CN101124245A - Neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc-binding proteins, and methods related thereto - Google Patents

Abstract

The compositions and methods of the present invention are based, in part, on our discovery that an effector function mediated by an Fc-containing polypeptide can be altered by modifying one or more amino acid residues within the polypeptide (by, for example, electrostatic optimization). The polypeptides that can be generated according to the methods of the invention are highly variable, and they can include antibodies and fusion proteins that contain an Fc region or a biologically active portion thereof.

Description

Neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc-binding proteins, and methods related thereto

related application

This application claims the benefit of US Provisional Application Serial No. 60/519,744, filed November 12, 2003, entitled "Antibodies Containing Altered Constant Regions and Variants Thereof." This application also claims the benefit of US Application Serial No. 60/519,743, filed November 12, 2003, entitled "Fc Receptor Binding Polypeptides, pH Specific Variants Derived by Electrostatic Optimization, and Uses Thereof." This application also claims the benefit of US Application Serial No. 60/519,733, filed November 12, 2003, entitled "Mutants Defined by Electrostatic Modeling in Human Fc Domains Capable of Modulating Human Antibody Half-Life." This application is also related to PCT application XXXXXXXX entitled "Fcγ receptor binding polypeptide variants and methods related thereto", filed on the same date as this application. The entire contents of all of these applications are hereby incorporated by reference.

Background technique

Many biological processes are mediated by the specific interaction of one protein with another. For example, enzymes are proteins that specifically bind their substrates, whereas ligands (such as neurotransmitters and hormones) bind to their cognate receptors when substantial transmission of information from cell to cell occurs. The most intriguing interactions occur in the context of an immune response, where antibodies (also called immunoglobulins) are produced to confer the body's defense against foreign substances capable of causing infection or disease. Antibodies contain distinct domains that specifically interact with antigens and with receptors on "effector" cells, such as phagocytes. Although binding to an antigen is useful (as this prevents the antigen from interacting with its endogenous target), the most effective immune response destroys the antigen. Thus, the most potent antibodies are those with domains that mediate high affinity antigen binding and domains that mediate potent effector functions.

Natural antibodies are usually heterotetramers; they contain two identical light (L) chains and two identical heavy (H) chains linked together by disulfide bonds. Each heavy chain has one variable domain (V _H ) followed by several constant domains (CH ₁ , CH ₂ and CH ₃ ), while each light chain has one variable domain (V H ) followed by only one constant domain. _L ). The constant region of the light chain is aligned with the first constant region of the heavy chain, and the _VL is aligned with the _VH . The variable region is so named because certain amino acids in the variable region vary widely between antibodies. These variable regions, also called complementarity determining regions (CDRs), are responsible for the binding specificity of each particular antibody for its particular antigen. Each variable domain contains 3 CDRs separated by highly conserved regions called framework regions, FRs. The CDRs in each chain are held close together by the FR regions and, along with the CDRs from the other chains, participate in the formation of the antigenic site of the antibody.

The constant regions are not directly involved in the binding of antibodies to antigens, but mediate various effector functions based on their binding to cell receptors or complement molecules. Depending on the amino acid sequence of the heavy chain constant region, antibodies or immunoglobulins can be assigned to different classes (A, D, E, G and M). The most commonly used therapeutic antibodies are of the "G" type (ie, they are IgG). These types can be further divided. For example, IgG antibodies are further divided into isotypes IgG1, IgG2, IgG3 and IgG4. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry 20:2361-2370, 1981; for a description of the Fc region, see Figure 1 of US Patent No. 6,242,195).

The Fc region mediates effector functions, which are divided into two categories. The first are functions that occur independently of antigen binding; these confer persistence in circulation and the ability to translocate across cellular barriers by transcytosis (see Ward and Ghetie, Therapeutic Immunology 2:77-94, 1995) . The second category is the functions performed by antibodies following antigen binding; these functions involve the complement cascade or the participation of cells bearing Fc receptors (FcR). FcRs are specialized cell surface receptors on hematopoietic cells that mediate the removal of antibody-coated pathogens (by phagocytosis of immune complexes) and the removal of corresponding antibody-coated red blood cells and various other cellular targets (such as tumor cells). crack. Lysis occurs by antibody-dependent cell-mediated cytotoxicity (ADCC; see Van de Winkel and Anderson, J. Leuk. Biol. 49:511-24, 1991). FcRs are defined based on their specificity for an immunoglobulin isotype. For example, the Fc receptors of IgG antibodies are called FcyRs.

Another Fc receptor, FcRn, regulates the serum half-life of IgG. An increase or decrease in Fc (or Fc-containing polypeptide) half-life can be reflected by an increase or decrease in the affinity of the Fc region for FcRn (neonatal Fc receptor), respectively (Ghetic et al., Nature Biotechnol. 15:637- 640, 1997; Kim et al., Eur.J.Immunol.24:542-548, 1994; Della'Acqua et al. (J.Immunol.169:5171-5180, 2002). Correlation between FcRn binding affinity and serum half-life Consistent with the proposed biological role of FcRn in rescuing antibodies from lysosomal degradation.Furthermore, FcRn transfers IgG from the mother to the fetus.

These apparently divergent roles are mediated by the ability of FcRn to transport bound IgG within and across cells. Antibodies are thought to be normally internalized by endothelial cells from circulation and targeted to the acidic endosomes and lysosomes of the cells for degradation. FcRn is able to bind antibody Fc regions at the acidic pH of endosomes (<6.5), fuse with endothelial cell membranes, and release antibodies at the neutral pH of the bloodstream (>7.4), thereby rescuing antibodies from destruction. When serum antibody levels decrease, more FcRn molecules are available for IgG binding, thereby salvaging increased amounts of IgG. Conversely, if serum IgG levels rise, FcRn becomes saturated, thereby increasing the rate of antibody that is internalized and degraded (Ghetie and Ward, Annu. Rev. Immunol. (2000), 18:739-66).

Consistent with the above model, altered polypeptides are expected to have longer half-lives if their binding affinity for neonatal Fc receptors is increased. Conversely, if an altered polypeptide has reduced binding affinity for neonatal Fc receptors, it is expected to have a shorter half-life.

Monoclonal antibodies (mAbs) are currently used to treat diseases in human patients (King and Adair, Curr. Opin. Drug Discovery Dev. 2: 110-117, 1999; Vaswani and Hamilton, Ann. Allergy Asthma Immunol. 81: 105- 119, 1998; and Hollinger and Hoogenboom, Nature Biotechnol. 16:1015-1016, 1998). This is not to say, however, that current antibody-based therapies have been completely successful; in some cases, limited circulation times and/or low bioavailability of therapeutic agents have resulted in a relatively low percentage of patients exhibiting a complete response to antibody-based therapy, or in other In some cases, toxicity due to prolonged circulating half-life or exposure to non-target tissues may render the antibody unusable as a therapeutic agent.

Thus, there is a need for antibodies (and other Fc-containing polypeptides, such as fusion proteins) whose antigen-independent effector functions have been tailored according to the antibody's intended use. Similarly, methods are needed that allow the prediction of antibody sequence changes that can lead to changes in antigen-independent effector function (thus avoiding reliance on tedious trial-and-error processes). In some instances, it may be desirable to increase the half-life of the antibody. For example, protein therapeutics with increased half-life in the blood have the advantage of simultaneously reducing the periodic dosing of the drug or alternatively reducing the dosage of the drug. Such antibodies also have the advantage of increased access to disease sites such as tumors. Conversely, a protein therapeutic with a reduced half-life would be expected to be less toxic, while maintaining the efficacy observed with higher and less well tolerated doses of the unchanged drug. These therapeutic agents and methods of making them would be of great benefit.

Summary of the invention

The invention relates to altered polypeptides (e.g., polypeptides with amino acid substitutions in the IgG constant region) having specific amino acid substitutions, e.g., within the Fc region or an FcRn-binding fragment thereof, that result in antigen-independent effector functions (e.g., Circulation half-life). The invention also provides methods of making the altered polypeptides and methods of using them as protein-based therapeutics. The invention also provides novel forms of FcRn and methods of their use.

The present invention is based at least in part on the identification of specific amino acid residues in the constant domain (Fc) of a human Fc region (in particular, from an IgG antibody) constant domain (Fc) which, when altered by one or more amino acid mutations Antigen-independent effector functions of the antibody will be altered, particularly the circulating half-life of the antibody. Accordingly, the present invention relates to polypeptides, such as antibodies and fusion proteins, comprising all or part of an Fc region, wherein the Fc region has been mutated at one or more amino acid residues to increase or decrease the antigen-independence of the polypeptide. Sexual effector function.

The invention also provides techniques for identifying desired amino acid mutations and methods for preparing polypeptides comprising such mutations. The methods include molecular modeling, which can be used to predict amino acid changes in the amino acid sequence, thereby altering (eg, enhancing or decreasing) binding to an Fc receptor (eg, a human neonatal Fc receptor). Generally, the method starts with a "starting" or "target" polypeptide or a complex (e.g., a crystal structure or a homology model) containing a first polypeptide that binds to FcRn, after which modification of the first polypeptide results in a binding to FcRn. A "second" or "altered" polypeptide that differs from the first polypeptide so as to have better properties in a particular therapeutic or diagnostic application. For example, the second polypeptide can more efficiently perform one or more antigen-dependent effector functions (eg, altered half-life). Modeling can be done on a computer (in silico).

In one aspect, the invention relates to an altered polypeptide comprising at least the FcRn binding portion of the Fc region, wherein said polypeptide comprises at least one mutation compared to the starting polypeptide, and wherein said at least one mutation is selected from:

A charged amino acid was substituted at position 248 of the EU amino acid position;

A positively charged amino acid was substituted at position 249 of the EU amino acid position;

A polar amino acid or lysine is substituted at position 251 of the EU amino acid position;

A polar amino acid was substituted at position 252 of the EU amino acid position;

A polar amino acid was substituted at position 255 of the EU amino acid position;

Lysine was substituted at position 256 of the EU amino acid position;

A charged amino acid was substituted at position 257 of the EU amino acid position;

Substitute a polar amino acid or a charged amino acid at position 258 of the EU amino acid position;

Substitution at EU amino acid position 277;

A charged amino acid was substituted at position 279 of the EU amino acid position;

A charged amino acid was substituted at position 280 of the EU amino acid position;

Substitute a charged amino acid or glutamine at position 281 of the EU amino acid position;

A charged amino acid was substituted at position 282 of the EU amino acid position;

Substitute a polar amino acid or a charged amino acid at position 284 of the EU amino acid position;

A positively charged amino acid, a polar amino acid or aspartic acid is substituted at position 285 of the EU amino acid position;

Substitution of glutamic acid, threonine or methionine at position 286 of the EU amino acid position;

Substitute a polar amino acid or a charged amino acid at position 287 of the EU amino acid position;

A charged amino acid was substituted at position 288 of the EU amino acid position;

Substitution at EU amino acid position 289;

A polar amino acid or a charged amino acid is substituted at position 304 of the EU amino acid position;

Substitute a polar amino acid or a charged amino acid at position 305 of the EU amino acid position;

Substitution at EU amino acid position 306;

A polar or charged amino acid is substituted at position 307 of the EU amino acid position;

A charged amino acid was substituted at position 308 of the EU amino acid position;

A charged amino acid was substituted at position 309 of the EU amino acid position;

Substitute a charged amino acid or a polar amino acid at position 310 of the EU amino acid position;

A positively charged amino acid was substituted at position 311 of the EU amino acid position;

Substitute a positively charged amino acid or a polar amino acid at position 312 of the EU amino acid position;

A charged amino acid was substituted at position 313 of the EU amino acid position;

A charged amino acid was substituted at position 315 of the EU amino acid position;

A positively charged amino acid was substituted at position 316 of the EU amino acid position;

Substitute a charged amino acid or a polar amino acid at position 317 of the EU amino acid position;

A charged amino acid was substituted at position 340 of the EU amino acid position;

A polar amino acid or a charged amino acid is substituted at position 343 of the EU amino acid position;

Leucine was substituted at position 344 of the EU amino acid position;

A polar amino acid or a charged amino acid is substituted at position 345 of the EU amino acid position;

A polar amino acid or a charged amino acid is substituted at position 376 of the EU amino acid position;

Serine was substituted at position 378 of the EU amino acid position;

A charged amino acid was substituted at position 383 of the EU amino acid position;

A charged amino acid was substituted at position 385 of the EU amino acid position;

A negatively charged amino acid was substituted at position 389 of the EU amino acid position;

A charged amino acid was substituted at position 424 of the EU amino acid position;

A charged amino acid was substituted at position 426 of the EU amino acid position;

A polar amino acid or a charged amino acid is substituted at position 430 of the EU amino acid position;

A charged amino acid was substituted at position 431 of the EU amino acid position;

A polar amino acid was substituted at position 432 of the EU amino acid position;

Substitute lysine, arginine or leucine at EU amino acid position 434;

substitution at EU amino acid position 436 with a negatively charged amino acid; and

A charged amino acid was substituted at EU amino acid position 438.

In another aspect, the invention relates to an altered polypeptide comprising at least the FcRn binding portion of the Fc region, wherein said polypeptide comprises at least one mutation compared to the starting polypeptide, and wherein said at least one mutation is selected from:

Replace the lysine at position 248 of the EU amino acid position with a charged amino acid;

Replace the aspartic acid at position 249 of the EU amino acid position with a positively charged amino acid;

Replace the leucine at position 251 of the EU amino acid position with a polar amino acid or lysine;

Substitution of methionine at EU amino acid position 252;

Arginine at position 255 of the EU amino acid position is replaced with a polar amino acid;

Replace threonine at position 256 of the EU amino acid position with lysine;

Proline at position 257 of the EU amino acid position is replaced with a charged amino acid;

Replace the glutamic acid at position 258 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace tryptophan at position 277 of the EU amino acid position;

Replacement of valine at EU amino acid position 279 with a charged amino acid;

Replace the aspartic acid at position 280 of the EU amino acid position with a charged amino acid;

Replace the glycine at position 281 of the EU amino acid position with a charged amino acid or glutamine;

Replacement of valine at position 282 of the EU amino acid position with a charged amino acid;

Replace the valine at position 284 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace the histidine or alanine at position 285 of the EU amino acid position with a charged amino acid or a polar amino acid;

Replace histidine or alanine at position 285 of the EU amino acid position with positively charged amino acids, polar amino acids or aspartic acid;

Replace asparagine or lysine at position 286 of the EU amino acid position with glutamic acid, threonine or methionine;

Replace the alanine at position 287 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace the lysine at position 288 of the EU amino acid position with a charged amino acid;

Substitution of threonine at EU amino acid position 289;

Replace the serine at position 304 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace the valine at position 305 of the EU amino acid position with a polar amino acid or a charged amino acid;

Substitution of leucine or valine at position 306 of the EU amino acid position;

replacing threonine or valine at position 307 of the EU amino acid position with a polar or charged amino acid;

Replacement of valine at EU amino acid position 308 with a charged amino acid;

replacing the leucine at position 309 of the EU amino acid position with a charged amino acid;

Replace the histidine at position 310 of the EU amino acid position with a charged amino acid or a polar amino acid;

Replace glutamine at EU amino acid position 311 with a positively charged amino acid;

Replace the aspartic acid or leucine at the 312th position of the EU amino acid position with a positively charged amino acid or a polar amino acid;

Substitution of asparagine at position 313 of the EU amino acid position with a charged amino acid;

Replace the asparagine at position 315 of the EU amino acid position with a charged amino acid;

Replace asparagine at position 316 of the EU amino acid position with a positively charged amino acid;

Replace the lysine at position 317 of the EU amino acid position with a charged amino acid or a polar amino acid;

Replace the lysine at position 340 of the EU amino acid position with a charged amino acid;

Replace the proline at position 343 of the EU amino acid position with a polar amino acid or a charged amino acid;

Arginine at position 344 of the EU amino acid position is replaced by leucine;

Replace the glutamic acid at position 345 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace the aspartic acid at position 376 of the EU amino acid position with a polar amino acid or a charged amino acid;

Replace alanine at position 378 of the EU amino acid position with serine;

Replace the serine at position 383 of the EU amino acid position with a charged amino acid;

Replace glycine at position 385 of the EU amino acid position with a charged amino acid;

Replace asparagine at position 389 of the EU amino acid position with a negatively charged amino acid;

Substituting the serine at position 424 of the EU amino acid position with a charged amino acid;

Replace the serine at position 426 of the EU amino acid position with a charged amino acid;

Replace the glutamic acid at position 430 of the EU amino acid position with a polar amino acid or a charged amino acid;

replacing the leucine at position 431 of the EU amino acid position with a charged amino acid;

Replace the histidine at position 432 of the EU amino acid position with a polar amino acid;

The asparagine at position 434 of the EU amino acid position is replaced by lysine, arginine or leucine;

replacing the tyrosine at EU amino acid position 436 with a negatively charged amino acid; and

Glutamine at EU amino acid position 438 was replaced with a charged amino acid.

In another aspect, the invention relates to an altered polypeptide according to claim 1 or 2, wherein the amino acid substitution at at least one of EU amino acid positions 277, 289, 306, 344 or 378 is a charged amino acid, a polar amino acid or a non- polar amino acids.

In one embodiment, the charged amino acid is a negatively charged amino acid.

In one embodiment, the negatively charged amino acid is selected from aspartic acid and glutamic acid.

In one embodiment, the charged amino acid is a positively charged amino acid.

In one embodiment, the positively charged amino acid is selected from arginine, histidine and lysine.

In one embodiment, the positively charged amino acid is lysine.

In one embodiment, the polar amino acid is selected from the group consisting of: methionine, phenylalanine, tryptophan, serine, threonine, tyrosine, asparagine, glutamine and cysteine.

In one embodiment, the non-polar amino acid is selected from the group consisting of alanine, leucine, isoleucine, valine, glycine and proline.

In one embodiment, the altered polypeptide is an antibody or fragment thereof.

In one embodiment, the altered polypeptide is a fusion protein.

In one embodiment, the Fc region or FcRn binding portion thereof is derived from a human antibody.

In one embodiment, the polypeptide comprises an entire Fc region.

In one embodiment, the starting polypeptide comprises the amino acid sequence of SEQ ID NO:2.

In one embodiment, the Fc region or Fc-binding portion thereof is of the IgG isotype.

In one embodiment, the IgG isotype is IgG1 subtype.

In one embodiment, the polypeptide comprises one or more non-human amino acid residues in a complementarity determining region ₍ CDR) of a VL or _VH .

In one embodiment, the polypeptide binds (a) an antigen and (b) an FcR.

In one embodiment, the antigen is a tumor-associated antigen.

In one embodiment, the polypeptide binds (a) a ligand and (b) an FcR.

In one embodiment, the FcR is FcRn.

In one embodiment, the polypeptide binds the FcR with a different binding affinity than the starting polypeptide without the mutation.

In one embodiment, the altered polypeptide has a binding affinity that is about 1.5-fold to about 100-fold higher than the starting polypeptide.

In one embodiment, the altered polypeptide has a binding affinity that is about 1.5-fold to about 100-fold lower than that of the starting polypeptide.

In one embodiment, the altered polypeptide exhibits a binding affinity for an FcR at a first pH and exhibits a different binding affinity for an FcR at a second pH.

In one embodiment, the binding affinity of the altered polypeptide is about 1.5-fold to about 100-fold higher at the first pH than at the second pH.

In one embodiment, the binding affinity of the altered antibody is about 1.5 to about 100 times lower at the first pH than at the second pH.

In one embodiment, the altered polypeptide exhibits a different circulating half-life when administered to a patient than the non-mutated starting polypeptide.

In one embodiment, the half-life of the altered polypeptide is about 1 hour to about 1 week longer than the half-life of the starting polypeptide without the mutation.

In one embodiment, the half-life of the altered polypeptide is about 1 hour to about 1 week shorter than the half-life of the starting polypeptide without the mutation.

In one embodiment, the altered polypeptide binds protein A or G.

In one embodiment, it relates to the altered polypeptide of claim 1 or 2.

In one embodiment, the invention relates to a nucleotide sequence encoding the polypeptide of claim 1 or 2.

In one embodiment, the nucleic acid is an expression vector.

In one embodiment, the expression vector is in a host cell.

In one embodiment, the present invention relates to a method of treating a patient suffering from a disease, said method comprising administering to the patient an altered polypeptide comprising at least the FcRn binding portion of the Fc region, wherein said polypeptide comprises at least one mutation selected from the group consisting of :

Glutamic acid was substituted at position 284 of the EU amino acid position;

Glutamic acid was substituted at position 285 of the EU amino acid position;

Substitute aspartic acid at position 286 of the EU amino acid position;

Glutamic acid or aspartic acid is substituted at position 288 of the EU amino acid position;

a glutamic acid substitution at EU amino acid position 290; and

Substitution of aspartic acid at EU amino acid position 304,

Among other things, the altered polypeptide exhibits a longer circulating half-life than the starting polypeptide without the mutation.

In one embodiment, the invention relates to a method of treating a patient suffering from a disease, said method comprising administering to the patient an altered polypeptide comprising at least the FcRn binding portion of the Fc region, said polypeptide comprising at least one mutation selected from the group consisting of:

Replace the valine at position 284 of the EU amino acid position with glutamic acid;

Replace histidine at position 285 of the EU amino acid position with glutamic acid;

The asparagine at position 286 of the EU amino acid position is replaced by aspartic acid;

Replace the lysine at position 288 of the EU amino acid position with glutamic acid or aspartic acid;

replacing lysine at EU amino acid position 290 with glutamic acid; and

The serine at position 304 of the EU amino acid position is replaced by aspartic acid,

Among other things, the altered polypeptide has a longer circulating half-life than the starting polypeptide without the mutation.

