amino acid residue 250 of the heavy chain constant region is glutamine and amino acid residue 428 of the heavy chain constant region is phenylalanine; or c) said amino acid residue 250 of the heavy chain constant region is glutamine and amino acid residue 428 of the heavy chain constant region is leucine;

7.

The antibody or fusion protein according to claim 1, wherein said amino acid residue 250 of the constant region of the heavy chain is glutamine and said amino acid residue 428 of the constant region of the heavy chain is leucine.

8.

The antibody or fusion protein according to claim 1, wherein the antibody or fusion protein has a binding affinity for FcRn greater at pH 6.0 than at pH 7.4.

9.

An antibody or fusion protein comprising a constant region substantially identical to the human antibody of natural IgG class, in which

a) at least the amino acid residues of the constant region of the heavy chain 250 and 428 are different from the residues present in the natural IgG antibody with glutamic acid or glutamine in the amino acid residue 250 and leucine or phenylalanine in the amino acid residue 428 b) the serum half-life in vivo of said antibody is increased in relation to the natural antibody, and c) in which the amino acid residues are numbered using the EU numbering system.

10.

The antibody or fusion protein according to claim 9, wherein said natural Ig class human antibody comprises a constant region of the heavy chain of a human IgG1, IgG2, IgG3 or IgG4 molecule.

eleven.

The antibody or fusion protein according to claim 9, wherein said natural Ig class human antibody comprises a constant region of the heavy chain of a human IgG1 molecule.

12.

The antibody or fusion protein according to claim 9, wherein said amino acid residue 250 of the heavy chain constant region is glutamine.

13.

The antibody or fusion protein according to claim 9, wherein said amino acid residue 428 of the heavy chain constant region is leucine.

14. The antibody or fusion protein according to claim 9, wherein a) said amino acid residue 250 of the constant region of the heavy chain is glutamic acid and said amino acid residue 428 of the constant region of the heavy chain is phenylalanine. b) said amino acid residue 250 of the constant region of the heavy chain is glutamine and said amino acid residue 428 of the constant region of the heavy chain is phenylalanine; or c) said amino acid residue 250 of the constant region of the heavy chain is glutamine and said amino acid residue 428 of the constant region of the heavy chain is leucine;

fifteen.

The antibody or fusion protein according to claim 9, wherein said amino acid residue 250 of the heavy chain constant region is glutamine and said amino acid residue 428 of the heavy chain constant region is leucine.

16.

A method for altering the binding affinity for FcRn and / or the serum half-life of an IgG class fusion antibody or protein comprising substituting amino acid residues 250 and 428 in a matural human IgG class antibody, unmodified in the that residue 250 is glutamic acid or glutamine, and amino acid residue 428 is leucine or phenylalanine, whereby the binding affinity for FcRn and / or serum half-life of the antibody is altered, in which the amino acid residues are numbered with the EU numbering system.

17.

A method of producing an IgG class antibody or fusion protein, comprising:

a) preparing an expression vector, comprising a suitable promoter operably linked to DNA encoding at least one constant region or an FcRn binding portion thereof of a human immunoglobulin heavy chain or an FcRn binding portion thereof, wherein amino acid residue 250 is substituted with glutamic acid or glutamine and amino acid residue 428 is substituted with phenylalanine or leucine; b) transforming the host cells with said vector; Y c) culturing said transformed host cells to produce said antibody or fusion protein, in which the amino acid residues are numbered by the EU numbering system.

18.

The method according to claim 17, further comprising: preparing a second expression vector comprising a promoter operably linked to

DNA encoding a complementary immunoglobulin light chain and transforming said host cells with said second expression vector.

19.

The process according to claim 17, wherein said amino acid residue 250 of the heavy chain constant region is substituted with glutamine.

twenty.

The method according to claim 17, wherein said amino acid residue 428 of the heavy chain constant region is substituted with leucine.

21. The method according to claim 17, wherein: a) said amino acid residue 250 of the heavy chain constant region is substituted with glutamic acid and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine. b) said amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with phenylalanine; or c) said amino acid residue 250 of the heavy chain constant region is substituted with glutamine and amino acid residue 428 of the heavy chain constant region is substituted with leucine.

22

The method according to claim 17, wherein said amino acid residue 250 of the constant region of the heavy chain is substituted with glutamine and said amino acid residue 428 of the constant region of the heavy chain is substituted with leucine.

2. 3.

The antibody or fusion protein or method of any of the preceding claims wherein the antibody or fusion protein is a therapeutic or prophylactic fusion antibody or protein.

24.

The antibody of claim 23, which is modified from daclizumab by glutamic acid or glutamine in amino acid residue 250 and leucine or phenylalanine in amino acid residue 428.

25. The antibody of claim 23, which is modified from any of the following antibodies: palivizumab, trastuzumab, infliximab, abciximab, bevacizumab, cetuximab, alemtuzumab, rituximab, efalizumab, visilizumab, adalimumab, natalizumab, or basiliximab, by glutamic acid or glutamine in amino acid residue 250 and leucine in amino acid 250.