In one embodiment, the invention relates to a method of treating a patient suffering from a disorder, said method comprising administering to the patient an altered polypeptide comprising at least the FcRn binding portion of the Fc region, said polypeptide comprising at least one mutation selected from the group consisting of:

Replace EU amino acid position 248 with aspartic acid;

Replace the EU amino acid position 249 with arginine or lysine;

Replace the EU amino acid position 250 with arginine or lysine;

Substitution of EU amino acid position 251 with arginine, lysine or asparagine;

Replace the EU amino acid position 252 with serine or threonine;

Replace the EU amino acid position 254 with serine or threonine;

Replace EU amino acid position 256 with arginine, glutamic acid, or lysine;

Substitution of EU amino acid position 255 with leucine, aspartic acid or methionine;

Replace EU amino acid position 260 with lysine;

Replace EU amino acid position 257 with arginine, aspartic acid, glutamic acid or lysine;

Replace EU amino acid position 277 with arginine, aspartic acid, glutamine or lysine;

Replace EU amino acid position 279 with glutamic acid;

Replace EU amino acid position 281 with glutamine;

Replace the EU amino acid position 282 with arginine, aspartic acid, glutamic acid or lysine;

Substitute EU amino acid position 287 with aspartic acid, glutamic acid, lysine, proline or threonine;

Replace EU amino acid position 284 with aspartic acid;

Replace EU amino acid position 285 with aspartic acid or phenylalanine;

Replace the EU amino acid position 286 with glutamic acid or methionine;

Replace EU amino acid position 288 with aspartic acid;

Replace EU amino acid position 290 with aspartic acid;

Replace EU amino acid position 304 with aspartic acid or glutamic acid;

Replace EU amino acid position 305 with arginine;

Substitute EU amino acid position 306 with arginine, aspartic acid, glutamic acid or lysine;

Substitute EU amino acid position 307 with arginine, aspartic acid or glutamic acid;

Substitute EU amino acid position 309 with arginine, aspartic acid, lysine or glutamic acid;

Substitute EU amino acid position 310 with arginine, leucine, lysine or asparagine;

Substitution of EU amino acid position 312 with arginine, asparagine, or lysine;

Substitute EU amino acid position 313 with aspartic acid, arginine or lysine;

Replace the EU amino acid position 315 with aspartic acid or glutamic acid;

Replace the EU amino acid position 343 with glutamine or lysine;

Replace the EU amino acid position 345 with arginine or glutamine;

Substitute EU amino acid position 374 with arginine, lysine or leucine;

Replace EU amino acid position 376 with asparagine;

Substitute EU amino acid position 426 with arginine, aspartic acid or glutamic acid;

Substitute EU amino acid position 428 with arginine, glutamine or lysine;

Replace EU amino acid position 430 with lysine;

Replace EU amino acid position 431 with proline;

Replace EU amino acid position 432 with arginine;

replacing EU amino acid position 434 with leucine or lysine; and

Replace EU amino acid position 438 with glutamic acid,

Wherein the altered polypeptide has a shorter circulating half-life than the starting polypeptide without the mutation.

In another aspect, the invention relates to a method of treating a patient suffering from a disorder, said method comprising administering to the patient an altered polypeptide comprising at least the FcRn binding portion of the Fc region, said polypeptide comprising at least one mutation selected from the group consisting of:

The lysine at position 248 of the EU amino acid position is replaced by aspartic acid;

Replace aspartic acid at position 249 of the EU amino acid position with arginine or lysine;

Replace threonine at position 250 of the EU amino acid position with arginine or lysine;

The leucine at position 251 of the EU amino acid position is replaced by arginine, lysine or asparagine;

Replace the methionine at position 252 of the EU amino acid position with serine or threonine;

Replace the methionine at position 254 of the EU amino acid position with serine or threonine;

Substituting threonine at position 256 of the EU amino acid position with arginine, glutamic acid or lysine;

Arginine at position 255 of the EU amino acid position is replaced by leucine, aspartic acid or methionine;

Replace threonine at position 260 of the EU amino acid position with lysine;

Proline at position 257 of the EU amino acid position is replaced by arginine, aspartic acid, glutamic acid or lysine;

Replace tryptophan at position 277 of the EU amino acid position with arginine, aspartic acid, glutamine, or lysine;

Replace the valine at position 279 of the EU amino acid position with glutamic acid;

Glycine at position 281 of the EU amino acid position is replaced by glutamine;

Substituting valine at position 282 of the EU amino acid position with arginine, aspartic acid, glutamic acid or lysine;

Replace the alanine at position 287 of the EU amino acid position with aspartic acid, glutamic acid, lysine, proline or threonine;

The valine at position 284 of the EU amino acid position is replaced by aspartic acid;

Replace histidine at position 285 of the EU amino acid position with aspartic acid or phenylalanine;

Replace asparagine at position 286 of the EU amino acid position with glutamic acid or methionine;

The lysine at position 288 of the EU amino acid position is replaced by aspartic acid;

The lysine at position 290 of the EU amino acid position is replaced by aspartic acid;

Replace the serine at position 304 of the EU amino acid position with aspartic acid or glutamic acid;

Replace the valine at position 305 of the EU amino acid position with arginine;

Substituting leucine at position 306 of the EU amino acid position with arginine, aspartic acid, glutamic acid or lysine;

Substituting threonine at position 307 of the EU amino acid position with arginine, aspartic acid or glutamic acid;

Substituting leucine at position 309 of the EU amino acid position with arginine, aspartic acid, lysine or glutamic acid;

Substituting histidine at position 310 of the EU amino acid position with arginine, leucine, lysine or asparagine;

Replace the aspartic acid at position 312 of the EU amino acid position with arginine, asparagine, or lysine;

Replace tryptophan at position 313 of the EU amino acid position with aspartic acid, arginine or lysine;

Replace asparagine at position 315 of the EU amino acid position with aspartic acid or glutamic acid;

Replace the proline at position 343 of the EU amino acid position with glutamine or lysine;

Replace the glutamic acid at position 345 of the EU amino acid position with arginine or glutamine;

Proline at position 374 of the EU amino acid position is replaced by arginine, lysine or leucine;

Replace aspartic acid 376 at the EU amino acid position with asparagine;

Substituting serine at position 426 of the EU amino acid position with arginine, aspartic acid or glutamic acid;

Replace the methionine at position 428 of the EU amino acid position with arginine, glutamine, or lysine;

Replace glutamic acid at position 430 of the EU amino acid position with lysine;

Replace the alanine at position 431 of the EU amino acid position with proline;

Replace the leucine at position 432 of the EU amino acid position with arginine;

replacing asparagine at EU amino acid position 434 with leucine or lysine; and

Replace glutamine at EU amino acid position 438 with glutamic acid,

Among other things, the altered polypeptide exhibits a shorter circulating half-life than the starting polypeptide without the mutation.

In one embodiment, the present invention relates to a method of preparing the altered polypeptide of claim 1 or 2, said method comprising:

(a) transfecting a cell with a nucleic acid molecule comprising a nucleotide sequence encoding said altered polypeptide; and

(b) purifying the altered polypeptide from said cells or cell supernatant.

In one embodiment, the invention relates to a method of preparing the antibody of claim 11 or 13, said method comprising:

(a) providing the first nucleic acid molecule comprising the nucleotide sequence of the variable region (V _L ) and the constant region ( _CL ) of the light chain encoding said antibody;

(b) providing a second nucleic acid molecule comprising nucleotide sequences encoding the variable region (V _H ) and constant regions (CH ₁ , CH ₂ and CH ₃ ) of the heavy chain of said antibody;

(c) transfecting a cell with the first and second nucleic acid molecules under conditions that permit expression of said altered antibody comprising said encoded light chain and heavy chain; and

(d) purifying said antibody from said cells or cell supernatant.

In one embodiment, the invention relates to a dimeric Fc-binding protein comprising first and second polypeptide chains, wherein each of said first and second polypeptide chains comprises at least one Fc operably linked to an Fc-binding domain region domain.

In one embodiment, the Fc domain is mutated to reduce or eliminate binding to FcRn.

In one embodiment, said first and second polypeptide chains are covalently linked.

In one embodiment, the Fc binding domain comprises the extracellular domain of FcRn.

In one embodiment, the Fc binding domain binds to β2 microglobulin.

In one embodiment, the Fc binding domain is derived from human FcRn.

In one embodiment, the binding protein comprises the amino acid sequence shown in SEQ ID NO: 10.

In one embodiment, the present invention relates to a method for measuring the binding affinity (affinity) of a polypeptide comprising at least an FcRn binding portion of an Fc region to FcR, said method comprising making a polypeptide comprising at least an FcRn binding portion of an Fc region combined with claim 44 contacts of dimeric Fc-binding proteins and determine the avidity of the interaction.

In one embodiment, the present invention relates to a method of screening a polypeptide having altered binding affinity for FcRn from a library of polypeptides comprising at least an FcRn-binding portion of an Fc region, the method comprising:

(a) contacting library members with the dimeric Fc binding protein of claim 44; and

(b) measuring the binding affinity of the polypeptide for the dimeric Fc-binding protein; and

(c) selecting polypeptides with altered binding affinity for FcRn.

In one embodiment, the present invention relates to a method for purifying a polypeptide comprising at least the FcRn-binding portion of an Fc region from a mixture of polypeptides, the method comprising loading the mixture onto an affinity column comprising a dimeric Fc-binding protein according to claim 44, The polypeptide is purified by eluting the polypeptide comprising at least the FcRn-binding portion of the Fc region.

In one embodiment, the mixture is loaded onto an affinity column at a first pH and the polypeptide is eluted from the affinity column at a second pH.

In one embodiment, the polypeptide is not denatured during purification.

In one embodiment, the invention relates to a method of identifying a polypeptide having an altered binding affinity for FcRn compared to a starting polypeptide, the method comprising:

(a) determining the spatial representation of the optimal charge distribution of the amino acids of the starting polypeptide upon binding to FcRn in a solvent and the associated change in the free energy of binding of the starting polypeptide;

(b) identifying at least one candidate amino acid residue position in the starting polypeptide to be modified to alter the binding free energy of the starting polypeptide when binding to FcRn; and

(c) identifying an amino acid selected at the amino acid position such that incorporation of the mutation in the starting polypeptide results in an altered polypeptide having an altered binding affinity for FcRn.

In one embodiment, the method further comprises incorporating the selected amino acid into the starting polypeptide.

In one embodiment, the method further comprises calculating the change in free energy of binding of the altered Fc-containing polypeptide compared to the starting polypeptide when binding to FcRn.

In one embodiment, the calculating step first comprises in silico modeling of mutations in the starting polypeptide and then calculating the change in free energy of binding.

In one embodiment, said calculation step uses at least one determination method selected from the group consisting of electrostatic binding energy and van der Waals binding energy determined using methods based on the Poisson-Boltzmann formula, and using methods based on solvent-accessible surface area method to determine the binding energy.

In one embodiment, the mutation is an amino acid substitution.

In one embodiment, amino acid substitutions result in the incorporation of a candidate amino acid having a different charge than the candidate amino acid.

In one embodiment, the mutation increases the free energy of binding of the altered Fc-containing polypeptide and FcRn in a solvent, thereby reducing the binding affinity of the altered Fc-containing polypeptide for FcRn.

In one embodiment, the mutation reduces the free energy of binding between the Fc-containing altered polypeptide and FcRn in a solvent, thereby increasing the binding affinity of the Fc-containing altered polypeptide for FcRn.

In one aspect, the invention relates to a method of identifying an altered Fc-containing polypeptide having an altered binding affinity for FcRn at two different pH levels, the method comprising:

(a) determining the spatial representation of the optimal charge distribution of the amino acids of the starting polypeptide and the associated change in the free energy of binding of the starting polypeptide upon binding to FcRn in a solvent at a first pH level;

(b) determining the spatial representation of the optimal charge distribution of the amino acids of the starting polypeptide upon binding to FcRn in a solvent at the second pH level and the associated change in the free energy of binding of the starting polypeptide;

(c) identifying at least one candidate in the starting polypeptide to be modified to alter the binding free energy of the starting polypeptide when binding to FcRn by comparing the charge distribution, residues exhibiting different charge distributions at the first and second pH levels amino acid residue positions; and

(d) selecting the selected amino acid at said amino acid position such that incorporation of the selected amino acid into the starting polypeptide results in an Fc-containing altered polypeptide having an altered binding affinity for FcRn.

In one embodiment, the first pH is about 7.4.

In one embodiment, the affinity of the polypeptide is about 1.5 to about 100 times higher at the first pH than at the second pH.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. The contents of all patents, patent applications and other references cited in this specification are hereby incorporated by reference in their entirety.

Brief description of the drawings

Figure 1A shows the DNA sequence of the mature huCBE11 heavy chain used as the starting polypeptide in the methods of the invention. This DNA sequence is encoded in the pEAG1787 expression vector. Figure IB shows the predicted amino acid sequence of the mature huCBE11 heavy chain.

Figure 2 shows the amino acid sequence of the Fc region of the huCBE11 heavy chain used as the starting polypeptide in the methods of the present invention. Amino positions are indicated by EU numbering in Kabat.

Figure 3A shows the DNA sequence of the huCBE11 light chain. This DNA sequence is encoded in the pEAG1754 expression vector. Figure 3B shows the predicted amino acid sequence of the huCBE11 light chain.

Figure 4A shows the DNA sequence of human beta microglobulin. This DNA sequence is encoded in the pEAG1761 expression vector. Figure 3B shows the predicted amino acid sequence of human beta microglobulin.

Figure 5A shows the nucleotide sequence of cDNA encoding human FcRn/Fc/fusion protein. This DNA sequence is encoded in the pEAG1761 expression vector. Figure 3B shows the predicted amino acid sequence of the FcRn/Fc/fusion protein.

Figure 6 shows the results obtained using a FRET-based assay to evaluate the FcRn binding affinity of the altered polypeptides of the invention. Mutations with measurable increases in binding affinity (H285E, N286D, K290E, and S304D) are indicated with downward pointing arrows. The mutation (N434L) with significantly reduced binding affinity is indicated with a left-pointing arrow.

Figure 7 shows the structure obtained using the AlphaScreen assay to evaluate the FcRn binding affinity of the altered polypeptides of the present invention. Mutations that measurably increased binding affinity (V284E, H285E, N286D, and K290E) are indicated with downward pointing arrows. The mutation (Q438E) with significantly reduced binding affinity is indicated with a left-pointing arrow.

Fig. 8 shows a structural model of the Fc region of the huCBE11 heavy chain used in the present invention. Specific "up mutants" with enhanced FcRn affinity (V284E, H285E, N286E, K290E) and specific "down mutants" with reduced FcRn affinity (V282E, M252T , N434L, Q438L) are indicated relative to the other domains of FcRn.

Panel A in FIG. 9 shows the location of the FcRn binding loop, which extends from aspartate 280 to threonine 299 shown in FIG. 9 for the FcRn domain. Panel B shows the positions of residues in the FcRn contact region of the relevant FcRn domain (e.g., 243F; 244P; 245P; 246K; 247P; 248K; 249D; 250T; 251L; 252M; ;258E; 259V; 260T; 261C; 275F; 276N; 277W; 278Y; 279V; 280D; 282V; 283E; 284V; 285H; 286N; 287A; ;305V; 306L; 307T; 308V; 309L; 310H; 311Q; 312D; 313W; 314L; 346P; 347Q; 348V; 367C; 369V; 372F; 373Y; 374P; 375S; 376D; 377I; 378A; 379V; ; 408S; 424S; 425C; 426S; 427V; 428M; 429H; 430E; 431A; 432L; 433H; 434N; 435H; 436Y; 437T; 438Q; 439K; and 440S).

Figure 10 shows the binding of biotinylated hIgG to hFcRnFc coated on ELISA plates at different concentrations of 3, 6 and 9 μg/ml. Concentrations of biotinylated hIgG were varied as indicated and HPR-streptavidin was used at a concentration of 1:20,000 and developed using standard protocols. After stopping the reaction, the absorbance at 450 nm was read. 9 μg/ml (□) and 6 μg/ml (o) hFcRnFc showed no change in the binding curve, while 3 μg/ml (Δ) hFcRnFc showed reduced binding to coated hIgG. Negative control (◇) shows background binding of streptavidin HRP in the absence of hFcRnFc.

Detailed description of the invention

The present invention is based at least in part on the identification of polypeptides (e.g., antibodies and fusion proteins) that include at least a portion of an Fc region that exhibits altered binding to neonatal Fc receptors (FcRn) (e.g., immunoglobulins such as constant region of IgG1). The altered polypeptide exhibits increased or decreased binding to FcRn compared to the wild-type polypeptide and thus has increased or decreased half-life in serum, respectively. Fc regions with increased affinity for FcRn are expected to have a longer serum half-life, and such molecules may be useful in methods of treatment of mammalian animals where a long half-life of the administered polypeptide is desired, eg, for the treatment of chronic diseases or conditions. Conversely, Fc region variants with reduced FcRn binding affinity are expected to have shorter half-lives, such molecules may also be useful, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, with In vivo diagnostic imaging or in cases where the starting polypeptide has toxic side effects if it persists in circulation for a long time. Fc region variants with reduced FcRn binding affinity are also less likely to cross the placenta and thus may also be useful in the treatment of diseases or conditions in pregnant women. Furthermore, other applications in which reduced FcRn binding affinity may be desired include those in which localization to the brain, kidney and/or liver is desired. In an exemplary embodiment, the altered polypeptide of the invention exhibits reduced transport from the vasculature across the glomerular epithelium. In another embodiment, the altered polypeptides of the invention exhibit reduced transport from the brain across the blood-brain barrier (BBB) into the vascular space.

The invention also relates to methods of making such altered polypeptides and methods of using such polypeptides. The invention also relates to novel forms of FcRn and methods of their use.

Various aspects of the invention are described in further detail in the following subsections:

I. Definition

The terms "protein", "polypeptide" and "peptide" are used interchangeably herein. A protein may contain one or more natural amino acids or unnatural amino acids.

A "starting polypeptide" or "first polypeptide" is a polypeptide comprising an amino acid sequence that lacks one or more Fc region modifications disclosed herein and that has a different effector function than the altered or modified polypeptide. The starting polypeptide is a natural or artificial polypeptide comprising an Fc region or an FcRn-binding portion thereof. The starting polypeptide may comprise a native Fc region sequence or an Fc region with pre-existing amino acid sequence modifications (eg additions, deletions and/or substitutions). The starting polypeptides of the invention are modified as indicated herein to modulate (increase or decrease) binding affinity to FcRn.

Herein, the term "altered polypeptide" or "second polypeptide" refers to a polypeptide comprising a non-native Fc-binding portion comprising at least one mutation in the Fc region. When referring to an altered polypeptide exhibiting "altered effector functions", it is meant that said altered polypeptide favors one or more of them (and possibly, but not required, all) effector functions.

Herein, the term "Fc region" includes amino acid sequences derived from the constant region of an antibody heavy chain. The Fc region is part of the constant region of the heavy chain of an antibody, starting at the N-terminus of the hinge region at the papain cleavage site (approximately position 216 according to the EU index), comprising the hinge, CH2 and CH3 domains.

The starting polypeptide may include at least a part of the Fc region that mediates binding to FcRn. For example, in one embodiment, the starting polypeptide is an antibody or an Fc fusion protein. As used herein, "fusion protein" refers to a chimeric polypeptide comprising a first amino acid sequence linked to a second amino acid sequence, wherein the first and second amino acid sequences are not naturally linked. For example, a fusion protein may comprise an amino acid sequence encoding at least a portion of an Fc region (e.g., the portion of the Fc region that confers binding properties to FcRn) and an amino acid sequence encoding a non-immunoglobulin polypeptide (e.g., the ligand binding domain of a receptor or the ligand binding domain of a ligand). amino acid sequence of the receptor binding domain). These amino acid sequences normally exist in separate proteins but are pulled together in the fusion polypeptide; or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be produced, for example, by chemical synthesis, or by construction and translation of polynucleotides encoding these peptide regions in the desired relationship.

Herein, the terms "linked", "fused" and "fused" are used interchangeably. These terms refer to two or more elements or components joined together by any means, including chemical conjugation or recombinant methods. "In-frame fusion" or "operably linked" refers to the joining of two or more reading frames (ORFs) in a manner that maintains the correct reading frames of the original ORFs to form a continuous longer ORF. Thus, the resulting recombinant fusion protein is a single protein comprising two or more segments corresponding to the polypeptide encoded by the original ORF (the segments are not normally so linked in nature). Although the reading frames thus produced are contiguous across the fused segments, these segments may be physically or spatially separated by, for example, in-frame linker sequences.

In one embodiment, a polypeptide of the invention comprises an immunoglobulin antigen binding site or the portion of a receptor molecule responsible for ligand binding or the portion of a ligand molecule responsible for receptor binding.

Herein, the term "effector function" refers to the functional ability of an Fc region or part thereof to bind proteins and/or cells of the immune system and mediate various biological effects. Effector functions can be antigen-dependent or antigen-independent.

Herein, the term "antigen-dependent effector function" refers to an effector function that is normally induced following binding of an antibody to the corresponding antigen. Typical antigen-dependent effector functions include the ability to bind complement proteins (eg, C1q). For example, binding of the C1 component of complement to the Fc region can activate the classical complement system, leading to opsonization and lysis of cellular pathogens, a process termed complement-dependent cytotoxicity (CDCC). Activation of complement also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity.

Other antigen-dependent effector functions are mediated by the binding of antibodies through their Fc regions to certain Fc receptors ("FcRs") on cells. There are several pairs of different antibody classes, including IgG (gamma receptor, or IgγR), IgE (ε receptor, or IgεR), IgA (alpha receptor, or IgαR), and IgM (mu receptor, or IgμR), with Specific Fc receptors. Binding of antibodies to Fc receptors on the cell surface triggers a number of important different biological responses, including endocytosis of immune complexes, ingestion and destruction of antibody-coated particles or microorganisms (also known as antibody-dependent phagocytosis, or ADCP ), clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, regulation of immune system cell activation, immune Placental transfer and control of globulin production.

Certain Fc receptors, Fc gamma receptors (FcγRs), play key roles in abolishing or enhancing immune recruitment. FcγRs are expressed on leukocytes and consist of three distinct types: FcγRI, FcγRII and FcγRIII. The Fc region of the IgG immunoglobulin isotype (Gessner et al., Ann. Hematol., (1998), 76:231-48). Structurally, these FcyRs are all members of the immunoglobulin superfamily with an IgG-binding alpha chain with an extracellular portion consisting of two or three Ig-like domains. Human FcγRI (CD64) is expressed on human monocytes and exhibits high affinity binding to monomeric IgG1, IgG3 and IgG4 (Ka=10 ⁸ -10 ⁹ M ⁻¹ ). Human FcyRII (CD32) and FcyRIII (CD16) have low affinity for IgGl and IgG3 (Ka<10 ⁷ M ⁻¹ ) and are only able to bind these IgG isotypes in complex or multimeric form. In addition, FcyRII and FcyRIII types include "A" and "B" forms. FcγRIIa (CD32a) and FcγRIIIa (CD16a) bind to the surface of macrophages, NK cells, and some T cells through transmembrane domains, while FcγRIIb (CD32b) and FcγRIIIb (CD16b) are selected through phosphatidylinositol (GPI) anchors Binds positively to the cell surface of granulocytes such as neutrophils. The corresponding murine homologues of human FcyRI, FcyRII and FcyRIII are FcyRIIa, FcyRIIb/1 and FcyR1o.

Herein, the term "antigen-independent effector function" refers to an effector function that can be induced by an antibody regardless of whether the antibody has bound its corresponding antigen or not. Typical antigen-independent effector functions include cellular transport, circulating half-life, and clearance rates of immunoglobulins. A structurally distinct Fc receptor, the "neonatal Fc receptor" or "FcRn", also known as the salvage receptor, has a key role in regulating these functions. Preferably, the FcRn to which the polypeptide of the present invention binds is human FcRn.

Unlike FcγRs belonging to the immunoglobulin superfamily, human FcRn is structurally similar to polypeptides of the class I major histocompatibility complex (MHC) (Ghetie and Ward, Immunology Today, (1997), 18(12):592 -8). FcRn is typically expressed as a heterodimer consisting of a complex of a transmembrane alpha or heavy chain and a soluble beta or light chain (beta2 microglobulin). FcRn shares 22-29% sequence identity with MHC class I molecules and has a non-functional form of the MHC peptide binding groove (Simister and Mostov, Nature, (1989), 337:184-7). Like MHC, the α-chain of FcRn consists of three extracellular domains (α1, α2, α3) and a short cytoplasmic tail anchors the protein to the cell surface. The α1 and α2 domains interact with the FcR binding site in the Fc region of antibodies (Raghavan et al., Immunity, (1994), 1:303-15).

FcRn is expressed in the maternal placenta or yolk sac of mammals and is involved in the transfer of IgG from the mother to the fetus. FcRn is also expressed in the small intestine of neonatal rodents, where FcRn is involved in the transfer of maternal IgG from ingested colostrum or milk across the brush border epithelium. FcRn is also expressed in many other tissues in many species, and in various endothelial cell lines. It is also expressed in adult serum endothelium, muscle vasculature and liver sinusoids. FcRn is also thought to be involved in maintaining the circulating half-life or serum levels of IgG by binding IgG and recycling IgG to serum. Binding of FcRn to IgG molecules is strictly pH dependent, with optimal binding at pH less than 7.0.

Herein, the term "half-life" refers to the biological half-life of a particular Fc-containing polypeptide in vivo. Half-life can be expressed by the time required to clear from the animal's circulation and/or other tissues half the amount administered to a subject. When the clearance profile for a given Fc-containing polypeptide was constructed as a function of time, the profile was generally biphasic, with a fast alpha phase and a longer beta phase. The alpha phase typically represents the balance of the administered Fc polypeptide between the intravascular space and the extravascular space, which depends in part on the size of the polypeptide. The beta phase typically represents the metabolism of Fc polypeptides in the intravascular space. Thus, in a preferred embodiment, the term "half-life" as used herein refers to the half-life of the beta-phase Fc polypeptide. The typical beta-phase half-life of a human antibody is 21 days.

Herein, the term "mutation" includes substitution, addition or deletion of amino acids in a starting polypeptide to obtain an altered polypeptide.

"Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with another different "replacement" amino acid residue. The one or more replacement residues may be "natural amino acid residues" (i.e., encoded by the genetic code) and are selected from: alanine (A); arginine (R); asparagine (N ); Aspartic acid (D); Cysteine (C); Glutamine (Q); Glutamic acid (E); Glycine (G); Histidine (H); Isoleucine (I ); Leucine (L); Lysine (K); Methionine (M); Phenylalanine (F); Proline (P); Serine (S); Threonine (T) ; tryptophan (W); tyrosine (Y); and valine (V). Substitution with one or more unnatural amino acid residues is also included within the definition of amino acid substitution herein. "Unnatural amino acid residue" refers to a residue other than the naturally occurring amino acid residues listed above that is capable of covalently binding adjacent amino acid residues in a polypeptide chain. Examples of unnatural amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogs, such as those described in Ellman et al. Meth. Enzym. 202:301-336 (1991) Those ones. To prepare these unnatural amino acid residues, the methods of, for example, Noren et al. Science 244:182 (1989) and Ellman et al., supra, can be used. Briefly, these methods involve in vitro transcription and translation of a suppressor tRNA following chemical activation of the suppressor tRNA with unnatural amino acid residues.

Herein, the term "non-polar" includes amino acids with uncharged side chains (eg, A, L, I, V, G, P). These amino acids generally participate in hydrophobic interactions.

Herein, the term "polar" includes amino acids with zero net charge but non-zero partial charges at different parts of their side chains (eg, M, F, W, S, Y, N, Q, C). These amino acids can participate in hydrophobic and electrostatic interactions.

Herein, the term "charged" includes amino acids that may have a non-zero net charge on the side chain (eg, R, K, H, E, D). These amino acids can participate in hydrophobic and electrostatic interactions.

"Amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. Although insertions are typically insertions of one or more amino acid residues, larger "peptide insertions" of the invention can be made, eg, insertions of about 3 to about 5 or even up to about 10 amino acid residues. As disclosed above, the inserted residues may be natural or non-natural.

"Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

Herein, the term "sufficiently large steric bulk" includes amino acids having side chains that occupy a relatively large 3-dimensional space. Exemplary amino acids with side chain chemistries of sufficiently large steric bulk include tyrosine, tryptophan, arginine, lysine, histidine, glutamic acid, glutamine, and methionine, or the like object or simulant.

Herein, the term "solvent-accessible surface area" refers to the surface area of atoms contacting solvent molecules. Solvent accessible surface area can be calculated using methods well known in the art. In short, an atom or a group of atoms is defined as accessible if it can make van der Waals contact with a solvent (water) molecule of a specified size. Van der Waals contact is the position of the center of the solvent molecule when the solvent molecule rolls along the protein causing the maximum allowable contact.

The term "binding affinity" herein includes the strength of the binding interaction and thus actual and apparent binding affinity. Actual binding affinity is the ratio of on rate to off rate. Thus, conferring or optimizing binding affinity includes altering one or both of these components to obtain a desired level of binding affinity. Apparent affinity can include, for example, avidity of an interaction.

The term "free energy of binding" or "free energy of binding" includes its art-recognized meaning herein and applies especially to Fc-Fc receptor interactions in a solvent. A decrease in binding free energy will increase affinity whereas an increase in binding free energy will decrease affinity.

The term "binding domain" or "binding site" refers herein to one or more regions of a polypeptide that mediate specific binding to a target molecule (eg, antigen, ligand, receptor, substrate or inhibitor). Exemplary binding domains include antibody variable regions, receptor binding domains of ligands, ligand binding domains of receptors, or enzyme domains. The term "ligand binding domain" refers herein to any native receptor (e.g. a cell surface receptor) or any region of a native receptor that retains at least qualitative ligand binding ability and preferably the biological activity of the corresponding native receptor or derivatives. The term "receptor binding domain" refers herein to any natural ligand, or any region or derivative of a natural ligand that retains at least a qualitative receptor binding ability and preferably the biological activity of the corresponding natural ligand. In one embodiment, the polypeptide has at least one binding domain specific for a molecule targeted for reduction or elimination, such as a cell surface antigen or a soluble antigen. In preferred embodiments, the binding domain is an antigen binding site.

In a preferred embodiment, a polypeptide of the invention comprises at least one binding site (eg, an antigen binding site, a receptor binding site, or a ligand binding site). In one embodiment, a polypeptide of the invention comprises at least two binding sites. In one embodiment, a polypeptide of the invention comprises three binding sites. In another embodiment, a polypeptide of the invention comprises four binding sites.

Polypeptides of the present invention may be monomeric or multimeric. For example, in one embodiment, a polypeptide of the invention is a dimer. In one embodiment, the dimers of the invention are homodimers, comprising two identical monomeric subunits. In one embodiment, the dimers of the invention are heterodimers comprising two different monomeric subunits. A subunit of a dimer may comprise one or more polypeptide chains. For example, in one embodiment, a dimer comprises at least two polypeptide chains. In one embodiment, the dimer comprises two polypeptide chains. In another embodiment, the dimer comprises four polypeptide chains (eg, in the case of antibody molecules).

The term "exposed" amino acid residue herein includes amino acid residues that, when present in a polypeptide in a solvent, have at least a portion of their surface exposed to some extent to the solvent. Preferably, the exposed amino acid residues are those for which at least about one-third of the surface area of the side chain is exposed to the solvent. Various methods are available for determining whether a residue is exposed, including analysis of molecular models or polypeptide structure.

The terms "variant", "altered polypeptide", "modified polypeptide", "polypeptide comprising modified amino acids" and like terms include herein polypeptides having an amino acid sequence that differs from the amino acids of the starting polypeptide. Typically, such polypeptides have one or more mutations, such as substitution of one or more amino acid residues by another amino acid residue or insertion or deletion of one or more amino acid residues. Preferably, the polypeptide comprises an amino acid sequence comprising at least a portion of a non-native Fc region. The variant necessarily has less than 100% sequence identity or similarity to the starting antibody. In a preferred embodiment, the variant has an amino acid sequence having about 75% to less than 100% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide, more preferably about 80% to less than 100%, more preferably Preferably about 85% to less than 100%, more preferably about 90% to less than 100%, most preferably about 95% to less than 100% amino acid identity or similarity. In one embodiment, there is one amino acid difference between the starting antibody and the modified antibody of the invention. Identity or similarity with respect to this sequence is defined herein as the amino acid residues in a candidate sequence that are identical to the starting amino acid residues after alignment of the sequences and introduction of gaps, if necessary, to achieve the maximum percent sequence identity (i.e., percentage of identical residues). Modified polypeptides of the invention can be expressed, or alternatively can be modeled in silico.

As used herein, the phrase "candidate amino acid residue position" includes an amino acid position identified in a polypeptide of the invention, wherein a substitution at a candidate amino acid position is modeled, predicted or empirically found to result in an alteration, deletion, insertion or substitution of another amino acid The FcRn binding affinity of the polypeptide is modulated after substitution.

The term "selected amino acid" is used herein to refer to an amino acid residue selected by the methods of the invention for incorporation as a replacement amino acid at a candidate amino acid position in a polypeptide. In one embodiment, the candidate amino acid residue position is replaced by an elected amino acid such that the role of electrostatics in the free energy of binding of the Fc-FcRn complex is reduced or increased.

The term "antibody" herein includes natural antibodies obtained or produced from an antibody-producing animal. For example, antibodies may be obtained from rodents such as mice, rats, gerbils, hamsters, or guinea pigs; from larger animals such as rabbits, cats, or dogs; from animals commonly raised as livestock (e.g., pigs, cows, horses, sheep) or goat); or antibodies obtained or produced from primates (including humans and non-human primates). The term "antibody" also includes immunoglobulin molecules and modified immunoglobulin molecules, eg, molecules that contain an antigen binding site that binds (immunoreacts with) an antigen and at least that portion of the Fc region that mediates binding to FcRn. Herein, the term "antibody" also includes modified or synthetic antibody molecules comprising at least a portion of an Fc region.

Herein, the term "hinge region" includes the part of the heavy chain molecule connecting the CH1 domain and the CH2 domain, for example, from about position 216 to position 230 according to the EU numbering system. The hinge region comprises approximately 25 residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol. 1998, 161:4083).

Herein, the term "CH2 domain" includes the portion of the heavy chain molecule extending from, for example, about EU position 231 to EU position 340. The CH2 domain is unique in that it is not closely paired with another domain. Instead, two N-linked branched sugar chains intervene between the two CH2 domains of the intact native IgG molecule.

Herein, the term "CH3 domain" includes that portion of the heavy chain molecule extending about 110 residues from the N-terminus of the CH2 domain (eg about residues 341 to 446, EU numbering system). The CH3 domain typically forms the C-terminal portion of the antibody. In some immunoglobulins, however, additional domains can extend from the CH3 domain to form the C-terminal portion of the molecule (eg, the CH4 domain in the mu chain of IgM and the delta chain of IgE).

In one embodiment, one or more amino acid mutations are made in the "FcRn binding loop" of the Fc region. The FcRn binding loop consists of amino acid residues 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298 and 299 (according to EU number) composition. This ring is depicted in FIG. 9 .

In another embodiment, one or more amino acid mutations are made in the 15A FcRn "contact region". As used herein, the term 15A FcRn "contact region" includes residues located at the following positions (exemplary amino acids for these positions are also listed): 243F; 244P; 245P; 246K; 247P; 248K; 249D; 250T; 251L ;252M; 253I; 254S; 255R; 256T; 257P; 258E; 259V; 260T; 261C; 275F; 276N; ;291P; 292R; 293E; 302V; 303V; 304S; 305V; 306L; 307T; 308V; 309L; ;340K; 341G; 342Q; 343P; 344R; 345E; 346P; 347Q; 348V; 367C; ;386Q; 387P; 388E; 389N; 391Y; 393T; 408S; 424S; 425C; 426S; 427V; 428M; 429H; EU number).

Reference herein to "computational analysis" refers to a computer-implemented process that performs all or some of the operations described herein. The process includes an output device (eg, CRT display, LCD, printer, communication device such as a modem, audio output, etc.) that presents information to the user. The computer-implemented process is not limited to a particular computer platform, a particular processor, or a particular high-level programming language.

The term "structure", or "structural data" herein includes structures occupied by atoms, molecules, compounds, amino acid residues or parts thereof, and macromolecules or parts thereof, and especially polypeptides bound to antigens in a solvent, already Known, predicted and/or modeled three-dimensional spatial positions. Various methods for identifying and/or predicting structure at the molecular/atomic level can be used, such as X-ray crystallography, NMR structure modeling, etc.

The phrase "spatial representation of an optimal charge distribution" as used herein includes modeling the electron distribution for the Fc region or Fc-FcRn complex, where relative to the known and/or The modeled charge distribution representation optimizes (minimizes) the electrostatic contribution in the free energy of polypeptide binding to FcRn. Modeling of the optimal charge distribution can be achieved by a program on a computer into which the known and/or modeled structure of the Fc region or Fc-FcRn complex is incorporated by input. The electrostatic binding free energy of the complex in solvent can be expressed as an Fc desolvation term, an Fc-FcRn interaction term, and an FcRn desolvation term using response continuum modeling (e.g., the linear Poisson-Boltzmann equation) The sum of the term. This in silico program is characterized by the ability to incorporate unipolar, bipolar and quadrupole terms when representing the charge distribution in the modeled charge distributions of the present invention, this in silico program allows for a broad evaluation of polypeptide amino acid residues in the Fc region or The solvation/desolvation energy of its fraction during the transition between the unbound and bound states. This method of modeling the spatial representation of the optimal charge distribution of the polypeptide-FcRn complex can also incorporate modeling of van der Waals forces, solvent accessible surface area forces, and the like.

The term "solvent" is used herein in its broad art-recognized sense to refer to any liquid in which a polypeptide of the invention is dissolved and/or present. Preferably, the solvent is a biologically compatible solvent. Preferred solvents include PBS, serum, and the like.

Preferred starting polypeptides comprise amino acid sequences derived from human antibodies. A polypeptide or amino acid sequence "derived from" a specified polypeptide or source species refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has substantially the same amino acid sequence as the starting sequence or a part thereof (wherein said part consists of at least 10 to 20 amino acids, preferably at least 20- 30 amino acids, more preferably at least 30-50 amino acids), or have an amino acid sequence that can be otherwise identified by a person of ordinary skill in the art as being derived from the starting sequence. For example, a polypeptide derived from a human polypeptide may comprise one or more amino acids from another mammalian species. For example, a primate heavy chain portion, hinge portion, or binding site may be included in a polypeptide of the invention. Alternatively, one or more murine amino acids may be present in the starting polypeptide, eg, the antigen binding site (CDR) of an antibody. Preferred polypeptides of the invention are not immunogenic.

The terms "PEGylated moiety," "polyethylene glycol moiety," or "PEG moiety" include poly(alkylene) glycol compounds or derivatives thereof, which may or may not have a coupling agent or be coupled or activated Partially derivatized (e.g., with a sulfhydryl, triflate, tresylate, aziridine, oxirane, or preferably with a maleimide moiety, such as PEG -maleimide). Other suitable poly(alkylene) glycol compounds include, but are not limited to, maleimido-methoxy PEG, activated PEG polypropylene glycol, and charged or neutral polymers of the following types: dextrose Sugars, polyacetylneuraminic acid, or other sugar-based polymers, amino acid polymers, and biotin derivatives.

The term "functional part" includes a part that preferably adds a desired function to a variant polypeptide. Preferably, functionality is added without substantially altering the inherent desired activity of the polypeptide, eg, in the case of an antibody, without substantially altering the antigen binding activity of the molecule. Variant polypeptides of the invention may comprise one or more functional moieties, which may be the same or different. Examples of useful functional moieties include, but are not limited to, PEGylation moieties, blocking moieties, detectable moieties, diagnostic moieties, and therapeutic moieties. Exemplary detectable moieties include fluorescent moieties, radioisotopic moieties, radiopaque moieties, and the like. Exemplary diagnostic sections include sections suitable for revealing the presence or absence of indications of a disease or condition. Exemplary therapeutic moieties include, for example, anti-inflammatory, anti-cancer, anti-neurodegenerative, and anti-infective agents. The functional part may also have one or more of the above-mentioned functions. Other useful functional moieties are known in the art and described below.

Herein, the term "anticancer agent" or "chemotherapy agent" includes agents that impair the growth and/or proliferation of neoplastic or tumor cells and that can be used to reduce, inhibit or destroy malignant tumors. Examples of such agents include, but are not limited to, cytostatic agents, alkylating agents, antibiotics, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, and the like. Any agent that can act to arrest or slow the growth of immunoreactive cells or malignant cells is within the scope of the invention.

The term "vector" or "expression vector" herein, for the purposes of the specification and claims, refers to a vector used according to the present invention as a vehicle for introducing a desired polynucleotide into a cell and allowing expression of the desired polynucleotide in the cell. As is known to those skilled in the art, the vector can easily be selected from: plasmids, bacteriophages, viruses and retroviruses. In general, vectors compatible with the present invention contain selectable markers, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

The term "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing methods for isolating proteins from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the protein unless clearly stated otherwise. In other words, recovery of protein from "cells" may refer to recovery from intact cells pelleted by centrifugation, or recovery from cell culture medium containing culture medium and suspended cells.

Herein, "tumor-associated antigen" refers to any antigen that is generally associated with tumor cells (ie, present to the same or greater extent than normal cells). The antigen may be relatively tumor specific, with expression restricted to the surface of malignant cells, but the antigen may also be present on non-malignant cells. In one embodiment, the altered polypeptide of the invention binds to a tumor-associated antigen. Thus, the starting polypeptide of the invention may be derived, generated or created from any antibody reactive with a tumor-associated molecule.

Herein, the term "malignant" refers to a non-benign tumor or carcinoma. As used herein, "cancer" includes malignant tumors characterized by deregulated or uncontrolled cell growth. Exemplary cancers include: carcinoma, sarcoma, leukemia and lymphoma. The term "cancer" includes primary malignancies (e.g., malignancies whose cells have not migrated to a site in the subject's body other than the location of the original tumor) and secondary malignancies (e.g., metastasized by tumor-tumor cells Migration to a second site different from the original tumor site - giving rise to malignancy).

Herein, the phrase "subject that will benefit from administration of a polypeptide" includes subjects, such as mammalian subjects, that will obtain a positive therapeutic or prophylactic result from administration of a polypeptide of the invention. Examples of beneficial uses of the polypeptides disclosed herein include, for example, detection of antigens recognized by the polypeptides (eg, in diagnostic methods) or treatment with the polypeptides to reduce or eliminate targets recognized by the polypeptides. For example, in one embodiment, a subject may benefit from reduction or clearance of soluble or particulate molecules (e.g., toxins or pathogens) from circulation or serum, or from a population of cells expressing a target (e.g., tumor cells). benefit from reduction or elimination. As described in more detail herein, polypeptides may be used in unconjugated form, or may be conjugated, for example, to a drug, prodrug, tag or isotope.

II. Fc-containing polypeptides for modification

In one embodiment, the starting polypeptide of the present invention comprises at least a portion of the Fc region sufficient to confer FcRn-binding properties. The part of the Fc region that binds to FcRn includes approximately 282 to 438 amino acids of IgG1 (EU numbering). Amino acid positions in the Fc region are numbered herein according to the EU index numbering system (see Kabat et al., "Sequences of Proteins of Immunological Interest", USDept. Health and Human Services, ^5th edition, 1991). "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

The Fc region of the invention is preferably of human origin. The nucleotide sequence encoding an exemplary Fc region (human IgG1 region) is shown in SEQ ID NO:1, and the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:1 is shown in SEQ ID NO:2. The amino acid sequence of the Fc region is also given in Table 1 below to illustrate the EU numbering of the amino acids.

Table 1: Human IgG1 CH2 and CH3 domains

CH2 domain (EU position 231-340)

231 APELLGG

238 PSVFLFPPKP

248 KDTLMISRTP

258 EVTCVVVDVS

268 HEDPEVKFNW

278 YVDGVEVHNA

288 KTKPREEQYN

298 STYRVVSVLT

308 VLHQDWLNGK

318 EYKCKVSNKA

328 LPAPIEKTIS

338 KAK

CH3 domain (EU position 341-446)

341 GQPREPQ

348 VYTLPPSRDE

358 LTKNQVSLTC

368 LVKGFYPSDI

378 AVEWESNGQP

388 ENNYKTTPPV

398 LDSDGSFFLY

408 SKLTVDKSRW

418 QQGNVFSCSV

428 MHEALHNHYT

438 QKSLSLSPG

In one embodiment, the starting polypeptide of the present invention comprises at least amino acids 282-340 of the CH2 domain. In another embodiment, the starting polypeptide of the invention comprises the entire CH2 domain (approximately amino acids 231-340 of the Fc region of an antibody according to EU numbering). In another embodiment, the starting polypeptide of the present invention at least comprises a CH2 domain and at least one hinge region (according to EU numbering, about 216-230 amino acids in the Fc region of an antibody) and a CH3 domain (according to EU numbering, the amino acid in the Fc region of an antibody) about amino acids 341-446). In one embodiment, the starting polypeptide of the invention comprises CH2 and CH3 domains. In one embodiment, the starting polypeptide of the invention comprises a hinge, CH2 and CH3 domains. In one embodiment, the starting polypeptide of the present invention comprises the sequence shown in SEQ ID NO:2. The Fc region or its FcRn binding portion may be derived from a heavy chain of any isotype including IgGl, IgG2, IgG3 and IgG4. In one embodiment, the human isotype IgGl is used.

The domains constituting the Fc region of the starting polypeptide may be derived from different immunoglobulin molecules. For example, a polypeptide may comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the starting polypeptide may comprise a hinge region derived partly from an IgG1 molecule and partly from IgG3. In another example, the starting polypeptide may comprise a chimeric hinge derived in part from an IgGl molecule and in part from an IgG4 molecule. As noted above, those of ordinary skill in the art will appreciate that the starting Fc domain (eg, in the non-FcRn binding portion of the molecule) may be modified so that it differs in amino acid sequence from a native antibody molecule.

The starting polypeptide of the invention may comprise at least one Fc region or an FcRn-binding portion thereof. Preferred starting polypeptides of the invention further comprise at least one binding domain, eg an antigen binding domain, a receptor binding domain or a ligand binding domain. In one embodiment, the starting polypeptide comprises at least one binding domain and at least one Fc portion. In one embodiment, the starting polypeptide consists of two binding domains and two Fc parts.

In one embodiment, the starting polypeptide of the invention has at least one binding specificity for a target molecule that mediates a biological effect (e.g., a ligand capable of binding a cell surface receptor or a cell surface receptor capable of binding a ligand). domain, the binding domain, together with at least one Fc portion, transmits a negative or positive signal to the cell. In one embodiment, the starting polypeptide has at least one binding domain specific for an antigen to be targeted for reduction or elimination (eg, a cell surface antigen or a soluble antigen) and at least one Fc region or FcRn binding portion thereof.

A. Antibodies

In one embodiment, the starting polypeptide of the invention is an antibody. Antibodies are raised in mammals using protocols known in the art, eg, preferably by multiple subcutaneous or intraperitoneal injections of the relevant antigen (eg, purified tumor-associated antigen or cells or cell extracts containing such antigen) and an adjuvant. The immunization typically elicits an immune response that includes the production of antigen-reactive antibodies from activated splenocytes or lymphocytes.

In embodiments where the Fc-containing polypeptide is an antibody, the antibody can be a monoclonal antibody or a polyclonal antibody. Methods for making monoclonal antibodies have been known for some time (see, e.g., Kohler and Milstein, Nature, 256:495-497, 1975), as has been known for the stable introduction of immunoglobulin-encoding DNA into myeloma cells (see, eg, Oi et al., Proc. Natl. Acad. Sci. USA 80:6351-6355, 1983). These techniques, including in vitro mutagenesis and DNA transfection, allow the construction of recombinant immunoglobulins and can be used to prepare polypeptides used in the methods of the invention or polypeptides generated therefrom (eg, therapeutic and diagnostic antibodies). Preparation methods, vectors and hosts are further described below.

The starting polypeptides used in the present invention can be produced in a non-human mammal such as a mouse, guinea pig, primate, rabbit or rat by immunizing the animal with an antigen or a fragment thereof. See Harlow & Lane, ibid., incorporated by reference for all purposes. Although the resulting antibodies can be harvested from animal serum to provide polyclonal antibody preparations, it is often desirable to isolate single lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogeneous monoclonal antibody (MAb) preparations. Polyclonal antibodies are typically prepared using rabbits or guinea pigs. Mice are typically used for the production of monoclonal antibodies. Monoclonal antibodies can be prepared against the fragment by injecting the fragment of the antigen into mice, preparing "hybridomas" and screening the hybridomas for antibodies that specifically bind the antigen. In this well-known method (Kohler et al. (1975), Nature, 256: 495), relatively short-lived or mortal lymphocytes from antigen-injected mice are combined with immortal tumor cell lines (e.g., myeloma cells). line) resulting in hybrid cells or "hybridomas" that are immortal and capable of producing genetically encoded B-cell antibodies. The resulting hybrids are segregated into individual genetic strains by selecting, diluting, and recultivating individual cell lines containing specific genes for the formation of individual antibodies. These genetic strains produce homogeneous antibodies to the desired antigen and are said to be "monoclonal" because of their pure genetic lineage.

The hybridoma cells thus prepared are seeded and cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and culture media for the formation, selection and cultivation of hybridomas are commercially available from a variety of sources, and standardized protocols are well established. Typically, the medium in which hybridoma cells are cultured is analyzed to check for the production of monoclonal antibodies directed against the desired antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). After identification of hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity, these clones can be subcloned by limiting dilution methods and processed by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp59-103 (Academic Press, 1986)) culture. It is also understood that the monoclonal secreted by these subclones can be isolated from culture medium, ascitic fluid or serum by conventional purification methods, for example, protein A, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Antibody.

Optionally, antibodies can be selected that bind to a specific region or desired fragment of the antigen but not to other non-overlapping fragments of the antigen. This latter selection can be achieved by measuring the binding of the antibody to a series of deletion mutants of the antigen and determining which deletion mutants bind to the antibody. Binding can be assessed by, for example, Western blot or ELISA. The smallest fragment showing specific binding to the antibody is defined as the epitope of the antibody. Alternatively, epitope specificity can be determined using a competition assay, in which a test antibody and a reference antibody compete for antigen binding. When a test antibody and a reference antibody compete, they bind to the same epitope, or are sufficiently close that binding of one antibody interferes with binding of the other.

In another embodiment, DNA encoding the desired monoclonal antibody can be readily isolated using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies) and sequencing. Isolated and subcloned hybridoma cells are a preferred source of this DNA. Once isolated, the DNA can be placed into an expression vector, which is then used to transfect prokaryotic or eukaryotic host cells, such as Escherichia coli (E.coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells or here Myeloma cells that do not produce immunoglobulins prior to vector transfection. More specifically, this isolated DNA (which, as described herein, may be synthetic) can be used to clone constant and variable region sequences to prepare antibodies as described in U.S. Patent No. 5,658,570, filed January 25, 1995 by Newman et al. , which is incorporated herein by reference. Basically, this requires RNA to be extracted from selected cells, converted to cDNA, and amplified using Ig-specific primers. Primers suitable for this purpose are also described in US Patent No. 5,658,570. As discussed in more detail below, transformed cells expressing the desired antibody can be grown to relatively large quantities to provide clinical and commercial immunoglobulin supplies.

Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (eg, antigen binding sites) can also be derived from antibody phage libraries, eg, using pd phage or Fd phagemid technology. Exemplary methods see e.g. EP 368 684 B1; US Patent 5,969,108, Hoogenboom, H.R. and Chames. 2000. Immunol. Today 21: 371; Nagy et al. 2002. Nat. Med. . Sci. USA 98: 2682; Lui et al. 2002. J. Mol. Biol. 315: 1063, all of which are incorporated herein by reference. Several publications (eg, Marks et al., Bio/Technology 10:779-783 (1992)) have described the production of high-affinity human antibodies by chain shuffling, combinatorial infection, and in vivo recombination (strategies for constructing large phage libraries). In another embodiment, ribosome display can be used instead of phage as a display platform (see, e.g., Hanes et al. 2000. Nat. Biotechnol. 18:1287; Wilson et al. 2001. Proc. Natl. or Irving et al. 2001 J. Immunol. Methods 248:31). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al., 2000, Proc. Natl. Acad. Sci. USA 97:10701; Daugherty et al., 2000 J. Immunol. Methods 243:211). These methods provide alternative techniques that can be used in place of traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.

Other embodiments of the invention include the production of human or substantially human antibodies in transgenic animals (eg, mice) incapable of producing endogenous immunoglobulins (see, eg, U.S. Pat. here for reference). For example, it has been described that homozygous deletion of the antibody heavy chain joining region in chimeric germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human immunoglobulin gene sets into this germline mutant mouse will result in the production of human antibodies following antigen challenge. Another preferred method of producing human antibodies using SCID mice is disclosed in US Patent No. 5,811,524, which is incorporated herein by reference. Obviously, the genetic material associated with these human antibodies can also be isolated and manipulated as described herein.

Another highly efficient method for making recombinant antibodies is disclosed in Newman, Biotechnology, 10:1455-1460 (1992). In particular, this technique has resulted in the production of primatized antibodies containing monkey variable regions and human constant regions. This reference is hereby incorporated by reference in its entirety. Additionally, this technology is also described in commonly assigned US Patent Nos. 5,658,570, 5,693,780, and 5,756,096, all of which are incorporated herein by reference.

In another embodiment, lymphocytes can be selected and the variable genes isolated by micromanipulation. For example, peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured in vitro for about 7 days. Cultures can be screened to detect specific IgG that meet the screening criteria. Cells from positive wells were isolated. Individual Ig-producing B cells can be isolated using FACS or by identifying B cells in a complement-mediated hemolytic plaque assay. IgG-producing B cells can be added to test tubes by micromanipulation, and the _VH and _VL genes can be amplified using, for example, RT-PCR. The _VH and _VL genes can be cloned into antibody expression vectors and transfected into cells (eg, eukaryotic or prokaryotic cells) for expression.

In addition, genetic sequences useful for producing polypeptides of the invention can also be obtained from a variety of different sources. For example, as discussed extensively above, various human antibody genes are available in publicly available deposits. Many antibody sequences and antibody-encoding genes have been published, and suitable antibody genes can be chemically synthesized from these sequences using techniques known in the art. Oligonucleotide synthesis techniques compatible with this aspect of the invention are well known to those skilled in the art and can be performed using several commercial automated synthesizers. In addition, DNA sequences encoding the several classes of heavy and light chains described herein can be obtained through the services of commercial DNA synthesizers. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to provide or obtain a polypeptide of the invention.

Variable and constant domains can be cloned separately, for example, using the polymerase chain reaction and primers selected to amplify the domain of interest. In addition, the sequences of many antibody variable and constant domains are known, and these domains can be synthesized using methods well known in the art. For example, constant region domains can be selected to have particular effector functions (or lack thereof) or have particular modifications to reduce immunogenicity. Alternatively, variable domains can be obtained from a library of variable gene sequences from an animal of choice. Libraries expressing random combinations of domains (eg, _VH and _VL domains) can be screened with the desired antigen to identify elements with the desired binding characteristics. Such screening methods are well known in the art. For example, repertoires of antibody genetic repertoires can be cloned into lambda phage expression vectors (Huse, WD et al. (1989), Science, 2476:1275). In addition, cells expressing antibodies on the surface can be screened (Francisco et al. (1994), PNAS, 90: 10444; Georgiou et al. (1997), Nat. Biotech., 15: 29; Boder and Wittrup (1997) Nat. Biotechnol. 15 : 553; Boder et al. (2000), PNAS, 97: 10701; Daugtherty, P. et al. (2000) J. Immunol. Methods. 243: 211) or viruses (eg, Hoogenboom, HR. (1998), Immunotechnology 4: 1 ; Winter et al. (1994). Annu. Rev. Immunol. 12: 433; Griffiths, AD. (1998). Curr. Opin. Biotechnol. 9: 102). It is also clear to those skilled in the art that the DNA encoding the antibody domain can also be derived from an antibody phage library, for example using pd phage or Fd phagemid technology. Exemplary methods see, eg, EP 368 684 B1; US Pat. No. 5,969,108; Hoogenboom et al. (2000) Immunol. Today 21:371; Nagy et al. (2002) Nat.Med.8:801; Huie et al. (2001), PNAS, 98:2682; Lui et al. (2002), J. Mol. Biol. 315:1063, all of which are incorporated herein by reference. Several publications (eg, Marks et al., Bio/Technology 10:779-783 (1992)) have described strand shuffling, combinatorial infection, and in vivo recombination as a strategy for the construction of large phage libraries to generate high affinity human antibodies. In another embodiment, ribosome display can be used instead of phage as a display platform (see, e.g., Hanes, et al. (1998), PNAS 95:14130; Hanes and Pluckthun. (1999), Curr. Top. Microbiol. Immunol. 243 : 107; He and Taussig. (1997), Nuc. Acids Res., 25: 5132; Hanes et al. (2000), Nat. Biotechnol. 18: 1287; Wilson et al. (2001), PNAS, 98: 3750; or Irving et al. (2001) J. Immunol. Methods 248:31).

A preferred library for screening is a human variable gene library. _VL and _VH domains from non-human sources can also be used. Libraries can be naive, derived from immunized subjects, or semi-synthetic (Hoogenboom and Winter. (1992). J. Mol. Biol. 227:381; Griffiths et al. (1995) EMBO J.13:3245 ; de Kruif et al. (1995). J. Mol. Biol. 248:97; Barbas et al. (1992), PNAS, 89:4457). In one embodiment, immunoglobulin domains can be mutated to generate libraries of nucleic acid molecules with greater heterogeneity (Thompson et al. (1996), J. Mol. Biol. 256:77; Lamminmaki et al. (1999), J. Mol. Biol. 291: 589; Caldwell and Joyce. (1992), PCR Methods Appl. 2: 28; Caldwell and Joyce. (1994), PCR Methods Appl. 3: S136). High affinity variants can be selected using standard screening methods. In another embodiment, the _VH and _VL sequences can be altered to increase antibody avidity, for example using information from crystal structures, using techniques known in the art.

Alternatively, antibody-producing cell lines can be selected and grown using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, techniques applicable to the present invention are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is fully (including Appendix) is hereby incorporated by reference.

Furthermore, all allelic variants, variants and mutants of the antigen-binding DNA sequences are clearly included within the scope of the invention.

RNA can be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidine isothiocyanate extraction and precipitation followed by centrifugation or chromatography, as is well known. When desired, mRNA can be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are well known in the art.

In one embodiment, cDNAs encoding antibody light and heavy chains can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated using consensus constant region primers or using more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, the library can be screened using consensus primers or larger homologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art and subjected to restriction mapping and sequencing according to standard well-known techniques (explained in detail, for example, in the aforementioned references relating to recombinant DNA technology). Of course, DNA may be synthesized according to the invention at any point during the isolation method or subsequent analysis. In many cases, immunoreactive antibodies against each of these antigens have been reported in the literature.

In another embodiment, binding of the initiating polypeptide to the antibody results in the reduction or elimination of the antigen, eg, from tissue or from circulation. In another embodiment, the starting polypeptide has at least one binding domain specific for an antigen, which can be used to detect the presence of a target molecule (eg, to detect a pollutant or to diagnose a disease or condition). In yet another embodiment, the starting polypeptide of the invention comprises at least one binding site which enables targeting of the molecule to a specific site in a subject (eg targeting a tumor cell or a blood clot).

In one embodiment, the starting polypeptide of the invention is immunoreactive with one or more antigen-related antigens. For example, for the treatment of cancer or neoplasia, the antigen-binding domain of the polypeptide preferably binds to a selected tumor-associated antigen. Given the number of antigens reported to be associated with neoplasia and the number of related antibodies, it will be apparent to those skilled in the art that the polypeptides of the invention may be derived from any of a variety of intact antibodies. More generally, starting antibodies useful in the present invention can be obtained or derived from any antibody (including those previously reported in the literature) that reacts with an antibody or marker associated with the condition of choice. Furthermore, the starting antibody or fragment thereof used to generate the polypeptides disclosed herein may be murine, human, chimeric, humanized, non-human primate, or primatized. Exemplary tumor-associated antigens to which the initiating antibody used in the invention binds include, for example, pan B antigen (e.g., CD20 found on the surface of malignant and non-malignant B cells (e.g., B cells in non-Hodgkin's lymphoma)) and pan T cell antigens (eg, CD2, CD3, CD5, CD6, CD7). Other exemplary tumor-associated antigens include, but are not limited to, MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 & E7, TAG-72, CEA, α-Lewis ^y , L6-antigen, CD19, CD22, CD25, CD30 , CD33, CD37, CD44, CD52, CD56, mesothelin, PSMA, HLA-DR, EGF receptor, VEGF receptor, and HER2 receptor.

Antibodies previously reported reactive with tumor-associated antigens can be altered as described herein to provide altered polypeptides of the invention. Exemplary target antibodies capable of reacting with tumor-associated antigens include: 2B8, Lym1, Lym2, LL2, Her2, B1, BR96, MB1, BH3, B4, B72.3, 5E8, B3F6, 5E10, α-CD33, α-CanAg , α-CD56, α-CD44v6, α-Lewis, and α-CD30.

More particularly, exemplary target antibodies include, but are not limited to, 2B8 and C2B8 (Zevalin ^(R) and Rituxan ^(R) , IDEC Pharmaceuticals Corp., San Diego), Lym1 and Lym2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), Trastuzumab ( Herceptin ^(R) , Genentech Inc., South San Francisco), Tositumomab (Bexxar ^(R) , Coulter Pharm., San Francisco), Alemtzumab (Campath ^(R) , Millennium Pharmaceuticals, Cambridge), Gemtuzumab ozogamicin (Mylotarg ^(R) , Wyeth-Ayerst, Philadelphia, C Erbitux ^(R) , Imclone Systems, New York), Bevacizumab (Avastin ^(R ), Genentech Inc., South San Francisco), BR96, BL22, LMB9, LMB2, MB1, BH3, B4, B72.3 (Cytogen Corp.), SS1 (NeoPharm ), CC49 (National Cancer Institute), Cantuzumab mertansine (ImmunoGen, Cambridge), MNL 2704 (Milleneum Pharmaceuticals, Cambridge), Bivatuzumab mertansine (Boehringer Ingelheim, Germany), Trastuzumab-DM1 (Genentech, South San Francisco), My9-6-DM1 (ImmunoGen, Cabridge), SGN-10, -15, -25 and -35 (Seattle Genetics, Seattle) and 5E10 (University of Iowa). In a preferred embodiment, the starting antibody of the invention binds to the same tumor-associated antigen as the antibody enumerated immediately above. In particularly preferred embodiments, the polypeptide is derived from or binds the same antigen as Y2B8, C2B8, CC49 and C5E10, and even more preferably comprises a domain-deleted antibody (i.e., a ΔCH2 antibody).

In a first preferred embodiment, the starting antibody binds to the same tumor-associated antigen as Rituxan ^(R) . Rituxan( ^R) (also known as rituximab, IDEC-C2B8 and C2B8) was the first FDA-approved monoclonal antibody for the treatment of human B-cell lymphoma (see, U.S. Patent Nos. 5,843,439; 5,776,456 and 5,736,137, all incorporated herein Reference). Y2B8 (90Y-labeled 2B8; Zevalin ^(R) ; ibritumomab tiuxetan) is the murine initiator of C2B8. Rituxan ^(R) is a chimeric anti-CD20 monoclonal antibody that is growth inhibitory and has been reported to sensitize certain lymphoma cell lines in vitro to the apoptotic effects of chemotherapeutic agents. The antibody effectively binds human complement, has strong FcR binding, and can effectively kill human lymphocytes in vitro through complement-dependent (CDC) and antibody-dependent (ADCC) mechanisms (Reff et al., Blood 83: 435-445 (1994 )). It will be apparent to those skilled in the art that C2B8 or 2B8 dimer variants (homodimer or heterodimer) synthesized according to the present disclosure can be conjugated with effector moieties according to the methods of the present invention to provide a therapeutic effect in the treatment of expressed CD20+ malignancies. The modified antibody had an even greater effect in patients.

In other preferred embodiments of the present invention, the starting polypeptide of the present invention is derived from CC49 or binds to the same tumor-associated antigen as CC49. CC49 binds the human tumor-associated antigen TAG-72, which is bound to the surface of certain tumor cells of human origin, especially the LS174T tumor cell line. LS174T [American Type Culture Collection (herein ATCC) number CL188] is a variant of the LS180 (ATCC No. CL187) colon adenocarcinoma line.

In addition, it is also clear that many murine monoclonal antibodies having binding specificity for TAG-72 have been developed. One of these monoclonal antibodies is called B72.3, which is a murine IgG1 produced by hybridoma B72.3 (ATCC No. HB-8108). B72.3 is the first generation monoclonal antibody developed using human breast cancer extracts as an immunogen (see Colcher et al., Proc. Natl. Acad. Sci. (USA), 78: 3199-3203 (1981); and U.S. Patent Nos. 4,522,918 and 4,612,282, all of which are incorporated herein by reference). Other monoclonal antibodies against TAG-72 are called "CC" (for colon cancer). CC monoclonals are a second generation murine monoclonal family prepared using TAG-72 purified by means of B72.3 as described by Schlom et al. (US Pat. No. 5,512,443, incorporated herein by reference). Due to their relatively good binding affinity for TAG-72, the following CC antibodies have been deposited with the ATCC and are available on request with restrictions: CC49 (ATCC No. HB 9459); CC 83 (ATCC No. HB 9453); CC46 (ATCC No. HB 9458); CC92 (ATCC No. HB 9454); CC30 (ATCC No. HB 9457); CC11 (ATCC No. 9455); and CC15 (ATCC No. HB9460). U.S.P.N. 5,512,443 also teaches that these disclosed antibodies can be altered to a chimeric form by recombinant DNA techniques known in the art by replacing the murine constant region, for example, with a human constant region (Fc) domain. In addition to disclosing murine and chimeric anti-TAG-72 antibodies, Schlom et al. also prepared humanized CC49 antibody variants (disclosed in PCT/US00/25552) and single-chain constructs (disclosed in U.S. Patent No. 5,892,019 ), all of which are also incorporated herein by reference. It will be clear to those skilled in the art that all of the aforementioned antibodies, constructs or recombinants and variants thereof may be synthetic and used to provide polypeptides according to the invention.

In addition to the anti-TAG-72 antibodies discussed above, various groups have reported the construction and partial characterization of domain-deleted CC49 and B72.3 antibodies (e.g., Calvo et al. Cancer Biotherapy, 8(1):95-109 (1993) , Slavin-Chiorini et al. Int. J. Cancer 53:97-103 (1993) and Slavin-Chiorini et al. Cancer. Res. 55:5957-5967 (1995).

In one embodiment, the starting polypeptide of the invention binds to CD23 (US Patent 6,011,138). In a preferred embodiment, the starting polypeptide of the present invention binds to the same epitope as the 5E8 antibody. In another embodiment, the starting polypeptide of the invention comprises at least one CDR from an anti-CD23 antibody (eg, 5E8 antibody).

In a preferred embodiment, the starting antibody of the invention binds to the CRIPTO-I antigen (WO02/088170A2 or WO03/083041A2). In a more preferred embodiment, the polypeptide of the present invention binds to the same epitope as the B3F6 antibody. In yet another embodiment, a polypeptide of the invention comprises at least one CDR from an anti-CRIPTO-1 antibody, eg, the B3F6 antibody.

Other embodiments of the invention include modified antibodies derived from C5E10 or that bind the same tumor-associated antigens as C5E10. As set forth in co-pending application 09/104,717, C5E10 is an antibody that recognizes an approximately 115 kDa glycoprotein determinant that appears to be specific for prostate tumor cell lines (eg, DU145, PC3 or ND1). Thus, in conjunction with the present invention, polypeptides that specifically bind to the same tumor-associated antigen as the C5E10 antibody can be used alone or conjugated to effector moieties by the methods of the present invention, thereby providing modified polypeptides useful for improving the treatment of neoplastic diseases . In an especially preferred embodiment, the starting polypeptide is derived from or comprises all or part of the antigen-binding region of the C5E10 antibody secreted by a hybridoma cell line having ATCC Accession No. PTA-865. The resulting polypeptide can then be conjugated with a therapeutic effector moiety (see below) and administered to a patient with prostate cancer according to the methods herein.

B. Antibody variants

In addition to natural antibodies, the starting antibodies of the invention may also include non-natural immunoreactive fragments or portions.

In another embodiment, the heavy chain variable part and the light chain variable part of the antigen binding domain of the target antibody of the present invention are present in the same polypeptide, for example present in a single chain antibody (ScFv) or a minibody (minibody) (see For example in US Patent No. 5,837,821 or WO 94/09817A1). Minibodies are dimeric molecules composed of two polypeptide chains, each containing a ScFv molecule (a single polypeptide containing one or more antigen-binding sites, e.g., domain-fused _VH domain-linked _VL domain). ScFv molecules can be constructed in _VH -linker- _VL orientation or _VL -linker- _VH orientation. The flexible hinge linking the VL and _VH domains that make up _the antigen binding site preferably comprises about 10 to about 50 amino acid residues. An exemplary connecting peptide for this purpose is (Gly4Ser)3 (Huston et al. (1988), PNAS, 85:5879). Other linker peptides are well known in the art.

Methods for preparing single-chain antibodies are well known in the art, for example, Ho et al. (1989), Gene, 77:51; Bird et al. (1988), Science 242:423; Pantoliano et al. (1991), Biochemistry 30:10117; Milenic et al. ( 1991), Cancer Research, 51:6363; Takkinen et al. (1991), Protein Engineering 4:837. Minibodies can be prepared by constructing the ScFv component and linking the peptide- _CH3 component using methods described in the art (see, eg, US Pat. No. 5,837,821 or WO94/09817A1). These components can be isolated from different plasmids as restriction fragments, ligated and recloned into appropriate vectors. Correct assembly can be verified using restriction digests and DNA sequence analysis. In one embodiment, the minibody of the invention comprises a linker peptide. In one embodiment, the connecting peptide comprises a Gly/Ser linker, eg, GGGSSGGGSGG.

In another embodiment, tetravalent minibodies can be constructed. Tetravalent minibodies can be constructed in the same manner as minibodies, except that a flexible linker (eg, having the amino acid sequence (G4S) _4G3AS ) is used to link two ScFv molecules.

In another embodiment, the starting antibody of the invention comprises a diabody. Diabodies are similar to scFv molecules, but typically have a short (less than 10, preferably 1-5) amino acid residue linker connecting the two variable domains so that the V _L and V _H are on the same polypeptide chain. Domains cannot interact. Conversely, the _VL and _VH domains of one polypeptide chain interact with the _VH and _VL domains (respectively) of a second polypeptide chain (WO 02/02781).

In another embodiment, the starting antibody of the invention comprises an immunoreactive fragment or portion thereof (eg, scFv molecule, minibody, tetravalent minibody, or diabody) operably linked to an FcR binding moiety. In an exemplary embodiment, the FcR binding portion is the entire Fc region.

In another embodiment, at least one antigen-binding domain of the starting antibody is catalytic (Shokat and Schultz. (1990). Annu. Rev. Immunol. 8:335). Antigen binding domains with catalytic binding specificities can be prepared using techniques known in the art (see eg, US Patent No. 6,590,080, US Patent No. 5,658,753). Catalytic binding specificity may function through many fundamental mechanisms similar to those identified for enzymes for stabilizing the transition state, thereby lowering the free energy of activation. For example, generally acidic and basic residues can be optimally placed to participate in the catalytic reaction in the catalytic active site; covalent enzyme-substrate intermediates can be formed; catalytic antibodies can also have the appropriate orientation for the reaction and increase The effective concentration of the reactants is at least 7 orders of magnitude (Fersht et al. (1968), J. Am. Chem. Soc. 90:5833) and thereby greatly reduces the entropy of the chemical reaction. Finally, the catalytic antibody can convert the energy gained upon substrate binding to distort the reactant toward a transition state-like structure.

Acidic or basic residues can be introduced into the antigen binding site by using complementary charged molecules as immunogens. This technique has been shown to successfully elicit antibodies using haptens containing positively charged ammonium ions (Shokat et al. (1988), Chem. Int. Ed. Engl. 27:269-271). In another approach, antibodies can be primed to stabilize compounds that are similar in size, shape and charge to the transition state of the desired reaction (ie, transition state analogs). See US Patent No. 4,792,446 and US Patent No. 4,963,355, which describe immunization of animals with transition state analogs and production of catalytic antibodies. Both patents are hereby incorporated by reference. These molecules can be administered as part of an immunoconjugate, eg, together with an immunogenic carrier molecule, eg KLH.

In one embodiment, the starting antibody of the invention is bispecific. A bispecific molecule can bind to two different target sites, eg, on the same target molecule or on different target molecules. For example, in the case of antibodies, a bispecific molecule may bind to two different epitopes, eg on the same antigen or on two different antigens. Bispecific molecules can be used, for example, in diagnostic and therapeutic applications. For example, they can be used with immobilized enzymes for use in immunoassays. They can also be used in the diagnosis and treatment of cancer, eg, by conjugation to tumor-associated molecules and detectable labels such as chelators that tightly bind radionuclides. Bispecific molecules can also be used in human therapy, for example, by directing cytotoxicity to a specific target (eg, by binding to a pathogen or tumor cell and a cytotoxicity triggering molecule, such as a T cell receptor). Bispecific molecules can also be used, for example, as fibrinolytic agents or vaccine adjuvants.

Examples of bispecific binding molecules include bispecific binding molecules having at least two arms directed against tumor cell antigens; bispecific binding molecules having at least one arm directed against tumor cell antigens and at least one arm directed against cytotoxicity triggering molecules (e.g., anti- CD3/anti-malignant B cells (1D10), anti-CD3/anti-p185.sup.HER2, anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti colon cancer), anti-CD3/anti-melanostimulating hormone analog, anti-EGF receptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti- CD3, anti-folate-binding protein (FBP)/anti-CD3, anti-pan cancer associated antigen (anti-pan carcinoma associated antigen) (AMOC-31)/anti-CD3); having at least one arm that specifically binds to a tumor antigen and at least one Bispecific binding molecules of the toxin-binding arm (e.g., anti-saporin/anti-Id-1, anti-CD22/anti-saporin, anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/ Anti-ricin A chain, anti-interferon alpha (IFN-α)/anti-hybridoma idiotype, anti-CEA/anti-vinca alkaloid); bispecific binding molecules for invertase-activated prodrugs (e.g., anti- CD30/anti-alkaline phosphatase (the enzyme that catalyzes the conversion of mitomycin phosphate prodrug to mitomycin alcohol); bispecific binding molecules that can be used as fibrinolytic agents (e.g., anti-fibrin/anti-tissue plasminogen activator (tPA), antifibrin/antiurokinase-type plasminogen activator (uPA)); bispecific binding molecules that direct immune complexes to cell surface receptors (e.g. anti-LDL protein (LDL)); bispecific binding molecules for the treatment of infectious diseases (e.g., anti-CD3/anti-herpes simplex virus (HSV), anti-T cell receptor:CD3 complex/anti-influenza virus, anti-FcγR/anti-HIV ); bispecific binding molecules for in vitro or in vivo detection of tumors (e.g. anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA, anti-p185HER2/anti-hapten); bispecific binding molecules as vaccine adjuvants (see Fanger et al. ibid); and bispecific binding molecules as diagnostic tools (e.g. anti-rabbit IgG/anti-ferritin, anti-horseradish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti-substance P, anti-HRP /anti-FITC, anti-CEA/anti-beta-galactosidase (see Nolan et al., loc. cit.)). Examples of trispecific antibodies include anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.

In a preferred embodiment, the bispecific molecule of the invention binds to CRIPTO-1.

Bispecific molecules can be monovalent for each specificity, or can be multivalent for each specificity. For example, an antibody molecule or fusion protein may comprise one binding site reactive with a first target molecule and one binding site reactive with a second target molecule, or it may comprise two binding sites reactive with a first target molecule and Two binding sites reactive with a second target molecule. Methods of making bispecific molecules are well known in the art. For example, bispecific molecules can be produced using recombinant techniques. Exemplary techniques for generating bispecific molecules are known in the art (e.g., Kontermann et al., Methods in Molecular Biology Vol. 248: Antibody Engineering: Methods and Protocols). Pp 227-242 US 2003 /0207346 A1 and references cited therein). In one embodiment, multimeric bispecific molecules are prepared using methods such as those described in US 2003/0207346A1 and US Patent 5,821,333 and US 2004/0058400.

The phrase "multispecific fusion protein" herein refers to a fusion protein (see definition above) that has at least two binding specificities (ie, combining the binding domains of two or more ligands or receptors). Multi-specific fusion proteins can be assembled into heterodimers, heterotrimers or heterotetramers, basically referring to WO89/02922 (published on April 6, 1989), EP 314,317 (published on May 3, 1989) , the disclosure in US Patent No. 5,116,964 (issued May 2, 1992). Preferably the multispecific fusion protein is bispecific. Examples of bispecific fusion proteins include CD4-IgG/TNF receptor-IgG and CD4-IgG/L-selectin-IgG. The last-mentioned molecule combines the lymph node-binding function of the lymphocyte homing receptor (LHR, L-selectin) with the HIV-binding function of CD4 and can potentially be used for the prevention or treatment of HIV infection, related disorders or as a diagnostic agent .

Target binding sites for multispecific binding molecules of the invention can be readily selected by one of ordinary skill in the art. Without limitation in any way, exemplary binding sites include one or more epitopes of tumor antigens. Other exemplary target molecules include, for example, heparan sulfate, one or more expressions of growth factors or their receptors (e.g., epidermal growth factor receptor, insulin-like growth factor receptor, hepatocyte growth factor (HGF/SF) receptor). position (see, eg, Cao et al. Proc. Natl. Acad. Sci 2001.98:7443; Lu et al. 2004. J. Biol. Chem. 279:2856).

In another embodiment, the antigen-binding domain of the starting antibody consists of a _VH domain that is stable in the absence of a _VL chain, such as a _VH domain derived from camelids (Hamers-Casterman et al. (1993). Nature, 363 : 446; Desmyter et al. (1996). Nat. Struct. Biol. 3: 803; Decanniere et al. (1999). Structure, 7: 361; Davies et al. (1996). . J. Protein Chem., 14: 167).

The non-human starting antibody, or a fragment or domain thereof, can be altered to reduce its immunogenicity using techniques known in the art. A humanized starting polypeptide is a starting polypeptide derived from a non-human protein that retains or substantially retains the properties of the starting antibody, but is less immunogenic in humans. For the humanized starting antibody, it can be achieved by various methods, including (a) grafting intact non-human variable domains on human constant regions to generate chimeric target antibodies; (b) grafting one or more non-human Grafting at least a portion of the complementarity determining regions (CDRs) onto human framework and constant regions with and without key framework residues; (c) grafting complete non-human variable domains, but replacing surface residues with human-like Partially "masking" it. These methods are disclosed in Morrison et al. (1984), PNAS.81:6851-5; Morrison et al., (1988), Adv.Immunol.44:65-92; Verhoeyen et al., (1988), Science 239:1534-1536; Padlan , (1991), Molec.Immun.28:489-498; Padlan, (1994), Molec.Immun.31:169-217; incorporated by reference.

Deimmunization can also be used to reduce the immunogenicity of the starting antibody. Herein, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see eg WO9852976A1, WO0034317A2). For example, analysis of the _VH and _VL sequences from the starting antibody "maps" the human T cell epitope from each V region, thereby showing the relative position of the epitope in the sequence relative to the complementarity determining regions (CDRs) and other key residues. base position. Each individual T-cell epitope in the T-cell epitope map was analyzed to identify alternative amino acid substitutions with the lowest risk of altering antibody activity. A series of alternative _VH and _VL sequences are designed to contain combinations of amino acid substitutions, and these sequences are subsequently incorporated into a series of polypeptides of the invention for functional examination. Typically, 12-24 variant antibodies are prepared and tested. Afterwards, the complete heavy and light chain genes containing the modified V and human C regions are cloned into expression vectors, and the resulting plasmids are introduced into cell lines to produce complete antibodies. These antibodies are then compared in appropriate biochemical and biological assays to identify the best variant.

In one embodiment, the starting polypeptide comprises a chimeric antibody. In the context of this application, the term "chimeric antibody" means that the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified according to the invention) is obtained from a second species any antibodies. In preferred embodiments, the target binding region or site is from a non-human source (eg, mouse) and the constant region is human. Preferably, the variable domains in the heavy and light chains of the target antibody are altered by at least partially replacing one or more CDRs and, if necessary, by partially replacing the framework regions and altering the sequence. Although the antibody from which the CDRs are derived may be of the same type or even the same subclass as the target antibody from which the framework regions are derived, it is conceivable that the CDRs are from a different type of antibody, and preferably from an antibody of a different species. In order to transfer the antigen binding capability of one variable domain to another, it may not be necessary to replace all CDRs with all CDRs from the donor variable region. Conversely, it may only be necessary to transfer those residues that are necessary to maintain the activity of the binding domain. Given the explanations set forth in US Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it is well within the ability of those skilled in the art to obtain functional antibodies with reduced immunogenicity by performing routine experiments or by trial and error.

In a preferred embodiment, the starting polypeptide of the invention does not elicit a deleterious immune response in humans. It will be apparent to those skilled in the art that chimeric starting polypeptides may also be prepared. In this application, the term "chimeric starting antibody" means that the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified according to the invention) is obtained from a second species. Any starting antibodies for the species. In preferred embodiments, the target binding region or site is from a non-human source (eg, mouse) and the constant region is human. Although the immunogenic specificity of a variable region is generally not affected by its source, human constant regions are less likely than constant regions from non-human sources to elicit an immune response in a human subject.

C. Fusion protein

The starting polypeptide of the present invention may also be a fusion protein comprising at least the FcRn-binding portion of the Fc region. Preferably, the fusion protein of the invention comprises a binding domain (comprising at least one binding site). Fusion proteins of the invention may be bispecific (having one binding site for a first target and a second binding site for a second target), or may be multivalent (having two binding sites for the same target) .

Examples of fusion proteins reported in the literature include T cell receptor (Gascoigne et al., Proc.Natl.Acad.Sci.USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989) ; Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990)); L-selectin (homing receptor) (Watson et al., J.Cell.Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991)); CD44 (Aruffo et al., Cell 61:1303- 1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 173: 721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174: 561-569 (1991) ); CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)); TNF receptor (Ashkenazi et al., Proc.Natl.Acad.Sci.USA 88:10535-10539 (1991); Lesslauer et al., Eur.J. Immunol. 27: 2883-2886 (1991); and Peppel et al., J. Exp. Med. 174: 1483-1489 (1991)); and the IgE receptor (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)) fusion.

Typically, the C-terminus of the binding domain is fused to the N-terminus of the Fc region and replaces the cellular anchoring region. For example, any transmembrane region or lipid or phospholipid anchor recognition sequence of the receptor-binding ligand is preferably inactivated or deleted prior to fusion. The DNA encoding the ligand or ligand-binding portion is cleaved with restriction enzymes at or near the 5' and 3' ends of the DNA encoding the desired ORF segment. The resulting DNA fragment can then be easily inserted into DNA encoding the heavy chain constant region. The precise location at which the fusion is made can be chosen empirically in order to optimize the secretion or binding characteristics of the soluble fusion protein. Then, the DNA encoding the fusion protein is transfected into host cells for expression.

In one embodiment, the fusion protein comprises a ligand or receptor binding domain (eg, the extracellular domain (ECD) of a receptor) and at least one Fc portion and optionally a synthetic linker peptide. In one embodiment, when preparing the fusion protein of the present invention, the nucleic acid encoding the ligand or receptor binding domain is fused with the nucleic acid encoding the N-terminus of the Fc region at the C-terminus. N-terminal fusions are also possible. Fusions to the C-terminus of the Fc portion of the constant domains or immediately N-terminal to the CH1 of the heavy chain or the corresponding region of the light chain can also be performed.

In one embodiment, the Fc region of the fusion protein comprises a substantially complete antibody Fc region, starting at the hinge region just upstream of the papain cleavage site that chemically defines the IgG Fc (approximately EU numbered residue 216, taking the heavy chain The first residue of the constant region is 114) and ends at its C-terminus. The precise location at which the fusion is made is not critical; specific sites are well known and can be chosen to optimize the biological activity, secretion or binding characteristics of the molecule. Methods of making fusion proteins are known in the art.

For bispecific fusion proteins, the fusion proteins can be assembled into multimers, especially heterodimers or heterotetramers. Generally, these assembled immunoglobulins have a known unit structure. The basic four-chain building block is the IgG, IgD and IgE forms. The four-chain unit repeats in higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units linked together by disulfide bonds. IgA globulins and occasionally IgG globulins can also exist in multimeric form in serum. In the case of multimers, each of the four units can be the same and different.

Other exemplary ligands and their receptors that may be included in fusion proteins of the invention include the following:

i) Cytokines and cytokine receptors

Cytokines have pleiotropic effects on the proliferation, differentiation and functional activation of lymphocytes. Various cytokines or their receptor binding portions can be used in the fusion proteins of the present invention. Exemplary cytokines include interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL -12, IL-13, and IL-18), colony-stimulating factors (CSFs) (eg, granulocyte-CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), and monocyte-macrophage Cellular CSF (M-CSF)), tumor necrosis factor (TNF) alpha and beta, and interferons such as interferon-alpha, beta, or gamma (US Patent Nos. 4,925,793 and 4,929,554).

Cytokine receptors typically consist of a ligand-specific alpha chain and a common beta chain. Examples of cytokine receptors include receptors for GM-CSF, IL-3 (US Patent No. 5,639,605), IL-4 (US Patent No. 5,599,905), IL-5 (US Patent No. 5,453,491), IFNγ (EP0240975), and TNF receptor family (eg, TNFR-1 (EP 417,563), TNFR-2 (EP 417,014) lymphotoxin beta receptor).

ii) Adhesin

Adhesion molecules are membrane-bound proteins that allow cells to interact with each other. Various adhesion proteins, including leukocyte homing receptors and cell adhesion molecules, or receptor binding portions thereof, can be incorporated into fusion proteins of the invention. Leukocyte homing receptors are expressed on the surface of leukocytes during inflammation, including β-1 integrins (e.g., VLA-1, 2, 3, 4, 5, and 6) that mediate binding to extracellular matrix components, and Cell adhesion molecule (CAM) bound β2 integrins (eg, LFA-1, LPAM-1, CR3 and CR4) on the vascular endothelium. Examples of CAMs include ICAM-1, ICAM-2, VCAM-1 and MAdCAM-1. Other CAMs include those of the selectin family, including E-selectin, L-selectin and P-selectin.

iii) Chemokines

Chemokines - chemoattractant proteins that stimulate the migration of leukocytes to the site of infection - may also be incorporated into fusion proteins of the invention. Examples of chemokines include macrophage inflammatory proteins (MIP-1-alpha and MIP-1-beta), neutrophil chemokine, and RANTES (activation-regulated, normally expressed and secreted by T cells) .

iv) Growth factors and growth factor receptors

Growth factors or their receptors (or their receptor-binding or ligand-binding portions) can be incorporated into fusion proteins of the invention. Examples of growth factors include vascular endothelial growth factor (VEGF) and its isoforms (U.S. Pat. No. 5,194,596); fibroblast growth factors (FGF), including aFGF and bFGF; atrial natriuretic peptide (ANF); liver growth factor ( HGFs; U.S. Pat. Nos. 5,227,158 and 6,099,841), neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT- 5, or NT-6), or nerve growth factor such as NGF-β, platelet-derived growth factor (PDGF) (U.S. Pat. Nos. 4,889,919, 4,845,075, 5,910,574, and 5,877,016); transforming growth factor (TGF) such as TGF-α and TGF-beta (WO 90/14359), osteoinductive factors including bone morphogenic protein (BMP); insulin-like growth factors-I and -II (IGF-I and IGF-II; U.S. Patent Nos. 6,403,764 and 6,506,874); erythrocyte-stimulating epoietin (EPO); stem cell factor (SCF), thrombopoietin (c-Mpl ligand), and Wnt polypeptides (US Patent No. 6,159,462).

Examples of growth factor receptors that may be used as targeting receptor domains in the present invention include EGF receptors; VEGF receptors (e.g., Flt1 or Flk1/KDR), PDGF receptors (WO 90/14425); HGF receptors ( U.S. Patent Nos. 5,648,273 and 5,686,292), and neurotrophic receptors including the low-affinity receptor (LNGFR) that binds NGF, BNDF, and NT-3, also known as p75 ^NTR or p75, and the trk family of receptor tyrosine kinases Members of high-affinity receptors (egtrkA, trkB (EP 455,460), trkC (EP522,530)).

v) Hormone

Examples of growth hormones useful as targeting agents in fusion proteins of the invention include renin, human growth hormone (HGH; U.S. Patent No. 5,834,598), N-methionyl human growth hormone; bovine growth hormone; growth hormone releasing factor; Parathyroid hormone (PTH); Thyroid stimulating hormone (TSH); Thyroxine; Proinsulin and insulin (US Pat. Nos. 5,157,021 and 6,576,608); Follicle stimulating hormone (FSH), Calcitonin, Luteinizing hormone (LH), leptin, glucagon; bombesin; somatotropin; Mullerian inhibitors; relaxin and prorelaxin; gonadotropin-related peptide; prolactin; placental lactogen; OB protein; and mullerian inhibitors.

vi) coagulation factors

Exemplary blood clotting factors for use as targeting agents in the fusion proteins of the invention include coagulation factors (e.g. factors V, VII, VIII, X, IX, XI, XII and XIII, von Wilebrand factors); tissue factor (U.S. Patent No. 5,346,991, 5,349,991, 5,726,147); thrombin and prothrombin; fibrin and fibrinogen; plasmin and plasminogen; plasminogen activators such as urokinase or human urine or tissue plasminogen Activator (t-PA).

Other exemplary fusion proteins are taught eg in WO0069913A1 and WO0040615A2. Another exemplary molecule that can be included in fusion proteins of the invention is IGSF9. Fusion proteins can be prepared using techniques well known in the art (see, eg, US Patent Nos. 5,116,964 and 5,225,538).

III. Methods of Identifying Candidate Amino Acids for Modification

The present invention provides methods for identifying specific amino acid residues in the Fc region (or FcRn-binding portion thereof) of an Fc-containing starting polypeptide that are altered by mutation (e.g., amino acid substitution) to be expected to result in binding to FcRn Modulation of affinity and modulation of the half-life of said polypeptide in serum.

The methods include molecular or computer modeling that can be used to predict amino acid changes in the Fc region that modulate (eg, enhance or decrease) FcRn binding. Typically, these methods start with a "first" or "starting" polypeptide or a complex containing it (e.g., a crystal structure or a homology model) and result in a protein that differs from the first polypeptide due to modulated FcRn binding affinity. A "second" or "altered" or "modified" polypeptide having better properties for a particular therapeutic or diagnostic application. The modeling can be performed on a computer.

These methods may include one or more steps. For example, the method may comprise providing structure or corresponding data of a complex of a target Fc polypeptide and FcRn. In another or subsequent step, the method may comprise identifying a defined residue or set of residues in the Fc region of the starting polypeptide that may be modified (e.g., mutated) and predicted to affect the binding affinity of the polypeptide for FcRn (ie, candidate amino acids).

Preferred mutations to introduce in the Fc region of the starting polypeptide include those mutations that alter the antigen-independent effector function (eg, half-life) of the starting polypeptide. In one embodiment, the mutation does not impair any other existing effector function of the starting polypeptide (e.g., antigen, ligand, or receptor binding or Fc-mediated effector function (non-FcRn-binding effector function)) or impair it in its intended use. Thus, the mutations introduced preferably maintain many of the other advantages offered by the Fc region. For example, Fc-containing polypeptides often have ADCC function. This important cell killing activity will be partially or completely lost in antibody constructs with truncated Fc regions. Preservation of Fc-dependent ADCC function may be important in certain applications, as it can elicit a cell-killing effect, thereby enhancing the efficacy of anticancer or other drugs that act through ADCC-dependent depletion mechanisms.

In preferred embodiments, the altered polypeptides of the invention contain mutations that do not abrogate or, more preferably, do not modulate other desired immune effector or receptor binding functions of the starting polypeptide. In an especially preferred embodiment, the altered polypeptide contains mutations that do not alter the binding of the altered polypeptide to an Fc-binding protein that facilitates the purification of the altered polypeptide, especially Staphylococcal protein A or G. The site on Fc responsible for binding to protein A is known in the art (Deisenhofer J. 1981, Biochemistry, Apr 28;20(9):2361-70).

A. Sequence-based analysis

In one embodiment, potential sites of alteration are predicted based on a sequence comparison of the Fc region of the starting polypeptide with mammalian Fc regions having different FcRn binding affinities. The sequences of the Fc region are aligned and one or more corresponding amino acids from sequences with different binding properties are substituted into the Fc region of the starting polypeptide.

In one embodiment, when a shorter half-life is desired, the corresponding amino acid is selected from an immunoglobulin of an unrelated mammalian species, wherein said immunoglobulin exhibits lower affinity for the FcRn receptor. In another embodiment, when a longer half-life is desired, the cognate amino acid is selected from an immunoglobulin of an unrelated mammalian species, wherein said immunoglobulin exhibits a higher affinity for the FcRn receptor.

For example, rabbit Fc regions show a high level of homology to human Fc regions, especially in regions that contact hFcRn. Furthermore, rabbit IgG binds hFcRn more tightly than human IgG (Ober et al., Int. Immunol. 13:1551-1559, 2001). Thus, in an exemplary embodiment, potential alteration sites are identified that are different in the Fc region of the polypeptide to be modified (e.g., in a human Fc region) than in a polypeptide having a desired biological property (e.g., a rabbit Fc region). those residues, and replace one or more of these amino acid residues in the human IgG1 Fc region with one or more corresponding amino acid residues from the rabbit IgG1 Fc region.

For example, a chimeric protein will exhibit a specific amino acid or combination of amino acids that have been replaced in human Fc with rabbit Fc amino acids. These rabbit Fc amino acids were determined from the interface of human Fc and neonatal Fc receptor (hFcRn) shown in a homology model developed from the ratIgG2a ratFcRn crystal structure. Specific amino acids determined to be present in the human Fc:human FcRn contact domain can then be changed from the human sequence to the rabbit sequence. Alternatively, one or more of these residues of the human Fc may be substituted by the corresponding amino acid residue from a guinea pig immunoglobulin of the same type.

Exemplary alteration sites include EU positions 280, 281, 282, 283, 284, 285, 288, 289, 290, 305, 307, 308, 309, 315, 340, 344, and 378.

More particularly, a polypeptide of the invention may contain at least one amino acid mutation selected from the group consisting of Asp280Asn (wherein, D indicates the amino acid position to be mutated (by substitution) at said EU position (278), N indicates待替代入该位置以形成改变的多肽的氨基酸)，Gly281Glu，Val282Glu，Glu283Gln，His285Arg，Asn286Thr， Lys288Arg，Thr289Pro，Lys290Pro，Val305Thr，Thr307Pro，Val308Ile，Leu309Thr，Asn315Arg，Lys340Arg，Arg344Leu，Ala378Ser，Ser383Lys，Glu386Lys， Pro387Ala, and Asn389Asp.

B. Conformational Analysis

In another embodiment, the method of identifying a target amino acid comprises analyzing (eg, visual observation or computational analysis) an initiating polypeptide (eg, an Fc-containing polypeptide) and/or an initiating polypeptide that binds to an Fc receptor (eg, FcRn).

The three-dimensional structure of a protein affects its biological activity and stability, and this structure can be determined or predicted in a variety of ways. Generally, empirical methods use physical biochemical analysis. Alternatively, the tertiary structure can be predicted by building a three-dimensional structure model of one or more homologous proteins (or protein complexes) with known three-dimensional structures. X-ray crystallography is probably the most familiar method for determining protein structure (thus, the term "crystal structure" may be used in place of the term "structure") (for example, the crystal structure of the human IgG1 Fc region has been determined (Disenhofer et al., Biochemistry( 1981), 20:2361-70)), but can also be estimated using circular dichroism, light scattering, or by measuring the absorption and release of radiant energy. Other useful techniques include neutron diffraction, nuclear magnetic resonance (NMR) and homology modeling. All these methods are known to those skilled in the art and they have been fully described in standard textbooks (see, for example, Physical Chemistry, 4 ^th Ed., WJ Moore, Prentiss-Hall, N, J., 1972, or Physical Biochemistry, KEVan Holde, Prentiss-Hall, NJ 1971)) and many publications. Any of these techniques can be used to determine the structure of an Fc region, a polypeptide comprising an Fc region (or an FcRn-binding portion thereof), or a complex of the polypeptide and FcRn (the structure can then be analyzed to predict amino acids for substitution) and and/or the techniques can be used to provide information about one or more steps of a method (eg, those described below).

Methods for forming crystals of antibodies, antibody fragments, or scFv-antigen complexes have been reported, for example, by van den Elsen et al. (Proc. Natl. Acad. Sci. USA 96:13679-13684, 1999, hereby incorporated herein Reference). These techniques known in the art can be used to determine the structure of complexes containing FcRn and Fc-containing polypeptides for analysis according to the methods of the invention. Alternatively, published complex structures or corresponding data are readily available from commercial or public databases, such as the Protein Data Bank. In addition, a co-crystal structure (2.8 Å) of rat FcRn and a heterodimeric rat Fc containing a single FcRn binding site was recently obtained (e.g., Martin et al., Molecular Cell, (2001), 7:867-77 ). When the structure of a complex (eg X-ray structure) or its corresponding data is unknown or unavailable, a homology model using a related complex (eg from another species or a homologous ligand/receptor complex) can be utilized. For example, the crystal structure of the rat Fc-FcRn complex can be used to model the interaction of human Fc with FcRn.

Data corresponding to Fc/FcRn complexes can be evaluated to identify potential sites of alteration. In another embodiment, the methods of the invention comprise analyzing (eg, structural analysis or computational analysis) the difference in conformation between a free (ie, unbound) Fc-containing polypeptide and an Fc-containing polypeptide bound to FcRn.

C. Electrostatic optimization

Basic calculations for carrying out the methods of the present invention are provided, for example, in US Patent No. 6,230,102, the contents of which are hereby incorporated by reference in their entirety. In one embodiment, the polypeptide is altered (or "modified") based on the results of computational analysis of the electrostatic forces between the polypeptide and FcRn, preferably according to the discrete criteria or rules of the invention described herein. This computational analysis allows prediction of the optimal charge distribution in the polypeptide receptor complex, one way of representing the charge distribution in a computer system is as a set of multipoles. Alternatively, the charge distribution can be represented by a set of point charges located at the positions of the polypeptide atoms. Once the charge distribution (preferably, the optimal charge distribution) has been determined, the polypeptide can be modified to match, or better match, the charge distribution.

Can be calculated by performing the calculations described in U.S. Patent No. 6,230,102 (or as described in Tidor and Lee, J. Chem. Phys. 106:8681, 1997; Kangas and Tidor, J. Chem. Phys. 109:7522, 1998) A computer-implemented method mediates the computational analysis. This computer program can be adapted to take into account the realities of polypeptide-FcRn binding (and, unlike other methods, the method of the invention takes into account, for example, the presence of polypeptide and FcRn in a solvent (e.g., an aqueous solvent such as water, phosphate-buffered saline (PBS) , plasma or blood), solvents, long-range electrostatic interactions, and dielectric effects when binding). This computer-implemented method can be used to identify modifications to the polypeptide structure that, on the modified polypeptide, will result in minimizing the contribution of electrostatic interactions to the free energy of binding of the modified polypeptide and FcRn (relative to the unmodified (“acting In the case of the original ") polypeptide) the charge distribution. Typically, a computer system (or device) implementing the operations described herein (see U.S. Patent No. 6,230,102 in more detail) will include an output device (e.g., a CRT monitor, LCD, printer, communication device such as a modem, audio output, etc.). In addition, an instruction to execute the method of the present invention may be partially or completely given to a medium suitable for use in an electronic device so that the instruction is executed. Thus, the methods of the invention can be modified into high-throughput methods involving software (eg, computer readable instructions) and hardware (eg, computers, robots, and chips). This computer-implemented method is not limited to a particular computer platform, a particular processor, or a particular high-level programming language. Useful methods are described in US Pat. No. 6,230,102 and described in more detail in Lee and Tidor (J. Chem. Phys. 106:8681-8690, 1997), all of which are hereby incorporated by reference.

The rules of the present invention can be applied as follows. To modulate the FcRn binding affinity of a polypeptide, eg, to reduce, improve or reestablish this binding, basic sequence and/or structural data are first obtained.

In one embodiment, candidate amino acid residues may be selected from those determined to have sub-optimal or optimal binding affinity. Alternatively or additionally, target amino acid residues may be selected from those residues in the Fc region that are adjacent to the residue with optimal or suboptimal binding affinity. Typically, electrostatic charge optimization is first used to determine the suboptimal position in the Fc region for binding (Lee and Tidor, J. Chem. Phys, 106:8681-8690, 1997; Kangas and Tidor, J. Chem. Phys .109:7522-7545, 1998).

The one or more mutations (ie, modifications) are then further computationally analyzed. Based on these calculations, binding affinities are then determined for a subset of modified polypeptides having one or more modifications according to the rules of the invention.

Using the continuum electrostatics model, electrostatic charge optimization can be performed on each side chain of amino acids in the polypeptide Fc. Charge optimization gives charge to the centers of atoms, but does not always produce actual mutations. Therefore, a round of charge optimization can be performed, during which various constraints are imposed to represent the natural side chain characteristics of the position of interest. For example, optimization can be performed for net side chain charges of -1, 0, and +1 with the additional constraint that no atoms have charges above a certain value (eg, 0.85 electron charge units). Candidate amino acid side chain positions and residue modifications at these positions were then identified based on the potential increase in electrostatic binding free energy observed in the optimization.

The difference in free energy of binding (in kcal/mol) from a native residue to a completely uncharged side chain isostere (ie, a residue with the same shape but no or partial charge on the atom) can be calculated. Negative numbers indicate predicted increases in binding affinity.

When the binding free energy difference is favorable (ΔG<-0.3kcal/mol) and isosteres from the native residue to a completely uncharged side chain (i.e., has the same shape but is atomically uncharged or partially charged The modification is selected from the set of amino acids with non-polar side chains (e.g., A, C, I, L, M, F, P, V) when transitions are relevant.

When the binding free energy difference that can be obtained using an optimal charge distribution in the side chains and a net side chain charge of -1 is favorable (ΔG < -0.3 kcal/mol), from amino acids with negatively charged side chains Select modifiers in a set (eg, D, E).

Similarly, when using the optimal charge distribution in the side chains and the binding free energy difference that can be obtained with a net side chain charge of +1 is favorable (ΔG < -0.3 kcal/mol), starting with positively charged side chains Modifications are selected from a set of amino acids (eg, R, H, K).

Finally, when the binding free energy difference that can be obtained using an optimal charge distribution in the side chains and a net side chain charge of 0 is favorable (ΔG < -0.3 kcal/mol), from the addition of C, G, M and F Modifications are selected from the set of amino acids with uncharged polar side chains (eg, N, C, Q, G, H, M, F, S, T, W, Y).

As described herein, the designed modified polypeptides can be constructed in silico and the binding energies recalculated. Modified side chains can be constructed by performing a rotamer dihedral scan in CHARMM using a dihedral increment of 60 degrees to determine the most desired position for each side chain. Binding energies were then calculated for wild-type (starting) and mutant (modified) complexes using the Poisson-Boltzmann electrostatic energy and additional terms for van der Waals energy and cryptic surface area.

The results obtained by calculating these computational modifications can then be re-evaluated in silico as desired, for example after subsequent repetitions of the method, or be informed by additional experimental structure/function data. Rules that can be divided into the following categories allow several predictions:

1) Modifications on the polypeptide at the interaction interface residues that are partially concealed when binding to FcRn (improving the interaction by forming hydrogen bonds);

2) modifying polar residues on the polypeptide that are sequestered upon binding and thus pay the price for desolvation but do not form any direct electrostatic interactions with the receptor (usually by modifying to wild-type residues have a similar shape to hydrophobic residues or are improved by adding residues that cause favorable electrostatic interactions); and

3) modifying surface residues on the polypeptide that exist in regions of uncomplementary potential. These modifications are thought to improve the long-range electrostatic interactions between the peptide and FcRn without disturbing the packing interactions at the binding interface.

So implemented, the rules of the invention allow the successful prediction of changes in affinity (eg decrease and increase), side chain modifications. These findings can be grouped into three broad categories of modifications. The first type of modification involves hydrogen-bond-forming interfacial residues located on the antigen spanned by self-charged groups; the second type involves cloaking that pays a desolvation penalty upon binding but does not cause back elctrostatic interactions. polar residues; and a third category involving long-range electrostatic interactions.

The first type of modification is identified by examining fundamental physical/chemical factors, since these residues essentially form hydrogen bonds with unsaturated hydrogen partners of the antigen. Unlike other approaches, the present rules allow for uncanny residue modifications where the cost of desolvation is allowed to outweigh the beneficial interaction energies.

The second class of modifications represents another group of modifications, since increased energy results primarily from eliminating unfavorable desolvation and maintaining nonpolar interactions.

A third class of modifications involves long-range interactions that exhibit the potential to significantly increase affinity. These types of modifications are of particular interest since they do not come into direct contact with the antigen and thus disturb the delicate interactions of the polypeptide-FcRn interface less.

Thus, once the desired side chain chemistry has been determined for a candidate amino acid position according to these rules, the residue position(s) may be modified or altered, for example by substitution, insertion or deletion (see further herein).

In addition to the above rules for polypeptide modification, note that certain determinants, such as solvent effects, may also be included in the initial (and subsequent) calculation of the optimal charge distribution.

In one embodiment, preferred mutations (e.g., amino acid substitutions) that alter the effector function of the target antibody are selected based on charge optimization data, and can be neutralized by predicting that the Fc region can be at different pH levels—for example, about 7.2-7.4. pH; an acidic pH of about 3.0-5.0 (eg, 4.0); or an alkaline pH of about 8.0-10.9 (eg, 9.0) - an amino acid change (eg, substitution) that alters (eg, enhances or decreases) binding to an Fc receptor to The mutations are identified.

Charge optimization results in an optimal set of charges at atomic centers, but does not generate actual mutation cues. Once charge optimization has been determined using the methods above, one or more target amino acid residues or any adjacent amino acid residues in the polypeptide can be altered (e.g., mutated) based on the results of charge optimization (e.g., FcRn binding in the CH2 domain or Fc region). residues in or around the loop). During this process, the optimal charge distribution is analyzed and mutations that are closer to the optimal situation than the current residue are selected. For example, amino acid substitutions can be selected that match, better match, or are closer to optimal than the current residue. One or more than one mutation may be selected so as to obtain an optimal charge distribution. Preferred mutations can be selected by observation of the data or by computational analysis of the data.

Currently, software for examining electrostatic forces can model an optimal charge distribution, after which the user can determine what amino acid substitutions or changes will improve this distribution. Accordingly, these steps (eg, examining the modeled optimal charge distribution and identifying sequence modifications for improving antigen binding) are, or can be, part of the presently claimed method. However, since the software can be easily modified in the future to include a selection of amino acid substitutions (or changes), it may only be necessary to examine the output and perform the suggested change (or, if desired, some variation of it) .

In one embodiment, the Fc region can be tested at different pH levels (for example, a neutral pH of about 7.2-7.4; an acidic pH of about 3.0-5.0 (such as 4.0); or a pH of about 8.0-10.9 (such as 9.0). Alkaline pH) Amino acid changes (eg, substitutions) that alter (eg, enhance or decrease) binding to Fc receptors, amino acids for substitution are identified.

In one embodiment, the present invention relates to a method for modulating the binding affinity of a polypeptide comprising an FcRn-binding portion of an Fc region to FcRn at two different pH levels, the method comprising: determining that the polypeptide binds to FcRn at a first pH level in a solvent The spatial representation of the optimal charge distribution of polypeptide amino acids and the associated change in binding free energy; determine the spatial representation of the optimal charge distribution of polypeptide amino acids and the associated change in binding free energy when the polypeptide binds to FcRn at a second pH level in a solvent ; based on comparing these charge distributions, identifying residues showing different charge distributions at the first and second pH levels, at least one candidate amino acid residue position in the polypeptide to be modified to change the binding free energy of the polypeptide when binding FcRn; and selecting options A predetermined amino acid residue is used for substitution at the amino acid position, so that the affinity of the polypeptide for FcRn is regulated after the substitution of the selected amino acid residue.

In one embodiment, the first pH is about 7.4. In one embodiment, the second pH is about 6.0.

In one embodiment, the uncharged amino acids of the starting polypeptide are replaced with charged amino acids. In another embodiment, an uncharged amino acid of the starting polypeptide is replaced with another uncharged amino acid. In another embodiment, amino acids (eg, uncharged or negatively charged amino acids) of the starting polypeptide are replaced with positively charged amino acids. Positively charged amino acids include histidine, lysine, asparagine. In another embodiment, amino acids (eg, uncharged or positively charged amino acids) of the starting polypeptide are replaced with negatively charged amino acids. Negatively charged amino acids include aspartate (aspartic acid) and glutamate (glutamic acid). In another embodiment, amino acids (eg, negatively or positively charged amino acids) of the starting polypeptide are replaced with uncharged amino acids. In another embodiment, an amino acid (eg, an uncharged amino acid) of a starting polypeptide is replaced with an uncharged amino acid having a different charge distribution.

In certain embodiments, when an amino acid is introduced into an altered polypeptide, the substituted amino acid alters the charge of the polypeptide such that the altered polypeptide has a different net charge than the starting polypeptide. In other embodiments, when an amino acid is introduced into an altered polypeptide, the substituting amino acid does not alter the charge of the polypeptide such that the altered polypeptide has the same net charge but a different charge distribution than the starting polypeptide.

In another embodiment, the amino acids are divided into the following three groups: (1) non-polar amino acids with uncharged side chains (eg, A, L, I, V, G, P). These amino acids typically participate in hydrophobic interactions; (2) polar amino acids with zero net charge but different parts of the side chains with non-zero partial charges (e.g., M, F, W, S, Y, N, Q, C) . These amino acids can participate in hydrophobic and electrostatic interactions. (3) Charged amino acids (eg, R, K, H, E, D) that may have a non-zero net charge on the side chain. These amino acids can participate in hydrophobic and electrostatic interactions.

In one embodiment, at least one mutation that alters the affinity of the polypeptide-Fc interaction is a mutation from one of the following three types:

(1) Mutations that change the charge distribution at the interaction interface or in the non-complementary electrostatic potential region between FcRn and polypeptides away from the interface. These changes can include: intergroup substitutions at polar, nonpolar, and charged amino acids (which will always change the position of the partial charge); and substitutions within the polar amino acid group and within the charged amino acid group, provided that They change the charge distribution (e.g. C has partial negative charges on SG atoms and partial positive charges on HG atoms. While N has partial positive charges on SG and HD atoms and partial negative charges on ND and OD atoms ; then, replacing N with C will change the charge distribution). For example, in one embodiment, polar amino acids (having zero net charge but partial charges on atoms in the side chain) are substituted for non-polar amino acids (zero charge on all atoms in the side chain) or vice versa;

(2) Polar or charged residues on the mutant antibody that are sequestered upon binding and thus pay a paydesolvation penalty (the energy cost of removing the solvent upon binding) but do not form any favorable electrostatic interactions with FcRn base. In this case, improvements are obtained by mutating to non-polar amino acids that do not interact with solvent and thus do not pay a desolvation penalty upon binding.

(3) Mutation of surface residues that change the shape of the molecule and thereby affect the dielectric properties of the mediator located between the polypeptide and FcRn. Since solvents have a higher shielding capacity (dielectric constant) than proteins, charges can interact more strongly through proteins than through solvents. Thus, filling (or clearing) the space between charges located on the polypeptide and FcRn with protein side chains will modulate the interaction of these charges. These mutations include amino acid substitutions with substitutes that have side chains of a different shape than the original amino acid (all changes except between isosteres: V to T, D to N, N to D, L to D , L to N, D to L, N to L, Q to E, and E to Q). For this set of substitutions on non-polar amino acids, this phenomenon would be the only effect on the electrostatic interaction between the polypeptide and FcRn.

In a particular embodiment, the altered polypeptide comprises a substitution at an amino acid position corresponding to an EU position selected from the group consisting of: 248, 249, 250, 251, 252, 254, 256, 255, 260, 257, 277, 281 , 282, 287, 284, 285, 286, 288, 290; 304, 305, 306, 307, 309, 310, 312, 313, 315, 343, 374, 426, 428, 430, 431, 432, 434, or 438.

In a more particular embodiment, the altered polypeptide may comprise any one or any combination (and up to all) of the following mutations: substitution of aspartic acid at EU position 248; substitution of arginine at EU position 249 or lysine; substitution at EU position 250 by arginine or lysine; substitution at EU position 251 by arginine, lysine or asparagine; substitution at EU position 252 by serine or threonine acid; substitution at EU position 254 by serine or threonine; substitution at EU position 256 by arginine, glutamic acid or lysine; substitution at EU position 255 by leucine, aspartic acid or Methionine; substitution at EU position 260 by lysine; substitution at EU position 257 by arginine, aspartic acid, glutamic acid, or lysine; substitution at EU position 277 by arginine, Aspartic acid, glutamine, or lysine; glutamic acid at EU position 279; glutamine at EU position 281; arginine, aspartate at EU position 282 acid, glutamic acid, or lysine; substitution at EU position 287 by aspartic acid, glutamic acid, lysine, proline, or threonine; substitution at EU position 284 by aspartic acid or glutamic acid; substitution at EU position 285 by aspartic acid, glutamic acid or phenylalanine; substitution at EU position 286 by aspartic acid, glutamic acid or methionine; at EU position Substitution of aspartic acid or glutamic acid at position 288; substitution of aspartic acid or glutamic acid at EU position 290; substitution of aspartic acid or glutamic acid at EU position 304; substitution of aspartic acid or glutamic acid at EU position 305 Substitution to arginine; substitution to arginine, aspartic acid, glutamic acid, or lysine at EU position 306; substitution to arginine, aspartic acid, or glutamine at EU position 307 acid; substitution at position 309 by arginine, aspartic acid, lysine or glutamic acid; substitution at EU position 310 by arginine, leucine, lysine or asparagine; at EU position Arginine, asparagine, or lysine at position 312; aspartic acid, arginine, or lysine at EU position 313; aspartic acid or glutamate at EU position 315 Amino acid; glutamine or lysine at EU position 343; arginine or glutamine at EU position 345; arginine, lysine or leucine at EU position 374 acid; substitution at EU position 376 by asparagine; substitution at EU position 426 by arginine, aspartic acid, or glutamic acid; substitution at EU position 428 by arginine, glutamine, or Lysine; substitution at EU position 430 by lysine; substitution at EU position 431 by proline; substitution at EU position 432 by arginine; substitution at EU position 434 by leucine or lysine acid; or substitution of glutamic acid at EU position 438.

In a more particular embodiment, a substitution is introduced in the Fc region of IgG1 selected from one of the following mutations: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L25 1N, M252S, M252T, M254S, M254T，T256R，T256E，T256K，R255D，R255L，R255M，T260K，T260R，T260K，T260Q，P257R，P257D，P257E，P257K，W277R，W277D，W277Q，W277K，V279E，G281Q，V282R，V282D，V282E，V282K， V282E，H287D，A287E，A287K，A287P，A287T，V284D，V284E，A287E，H287D，H285E，H285F，N286D，N286E，N286M，K288D，K288E，K290D，K290E，S304D，S304E，V305R，V306E，L306R，L306D， L306E，L306K，V307E，T307R，T307D，L309R，L309D，L309E，L309K，H310R，H310N，H310L，H310K，L312K，D312R，D312N，D312K，N313R，W313D，N313K，W313K，N315D，N315E，P343Q，P343K， E345R, P374R, P374L, P374K, D376N, S426R, S426D, S426E, E430K, A431P, L432R, N434K, N434L, or Q438E.

D. Side chain re-accumulation

In another embodiment, the method of selecting preferred amino acid substitutions comprises applying sidechain repacking techniques on a structure (eg, a crystal structure or model) of a complex comprising FcRn and an Fc-containing polypeptide. In side chain restacking calculations, target residues can be computationally modified and the resulting Fc polypeptide mutants evaluated computationally for stability in a conformation bound to FcRn. The side chain re-stacking calculation produces a ranked list of variants with altered stability (ie, altered intramolecular energy).

In another embodiment, a method of selecting preferred amino acid substitutions comprises applying side chain repacking techniques to a structure (eg, a crystal structure or model) of a complex comprising two polypeptides (eg, an Fc-containing polypeptide and FcRn). Mutations that result in a desired change (eg, increase or decrease) in receptor binding affinity can then be selected for experimental expression.

The number of protein mutants evaluated computationally can be enormous, since every variable amino acid position can be mutated to all 20 standard amino acids. Exemplary computational algorithms for ranking computational analysis results include dead-end elimination and tree retrieval algorithms (see, e.g., Lasters et al. (Protein Eng. 8:815-822, 1995), Looger and Hellinga ( J. Mol. Biol. 307:429-445, 2001), and Dahiyat and Mayo (Protein Sci. 5:895-903, 1996)).

E.3-D display

In one embodiment, the three-dimensional structure and/or model of the polypeptide-FcRn complex can be analyzed visually (eg, using a 3-D molecular imager) to predict mutations that favor or disfavor a particular molecular conformation.

In one embodiment, the mutation results in an increased affinity of the Fc-containing polypeptide for an Fc receptor by introducing additional contacts between the mutated Fc-containing polypeptide and the amino acid residues of FcRn. This can be achieved by, for example, replacing smaller amino acid side chains (e.g., alanine, glycine, serine, aspartic acid, asparagine, valine, cysteine) of an Fc-containing polypeptide with larger amino acid side chains. chains (e.g., methionine, tryptophan, phenylalanine, tyrosine, leucine, isoleucine, lysine, arginine, glutamine, glutamic acid, proline , threonine, histidine) to achieve. In another embodiment, the mutation results in a reduced affinity of the Fc-containing polypeptide for an Fc receptor by altering an amino acid in the Fc-containing polypeptide that contacts FcRn. This can be done, for example, by incorporating larger amino acid side chains (e.g., methionine, tryptophan, phenylalanine, tyrosine, leucine, isoleucine, lysine, arginine, amino acid, glutamine, glutamic acid, proline, threonine, histidine) with smaller amino acid side chains (e.g., alanine, glycine, serine, aspartic acid, asparagine, valine, cysteine) to obtain.

In one embodiment, one or more mutations are made in the FcRn binding loop region. The FcRn binding loop comprises amino acid residues 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298 and 299 (according to EU numbering ). This ring is shown in Figure 1. In one embodiment, one or more mutations are carried out in amino acids selected from the group consisting of: 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 291, 292, 293, 294, 295, 296, 297, 298 and 299 (according to EU number). In one embodiment, mutations to one or more amino acids in the FcRn binding loop result in reduced binding affinity to FcRn. Preferably, mutations to one or more amino acids in the FcRn binding loop result in increased FcRn binding affinity. In one embodiment, the invention relates to a method of increasing the half-life of a polypeptide of the invention comprising mutating at least one amino acid residue in the FcRn binding loop. In another embodiment, the present invention relates to compositions for use in the treatment of a patient who would benefit from treatment with an altered polypeptide of the present invention having an increased half-life.

In another embodiment, one or more mutations are made in the 15A FcRn contact region, for example at one or more of the following positions (exemplary amino acids for these positions are also listed): 243F; 244P; 245P ;246K; 247P; 248K; 249D; 250T; 251L; 252M; 253I; 254S; 255R; ;285H; 286N; 287A; 288K; 289T; 290K; 291P; 292R; 293E; 302V; 303V; 304S; 318E; 319Y; 336I; 337S; 338K; 339A; 340K; 341G; 342Q; 343P; 344R; 345E; 346P; ;380E; 381W; 382E; 383S; 384N; 385G; 386Q; 387P; 388E; 389N; 391Y; 393T; ; 436Y; 437T; 438Q; 439K; and 440S (according to EU numbering).

F. Panning FcR mutant libraries

In another embodiment, the members of any available Fc polypeptide library can be assayed for altered effector function using the multimeric FcRn receptors of the invention (ie, the FcRn-Fc fusion proteins described in further detail herein). For example, a phage library of mutated Fc-containing polypeptides can be screened or "panned" using the multimeric Fc receptors of the invention. Phage display libraries typically comprise phage particles expressing the mutated Fc-containing polypeptide, or Fc region, or a mutated region or portion thereof comprising a polynucleotide sequence inserted into the phage genome. Thus, a phage library may include therein phage particles expressing a repertoire of mutated Fc-polypeptides or each member of a combinatorial library. Phage used in these methods are typically filamentous phage, including fd and M13. The mutated Fc portion is typically expressed on the surface of the phage as a fusion with a viral coat protein (eg, phage gene III or VIII protein). Methods for preparing phage libraries are known in the art (see, Brinkman et al., J. Immunol. Methods, (1995), 182:41-50; Ames et al., J. Immunol. Methods, (1995), 184:177- 86; Kettleborough et al., (1994), 24:952-8; Persic et al., Gene, (1997), 187:9-18; Burton et al., Advances in Immunol., (1994), 57:191-280. Or, can Screening of existing phage display libraries. Available libraries include the library of altered Fc polypeptides described in WO 02/060919.

Phage expressing an Fc region that associates with a multimeric Fc receptor of the invention at a higher ratio can be selected or identified, for example using labeled multimeric FcRn (bound to or immobilized on a solid surface such as a bead). Binding occurs at or below the affinity of the starting polypeptide. The phage can also be used in FACS sorting to select for cells expressing a higher affinity form of the Fc region.

Using these methods, altered forms of Fc molecules can be detected for changes in FcRn binding affinity and those molecules selected with the desired increase or decrease in binding affinity.

G. Further optimization of FcRn binding affinity

Altered polypeptides produced by the methods of the invention can be reconstituted and further altered to further modulate FcR binding (eg, to further increase or further decrease binding). Accordingly, additional steps may be performed after the above steps, including, for example: (a) obtaining data corresponding to the structure of the complex of the altered or "second" polypeptide and receptor; (b) using this data (we can This is referred to as "additional data" to distinguish it from the data obtained and used in the first "round", determining the behavior of the distribution of additional charges of the constant region of the second polypeptide, wherein the additional charges distributed such that electrostatic interactions contribute minimally to the free energy of binding between the second polypeptide and the receptor; and (c) expressing a third polypeptide that binds to the receptor having at least one amino acid residue different from the sequence of the second polypeptide. In addition, empirical binding data can be used to inform further optimization. Additional rounds of optimization can then be performed.

IV. Methods of Altering Polypeptides

Once the desired mutation to be made in the starting polypeptide has been obtained, any of a number of available methods can be used to generate an altered polypeptide comprising the mutation. For example, the polynucleotides can be produced by recombinant methods. Furthermore, due to the degeneracy of the genetic code, multiple nucleic acid sequences can be used to encode each desired polypeptide.

Exemplary art-known methods for preparing nucleic acid molecules encoding amino acid sequence variants of a starting polypeptide include, but are not limited to, site-directed (or oligonucleotide-mediated) manipulation of earlier prepared DNA encoding the polypeptide. of) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

Site-directed mutagenesis is the preferred method for making substitutional variants. This technique is well known in the art (see, eg, Carter et al. Nucleic Acids Res. 13:4431-4443 (1985) and Kunkel et al. Proc. Natl. Acad. Sci. USA 82:488 (1987)). Briefly, in performing site-directed mutagenesis on DNA, the parental DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of the parental DNA. Following hybridization, DNA polymerase is used to synthesize an intact second strand using the hybridized oligonucleotide as a primer and this single strand of parental DNA as a template. In this way, oligonucleotides encoding the desired mutations are incorporated into the resulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See, Higuchi, "PCR Protocols", pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17: 723-733 (1989). In short, when a small amount of template DNA is used as a starting material in PCR, primers that differ slightly in sequence from the corresponding regions in the template DNA can be used to generate a relatively large number of specific DNA fragments that will interact with The template sequence differs only at the positions where the primers differ from the template.

Another method of making variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34:315-323 (1985). The starting material is a plasmid (or other vector) containing the starting polynucleotide DNA to be mutated. The codons in the parental DNA to be mutated are identified. There must be a single restriction enzyme site on each side of the identified mutation site. If no such restriction sites exist, the oligonucleotide-mediated mutagenesis methods described above can be used to create such sites by introducing them at appropriate locations in the starting polypeptide DNA. Plasmid DNA is cut at these sites to linearize it. Double-stranded oligonucleotides encoding the DNA sequence between the restriction sites but containing the desired mutation are synthesized using standard methods, in which the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques . This double-stranded oligonucleotide is called a cassette. The cassette is designed with 5' and 3' ends compatible with the ends of the linearized plasmid so that the cassette can be ligated directly to the plasmid. The plasmid thus contains the mutated DNA sequence.

Alternatively, or in addition, a desired amino acid sequence encoding a polypeptide variant can be determined, and a nucleic acid sequence encoding the amino acid sequence variant can be prepared synthetically.

Those of ordinary skill in the art will appreciate that the polypeptides of the invention with altered FcRn binding can be further modified so as to alter their amino acid sequence without altering the desired activity. For example, other nucleotide substitutions may be made in the protein to result in amino acid substitutions at "non-essential" amino acid residue positions. For example, nonessential amino acid residues in an immunoglobulin polypeptide may be replaced with other amino acid residues from the same side chain family. In another embodiment, a stretch of amino acid chain can be replaced with a structurally similar chain (distinguished by the type and/or composition of side chain family members), i.e., a conservative substitution can be made, wherein an amino acid residue is replaced by a chain with a similar side chain Amino acid residue substitutions.

Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine acid, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

In addition to amino acid substitutions, the invention also contemplates other modifications to the starting Fc region amino acid sequence to generate Fc region variants with altered effector function. For example, one or more amino acid residues of the Fc region can be deleted to reduce or enhance binding to the FcR. In one embodiment, one or more residues of the Fc region may be modified to generate such Fc region variants. Typically, no more than 1 to about 10 Fc region residues are deleted according to this embodiment of the invention. An Fc region comprising one or more amino acid deletions herein preferably retains at least about 80%, preferably at least about 90%, most preferably at least about 95% of the starting Fc region having the native sequence of a human Fc region.

Amino acid insertion Fc region variants with altered effector functions can also be prepared. For example, at least one amino acid residue (eg, one to two amino acid residues, generally no more than 10 residues) can be introduced adjacent to one or more Fc region positions identified herein as affecting FcR binding. "Adjacency" means within one to two amino acid residues from the Fc region residues identified herein. Such Fc region variants may exhibit enhanced or reduced FcRn binding.

The Fc region variant generally comprises at least one amino acid modification in the Fc region. In one embodiment, amino acid modifications may be combined. For example, a variant Fc region may include therein two, three, four, five, etc. substitutions, for example, of particular Fc region positions identified herein. In another embodiment, an altered polypeptide may have altered binding to FcRn as well as to another Fc receptor.

The Fc region consists of two identical protein chains. Thus, in one embodiment, mutations occur in both protein chains. In another embodiment, the mutation occurs in only one protein chain.

V. Preferred Changes

An altered polypeptide of the invention contains at least one mutation (eg, amino acid substitution) in its Fc region. In one embodiment, the substituted amino acid is located in the CH2 domain of the Fc region. In another embodiment, the substituted amino acid is located in the CH3 domain of the Fc region. In another embodiment, the substituted amino acids are located in both the CH2 and CH3 domains of the Fc region.

In one embodiment, the altered polypeptide of the invention comprises at least one amino acid mutation in the Fc region, the mutation acting to enhance the half-life of the molecule in blood. Molecules with increased half-life have the advantage of simultaneously reducing the periodic dosing of the drug, or alternatively reducing the dose of the drug, and maintaining the same pharmacokinetic profile.

In another embodiment, the altered polypeptide of the invention comprises at least one amino acid mutation in the Fc region, which mutation acts to reduce the half-life of the antibody in blood. Molecules with reduced half-life have the advantage of reduced patient exposure time and dosage. The reduced half-life is especially important when the altered antibody is conjugated to a toxic or radiopharmaceutical (eg, anticancer therapeutic) diagnostic marker, since nonspecific and specific binding of the antibody must be balanced.

Alterations in half-life or other antigen-independent effector functions can be predicted from the difference in FcRn binding affinity of the starting antibody and the altered antibody.

In another exemplary embodiment, the tissue distribution or bioavailability of the polypeptide is modulated by modulating FcRn binding affinity. In one embodiment, the altered polypeptide of the invention comprises at least one amino acid in the Fc region that results in increased localization of the polypeptide to a particular target tissue, such as a mucosal surface or a site of disease, such as a tumor or other specific disease characterized by pathology mutation.

In another embodiment, the altered polypeptide of the invention comprises at least one amino acid mutation in the Fc region that results in reduced localization of the altered polypeptide in tissues that are susceptible to the effects of the unaltered starting polypeptide. In an exemplary embodiment, the altered polypeptide exhibits reduced placental transfer from the maternal circulatory system to the fetus. Other sensitive tissues that may benefit from reduced (or increased) localization include brain, kidney, and liver. In an exemplary embodiment, the altered polypeptide of the invention exhibits reduced transport from the vasculature across the glomerular epithelium. In another embodiment, the altered polypeptides of the invention exhibit reduced transport from the brain across the blood-brain barrier (BBB).

In another embodiment, the altered polypeptides of the invention comprise at least one amino acid mutation in the Fc region that results in reduced binding of accumulated IgG mediated by FcRn to glomerular epithelial cells and results in a protein that is associated with the absence of the Fc region. Mutant starting polypeptides had less membranous nephropathy compared to (Haymann et al. 2004. Nephron Exp. Nephrol. 90: e13-e21)

An increase or decrease in the tissue localization of an Fc (or Fc-containing polypeptide) may be reflected by an increase or decrease in the affinity of the Fc region for FcRn (neonatal Fc receptor), respectively. Similarly, the correlation between the FcRn binding affinity of Fc polypeptides and the tissue distribution or bioavailability of Fc polypeptides is also consistent with the biological role of FcRn in promoting the transport of antibodies across epithelial barriers through endocytosis.

In some embodiments, the altered polypeptides of the invention exhibit altered antigen-independent effector function but do not alter antigen-dependent effector function (eg, ADCC or CDC). In other embodiments, the altered polypeptides of the invention have altered antigen-independent effector functions and altered antigen-dependent effector functions. In one embodiment, one or more mutations disclosed herein may confer increased antigen-dependent effector function and reduced half-life.

In another embodiment, one or more mutations disclosed herein may confer increased antigen-dependent effector function and increased half-life. In another embodiment, one or more mutations disclosed herein may confer reduced antigen-dependent effector function and reduced half-life. In another embodiment, one or more mutations disclosed herein may confer reduced antigen-dependent effector function and increased half-life.

In particular embodiments, the altered polypeptides of the invention comprise substitutions at amino acid positions corresponding to EU amino acid positions selected from the group consisting of: 248, 249, 250, 251, 252, 255, 256, 257, 258, 260, 277 , 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 304, 305, 306, 307, 309, 310, 311, 312, 313, 314, 315, 316, 317 , 340, 343, 344, 345, 374, 376, 378, 383, 386, 387, 389, 426, 428, 430, 431, 432, 434, 436, or 438.

In an exemplary embodiment, the altered polypeptide has amino acids in the 15A FcRn contact region or "band" (e.g., from EU position 243 to 261, from EU position 275 to EU position 280, from EU position 282 to EU Position 293, from EU position 302 to EU position 319, from EU position 336 to EU position 348, EU position 367, EU position 369, from EU position 372 to EU position 389, EU position 391, EU position 393, EU position 408, and from EU location 424 to EU location 440) contain substitutions.

In another exemplary embodiment, the altered polypeptide comprises EU amino acid positions 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, and 299 contain substitutions in the FcRn contact loop.

Exemplary Fc region (or FcRn binding portion thereof) sites for mutation include EU positions 280, 281, 282, 283, 285, 286, 288, 289, 290, 305, 307, 308, 309, 315, 340, 344, and 378.

In another embodiment, the altered polypeptide may comprise any one or any combination (up to all) of the following mutations: substitution of EU position 248 with aspartic acid; substitution of EU position 249 with arginine or lysine; substitution of Substitute EU position 250 with arginine or lysine; EU position 251 with arginine, lysine, or asparagine; EU position 252 with glutamine, asparagine, serine, or threonine acid; substitute EU position 255 for methionine, aspartic acid, or leucine; substitute EU position 256 for arginine, glutamic acid, or lysine; substitute EU position 257 for arginine acid, aspartic acid, glutamic acid, or lysine; substitute EU position 258 for arginine, glutamine, or lysine; substitute EU position 260 for lysine; substitute EU position 277 is arginine, aspartic acid, glutamine, or lysine; substitute EU position 279 for arginine, aspartic acid, glutamic acid, or lysine; substitute EU position 280 for day Paragine; substitute EU position 281 for aspartic acid, glutamic acid, or glutamine; substitute EU position 282 for arginine, aspartic acid, glutamic acid, or lysine; substitute EU position 283 for glutamine; EU position 284 for arginine, lysine, aspartic acid, or glutamic acid; EU position 285 for arginine, aspartic acid, glutamic acid, lysine amino acid, proline, threonine, or phenylalanine; EU position 286 is replaced by aspartic acid, glutamic acid, threonine, or methionine; EU position 287 is replaced by aspartic acid amino acid, glutamic acid, lysine, proline, or threonine; substitute EU position 288 for methionine, arginine, aspartic acid, or glutamic acid; substitute EU position 289 for Proline; substitute EU position 290 for proline, aspartic acid, or glutamic acid; EU position 304 for aspartic acid or glutamic acid; EU position 305 for arginine or threonine acid; EU position 306 is replaced by arginine, aspartic acid, glutamic acid, or lysine; EU position 307 is replaced by arginine, proline, aspartic acid, or glutamic acid; Replace EU position 308 with arginine, aspartic acid, glutamic acid, lysine, or isoleucine; replace position 309 with threonine, arginine, aspartic acid, lysine or glutamic acid; substitute EU position 310 for arginine, leucine, lysine, or asparagine; substitute EU position 311 for arginine or lysine; substitute EU position 312 for arginine , asparagine, leucine, or lysine; substitute EU position 313 for aspartic acid, arginine, or lysine; substitute EU position 314 for arginine, lysine, or lysine Paragine; substitute EU position 315 for arginine, aspartic acid, or glutamic acid; substitute EU position 316 for aspartic acid or lysine; substitute EU position 317 for aspartic acid or glutamic acid acid; substitute EU position 340 for arginine; EU position 343 for glutamine or lysine; EU position 344 for leucine; EU position 345 for arginine, lysine, or glutamine; substitute EU position 374 for arginine, lysine, or leucine; substitute EU position 376 for asparagine, arginine, leucine, or lysine; substitute EU position Replace 378 with serine; replace EU position 383 with lysine; replace glycine with lysine at EU position 385; replace glutamine with alanine or lysine at EU position 386; replace EU position 387 to alanine; replace EU position 389 with aspartic acid; replace EU position 426 with arginine, aspartic acid, or glutamic acid; replace EU position 428 with aspartic acid, glutamic acid , arginine, glutamine, or lysine; substitute EU position 430 for arginine, glutamine, methionine, or lysine; substitute EU position 431 for lysine or proline acid; replace EU position 432 with arginine and phenylalanine; replace EU position 434 with leucine, arginine, or lysine; replace EU position 436 with arginine or glutamic acid; Or substitute glutamic acid at EU position 438.

More particularly, the altered polypeptide may comprise any one or any combination (up to all) of the following mutations: substitution of lysine at EU position 248 for aspartic acid; substitution of aspartic acid at EU position 249 for arginine acid or lysine; replace threonine at EU position 250 with arginine or lysine; replace leucine at EU position 251 with arginine, lysine, or asparagine; replace methylthio Amino acid is replaced by glutamine, asparagine, serine or threonine at EU position 252; arginine is replaced by methionine, aspartic acid, or leucine at EU position 255; threonine is replaced by Substitution of acid at EU position 256 by arginine, glutamic acid, or lysine; substitution of proline at EU position 257 by arginine, aspartic acid, glutamic acid, or lysine; substitution of glutamic acid Amino acid is replaced by arginine, glutamine, or lysine at EU position 258; threonine is replaced by lysine at EU position 260; tryptophan is replaced by arginine at EU position 277, day Aspartic acid, glutamic acid, glutamine, or lysine; substitute valine at EU position 279 for arginine, aspartic acid, glutamic acid, or lysine; substitute aspartic acid Substitution of asparagine at EU position 280; substitution of glycine at EU position 281 by aspartic acid, glutamic acid, or glutamine; substitution of valine at EU position 282 by arginine, aspartic acid , glutamic acid, or lysine; substitute glutamic acid at EU position 283 for glutamine; substitute valine at EU position 284 for arginine, lysine, aspartic acid, or glutamic acid ; substitution of histidine or alanine at EU position 285 by arginine, aspartic acid, glutamic acid, lysine, proline, threonine, or phenylalanine; substitution of asparagine or lysine at EU position 286 with aspartic acid, glutamic acid, threonine, or methionine; alanine at EU position 287 with aspartic acid, glutamic acid, lysine acid, proline, or threonine; substitute lysine at EU position 288 for methionine, arginine, aspartic acid, or glutamic acid; substitute threonine for pro at EU position 289 amino acid; replace lysine at EU position 290 with proline, aspartic acid, or glutamic acid; replace serine at EU position 304 with aspartic acid or glutamic acid; replace valine at EU position Substitution of 305 by arginine or threonine; substitution of leucine or valine by arginine, aspartic acid, glutamic acid, or lysine at EU position 306; substitution of threonine or valine Acid substitution at EU position 307 by arginine, proline, aspartic acid, or glutamic acid; substitution of valine at EU position 308 by arginine, aspartic acid, glutamic acid, lysine acid, or isoleucine; substitute leucine at EU position 309 for threonine, arginine, aspartic acid, lysine, or glutamic acid; substitute histidine for arginine at EU position 310 amino acid, leucine, lysine or asparagine; substitute glutamine at EU position 311 for arginine or lysine; substitute aspartic acid or leucine for arginine at EU position 312 acid, asparagine, leucine, or lysine; substitute asparagine at EU position 313 with aspartic acid, arginine, or lysine; substitute leucine at EU position 314 with arginine amino acid, lysine, or asparagine; substitute asparagine at EU position 315 for arginine, aspartic acid, or glutamic acid; substitute asparagine or glycine for asparagine at EU position 316 acid or lysine; substitute lysine for aspartic acid or glutamic acid at EU position 317; substitute lysine for arginine at EU position 340; substitute proline for glutamic acid at EU position 343 Aminoamide or lysine; substitute arginine at EU position 344 for leucine; substitute glutamic acid at EU position 345 for arginine, lysine, or glutamine; substitute proline at EU position Replacement of position 374 with arginine, lysine, or leucine; replacement of aspartic acid at EU position 376 with asparagine, arginine, leucine, or lysine; replacement of alanine at Replacement of serine at EU position 378 with serine; substitution of serine at EU position 383 by lysine; substitution of glycine at EU position 385 by lysine; substitution of glutamine at EU position 386 by alanine or lysine; Proline is replaced by alanine at EU position 387; asparagine is replaced by aspartic acid at EU position 389; serine is replaced by arginine, aspartic acid, or glutamic acid at EU position 426; Substitution of methionine at EU position 428 by aspartic acid, glutamic acid, arginine, glutamine, or lysine; substitution of glutamic acid at EU position 430 by arginine, glutamine , methionine, or lysine; replace leucine at EU position 431 with lysine or proline; replace histidine at EU position 432 with arginine for phenylalanine; replace Substitution of asparagine with leucine, arginine, or lysine at EU position 434; substitution of tyrosine with arginine or glutamic acid at EU position 436; or substitution of glutamine at EU position 438 for glutamic acid.

In another embodiment, the polypeptide of the present invention may contain at least one amino acid mutation selected from the group consisting of replacing EU position 281 with E, EU position 282 with E, EU position 283 with Q, according to the EU numbering system, Replace EU position 285 with R, EU position 286 with T, EU position 288 with R, EU position 289 with P, EU position 290 with P, EU position 305 with T, EU Replace position 307 with P, EU position 308 with I, EU position 309 with T, EU position 315 with R, EU position 340 with R, EU position 344 with L, EU position 378 Substitute S, replace EU position 383 with K, EU position 385 with K, EU position 386 with A, and EU position 389 with D.

More particularly, a polypeptide of the invention may contain at least one amino acid mutation selected from the group consisting of Asp280Asn according to the EU numbering system (wherein D indicates the amino acid position to be mutated at said EU position (280), and N indicates the position to be mutated in order to achieve said EU numbering system).改变的多肽在该位置待替代的氨基酸)，Gly281Glu，Val282Glu，Glu283Gln，His285Arg，Asn286Thr，Lys288Arg，Thr289Pro，Lys290Pro，Val305Thr，Thr307Pro，Val308Ile，Leu309Thr，Asn315Arg，Lys340Arg，Arg344Leu，Ala378Ser，Ser383Lys，Glu386Lys，Pro387Ala， and Asn389Asp.

In a particular embodiment, the altered polypeptide comprises a substitution at an amino acid position corresponding to an EU position selected from the group consisting of: 248, 249, 250, 251, 252, 256, 255, 260, 257, 277, 281, 282 , 287, 284, 285, 286, 288, 290; 304, 305, 306, 307, 309, 310, 312, 313, 315, 343, 374, 426, 428, 430, 431, 432, 434, or 438.

In another embodiment, the altered polypeptide may comprise any one or any combination (up to all) of the following mutations: substitution of EU position 248 with aspartic acid; substitution of EU position 249 with arginine or lysine; substitution of Substitute EU position 250 with arginine or lysine; EU position 251 with arginine, lysine, or asparagine; EU position 252 with serine or threonine; EU position 256 with Arginine, glutamic acid, or lysine; substitute EU position 255 for leucine, aspartic acid, or methionine; substitute EU position 260 for lysine; substitute EU position 257 for arginine , aspartic acid, glutamic acid, or lysine; substitute EU position 277 for arginine, aspartic acid, glutamine, or lysine; substitute EU position 279 for glutamic acid; substitute EU position 281 is replaced by glutamine; EU position 282 is replaced by arginine, aspartic acid, glutamic acid, or lysine; EU position 287 is replaced by aspartic acid, glutamic acid, lysine acid, proline, or threonine; replace EU position 284 with aspartic acid or glutamic acid; replace EU position 285 with aspartic acid, glutamic acid or phenylalanine; replace EU position 286 Substitute with aspartic acid, glutamic acid, or methionine; replace EU position 288 with aspartic acid or glutamic acid; replace EU position 290 with aspartic acid or glutamic acid; replace EU position Replace 304 with aspartic acid or glutamic acid; replace EU position 305 with arginine; replace EU position 306 with arginine, aspartic acid, glutamic acid, or lysine; replace EU position 307 Substitution to arginine, aspartic acid, or glutamic acid; substitution to arginine, aspartic acid, lysine, or glutamic acid at position 309; substitution to arginine, leucine at EU position 310 acid, lysine, or asparagine; replace EU position 312 with arginine, asparagine, or lysine; replace EU position 313 with aspartic acid, arginine, or lysine; replace Substitute EU position 315 with aspartic acid or glutamic acid; EU position 343 with glutamine or lysine; EU position 345 with arginine or glutamine; EU position 374 with arginine acid, lysine, or leucine; substitute EU position 376 for asparagine; substitute EU position 426 for arginine, aspartic acid, or glutamic acid; substitute EU position 428 for arginine , glutamine, or lysine; substitute EU position 430 for lysine; EU position 431 for proline; EU position 432 for arginine; EU position 434 for leucine or lysine; or substitute glutamic acid at EU position 438.

In another embodiment, a substitution is introduced in the Fc region of IgG1 selected from: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L251N, M252S, M252T, M252Q, M252N, R255D, R255L, R255M ，T256R，T256E，T256K，P257R，P257D，P257E，P257K，E258R.E258Q.E258K，T260K，T260R，T260K，T260Q，W277R，W277D，W277Q，W277E，W277K，V279R，V279D，V279E，V279K，D280N，G281D ，G281E，G281Q，V282R，V282D，V282E，V282K，E283Q，V284R，V284D，V284E，V284K，H285R，H285D，H285E，H285K，H285P,H285T，H285F，A285D，A285E，N286D，N286E，N286T，N286M，A286M ，A286E，A286D，A287D，A287E，A287K，A287P，A287T，K288D，K288E，K288M，K288R，T289P，K290D，K290E，K290P，S304D，S304E，V305T，V305R，V306D，V306E，L306R，L306D，L306E，L306K ，V307E，T307R，T307D，T307P，V308R，V308D，V308E，V308K，V308I，L309R，L309D，L309E，L309K，L309T，H310R，H310N，H310L，H310K，L312K，Q311R，Q311K，D312R，D312N，D312L，D312K ，L312K，N313R，N313K，W313D，N313K，W313K，L314R，L314N，L314K，N315R，N315D，N315E，N316D，N316K，K317D，K317E，K340R，P343Q，P343K，R344L，E345R，E345Q，E345R，P374R，P374L ，P374K，D376R，D376L，D376K，D376N，A378S，S383K，G385K，Q386A，Q386A，P387A，N389D，S426R，S426D，S426E，M428R，M428D，M428E，E430R，E430Q，E430K，E430M，A431P，L431K，H432F , L432R, N434R, N434K, N434L, Y436R, Y436E or Q438E.

As noted above, it will be appreciated that compositions of the invention may comprise one or more of the mutations described herein. In one embodiment, an altered polypeptide of the invention comprises only one of the mutations listed herein. In one embodiment, an altered polypeptide of the invention comprises only two of the mutations listed herein. In one embodiment, the altered polypeptide of the invention comprises only three of the mutations listed herein. In one embodiment, an altered polypeptide of the invention comprises only four of the mutations listed herein.

A. Altered polypeptides with enhanced FcRn binding affinity

In one embodiment, the invention provides an altered polypeptide having enhanced neonatal Fc receptor affinity compared to the corresponding starting polypeptide. Preferably, the altered polypeptide exhibits a longer circulating half-life than the corresponding polypeptide without the mutation.

In one embodiment, the altered polypeptide with enhanced FcRn binding affinity may comprise at least one amino acid substitution at one of the following EU positions: 284, 285, 286, 288, 290, and 304.

In another embodiment, the altered polypeptide with enhanced FcRn binding affinity may comprise at least one of the following amino acid substitutions: EU position 284 is replaced by glutamic acid; EU position 285 is replaced by glutamic acid; EU position 286 is replaced by Aspartic acid; EU position 288 was substituted for glutamic acid; EU position 290 was substituted for glutamic acid.

In an exemplary embodiment, the altered polypeptide comprises an Fc region of an IgG1 molecule. Preferably, the molecule contains at least one of the following mutations: V282E, V284E, H285E, N286D, N286E, K288E, K290E, and S304D.

In a preferred embodiment of the invention, the FcRn binding affinity of the modified polypeptide is increased by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold compared to the starting polypeptide, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x.

When administered to a patient, the altered polypeptides of the present invention may have a duration in the patient of more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days , 5 days, 10 days, 12 days, 2 weeks, 3 weeks, or 1 month circulation half-life. In an exemplary embodiment, the altered polypeptide of the invention has a circulating half-life in a patient of greater than 21 days.

In one embodiment, the altered binding molecule may have increased localization or bioavailability in a particular target tissue, eg, diseased tissue. Exemplary diseased tissues include neoplastic tissue or tumors or other tissues or organs characterized by the pathology of any of the disorders described herein, including the brain or CNS, lung, heart, pancreas, liver, kidney, bladder, stomach, large or small intestine , respiratory tract, lymph nodes, muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow, prostate, ovaries or uterus.

B. Altered Polypeptides with Reduced FcRn Binding Affinity

In another embodiment, the invention provides an altered polypeptide having reduced affinity for a neonatal Fc receptor compared to the corresponding starting polypeptide. Preferably, said altered polypeptide exhibits a shorter circulating half-life compared to the corresponding polypeptide without the mutation.

The altered polypeptide having reduced FcRn binding affinity may comprise at least one amino acid substitution at the EU position, wherein said EU position is selected from one of the following regions: a) from position 248 to position 260; b) from position 277 to position 315; c) from position 343 to position 374; and d) from position 426 to position 438.

In one embodiment, the altered polypeptide having reduced FcRn binding affinity may comprise at least one amino acid substitution at one of the following EU positions: 248, 249, 250, 251, 252, 256, 255, 260, 257, 277, 281 , 282, 287, 284, 285, 286, 288, 290; 304, 305, 306, 307, 309, 310, 312, 313, 315, 343, 374, 426, 428, 430, 431, 432, 434, or 438.

In another embodiment, the altered Fc polypeptide with reduced FcRn binding affinity may comprise at least one of the following amino acid substitutions: substitution of EU position 248 with aspartic acid; substitution of EU position 249 with arginine or lysine ; replace EU position 250 with arginine or lysine; replace EU position 251 with arginine, lysine, or asparagine; replace EU position 252 with serine or threonine; replace EU position 256 Substitution to arginine, glutamic acid, or lysine; substitution of EU position 255 to leucine, aspartic acid, or methionine; substitution of EU position 260 to lysine; substitution of EU position 257 is arginine, aspartic acid, glutamic acid, or lysine; substitute EU position 277 for arginine, aspartic acid, glutamine, or lysine; substitute EU position 279 for glutamic acid amino acid; substitute EU position 281 for glutamine; substitute EU position 282 for arginine, aspartic acid, glutamic acid, or lysine; substitute EU position 287 for aspartic acid, glutamine acid, lysine, proline, or threonine; substitute EU position 284 for aspartic acid; EU position 285 for aspartic acid or phenylalanine; EU position 286 for glutamine acid or methionine; replace EU position 288 with aspartic acid; replace EU position 290 with aspartic acid or glutamic acid; replace EU position 304 with aspartic acid or glutamic acid; replace EU Substitution of arginine at position 305; substitution of arginine, aspartic acid, glutamic acid, or lysine at EU position 306; substitution of arginine, aspartic acid, or glutamine at EU position 307 acid; replace position 309 with arginine, aspartic acid, lysine or glutamic acid; replace EU position 310 with arginine, leucine, lysine or asparagine; replace EU position 312 Substitution to arginine, asparagine, or lysine; substitution of EU position 313 to aspartic acid, arginine, or lysine; substitution of EU position 315 to aspartic acid or glutamic acid; Replace EU position 343 with glutamine or lysine; replace EU position 345 with arginine or glutamine; replace EU position 374 with arginine, lysine, or leucine; replace EU position Replace 376 with asparagine; replace EU position 426 with arginine, aspartic acid, or glutamic acid; replace EU position 428 with arginine, glutamine, or lysine; replace EU position 430 Substitute lysine; EU position 431 to proline; EU position 432 to arginine; EU position 434 to leucine (leucine) or lysine; or EU position 438 Substitute for glutamic acid.

In another embodiment, the altered polypeptide may comprise any one or any combination (up to all) of the following mutations: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L251N, M255S, M255T, T256R, T256E, T256K, R255D，R255L，R255M，T260K，T260R，T260K，T260Q，P257R，P257D，P257E，P257K，W277R，W277D，W277Q，W277K，V279E，G281Q，V282R，V282D，V282E，V282K，V282E，A287D，A285E，A287K， A287P，A287T，V284D，，H285D，H285F，N286E，N286M，K288D，K290D，K290E，S304D，S304E，V305R，V306E，L306R，L306D，L306E，L306K，V307E，T307R，T307D，L309R，L309D，L309E，L309K ，H310R，H310N，H310L，H310K，L312K，D312R，D312N，D312K，N313R，W313D，N313K，W313K，N315D，N315E，P343Q，P343K，E345R，P374R，P374L，P374K，D376N，S426R，S426D，S426E，E430K , A431P, L432R, N434K, N434L, or Q438E.

In certain preferred embodiments, the altered Fc with reduced FcRn binding affinity may comprise at least one mutation: M252S, M252T, V282E, K288E; V308E, V308D, L314K, N434L, or Q438E. In a more preferred embodiment, the altered polypeptide with reduced FcRn binding affinity may comprise at least one mutation: M252S, M252T, N434L or Q438E. In an even more preferred embodiment, the altered Fc polypeptide having reduced FcRn binding comprises the mutation N434L or Q438. In another embodiment, the altered polypeptide with reduced FcRn binding affinity may comprise at least one of the following amino acid substitutions: EU position 252 is replaced by threonine; EU position 255 is replaced by aspartic acid; EU position 282 is replaced is arginine, aspartic acid, glutamic acid or lysine; position 309 is replaced by arginine, aspartic acid, lysine or glutamic acid; and position 434 is replaced by leucine. .

In an exemplary embodiment, the altered polypeptide comprises an Fc region of an IgG1 molecule. Preferably, the molecule comprises at least one of the following mutations: M252T, R255D, V282R, V282D, V282E, V282K, L309R, L309D, L309K, L309E, or N434L.

In a preferred embodiment of the invention, the binding affinity of the modified polypeptide for FcRn is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, compared to the starting polypeptide, 95%, 97%, 98%, or 99%.

When administered to a patient, the altered polypeptides of the present invention may have less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 4 hours in the patient. Circulating half-lives of days, 5 days, 10 days, 12 days, 2 weeks, 3 weeks, or 1 month. In an exemplary embodiment, the altered polypeptides of the invention have a circulating half-life in a patient of less than about 21 days.

In one embodiment, the altered polypeptide may have reduced localization or bioavailability in a particular tissue, eg, a tissue that is susceptible to toxicity by the unaltered binding molecule. Exemplary tissues that are typically vulnerable to the toxic effects of therapeutic agents include the brain or CNS, heart, liver and kidney. In another embodiment, the altered polypeptide may have reduced localization to the fetus or placental transfer. In another embodiment, the altered polypeptide may have a reduced neonatal circulatory localization when ingested through milk or colostrum.

V. Expression of Altered Polypeptides

Polypeptides of the invention, such as starting polypeptides and modified polypeptides, can be produced by recombinant methods.

For example, a polynucleotide encoding a polypeptide can be inserted into an appropriate expression vector for recombinant expression. When the polypeptide is an antibody, the polynucleotides encoding the additional light and heavy chain variable regions, optionally linked to constant regions, can be inserted into the same or different expression vectors. An affinity tag sequence (eg, a His(6) tag) can optionally be attached to or included in the starting polypeptide sequence to facilitate downstream purification. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of the immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the polypeptide.

These expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain a selectable marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, or neomycin resistance) to allow detection of those cells transformed with the desired DNA sequence (see, e.g., U.S. Pat. No. 4,703,362).

Escherichia coli (E. coli) is a particularly useful prokaryotic host for cloning polynucleotides (eg, DNA sequences) of the invention. Other suitable microbial hosts include bacilli such as Bacillus subtilus and other Enterobacteriaceae such as Salmonella, Serratia and various Pseudomonas species (Pseudomonas) species.

Other microorganisms such as yeast can also be used for expression. Saccharomyces and Pichia are exemplary yeast hosts, and suitable vectors have expression control sequences (eg, promoters), origins of replication, termination sequences, and the like, as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzyme promoters. Inducible yeast promoters include, for example, promoters from aldehyde dehydrogenase, isocytochrome C, and enzymes responsible for methanol, maltose, and galactose utilization.

In addition to microorganisms, mammalian tissue culture can also be used to express and produce polypeptides of the invention (eg, polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells are actually preferred as a number of suitable host cell lines capable of secreting heterologous proteins such as intact immunoglobulins have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, 293 cells , myeloma cell lines, transformed B cells and hybridomas. Expression vectors for these cells include expression control sequences, such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and essential processing information sites such as ribosome binding sites , RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus. Promoters of cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

Vectors containing polynucleotide sequences of interest (eg, heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, which may vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biobombardment or virus-based transfection can be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press), Second Edition, 1989). Other methods for transforming mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation and microinjection (see generally, Sambrook et al., supra). For the production of transgenic animals, the transgene can be microinjected into a fertilized oocyte, or it can be incorporated into the genome of an embryonic stem cell and the nucleus of that cell is transferred into an enucleated oocyte.

Polypeptides of the invention can also be incorporated into transgenes for introduction into the genome of a transgenic animal and subsequent expression, e.g., in the milk of the transgenic animal (see, e.g., Deboer et al., 5,741,957; Rosen 5,304,489; and Meade 5,849,992). Suitable transgenes include coding sequences for the light and/or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes such as casein or beta-lactoglobulin.

Altered polypeptides (eg, polypeptides) can be expressed using a single vector or two vectors. For example, antibody heavy and light chains can be cloned on separate expression vectors and co-transfected into cells.

In one embodiment, a signal sequence may be used to facilitate expression of the polypeptides of the invention.

Once expressed, the polypeptide can be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity columns (e.g., protein A or protein G), column chromatography, HPLC purification, gel electrophoresis, etc. (see generally Scopes, Protein Purification (Springer - Verlag, N.Y., (1982)). In a preferred embodiment, the purification method can use the multimeric Fc receptor of the present invention as described below.

VI. Analysis of Binding Affinity

Binding affinity can be measured in a variety of ways. In general, and regardless of the exact means used to determine or measure affinity, the methods of the invention modulate binding affinity to FcRn, and when the methods of the invention result in a polypeptide, the polypeptide is superior in any respect to the clinical utility from which it was derived. The starting polypeptide (e.g., when the modified polypeptide can be administered at a lower dose or less frequently or with a more convenient route of administration or when the modified polypeptide has reduced side effects or altered biodistribution, the methods of the invention are considered to be valid or successful).

Alterations in effector function of an altered polypeptide can be determined by determining its binding affinity for a particular Fc receptor. In one embodiment, alterations in antigen-dependent effector function can be determined by measuring the binding affinity of the altered polypeptide for Fc gamma receptors. In another embodiment, antigen-independent effector function (eg, half-life or biodistribution) can be determined by measuring binding affinity to other Fc receptors, particularly neonatal Fc receptors (eg, human FcRn).

The change in binding affinity of an altered polypeptide of the invention can be determined by comparing the binding affinity of the altered polypeptide to a suitable control polypeptide (eg, the corresponding starting polypeptide). In one embodiment, an alteration in binding affinity can be determined by comparing the binding affinity of the altered polypeptide in a first assay to the binding affinity of a control polypeptide in a second binding assay. In alternative embodiments, changes in binding affinity can be determined by comparing the binding affinity of the altered polypeptide to a control polypeptide in the same assay. For example, the assay can be a competitive binding assay in which the binding affinity of the altered polypeptide is assessed using increasing concentrations of a control polypeptide. In a particular embodiment, binding affinity for an Fc receptor (eg, FcRn) can be determined at a first pH (eg, acidic pH) and a second pH (eg, basic pH).

More particularly, it can be tested by various assays including, for example, surface plasmon resonance (e.g., BiaCore assay), analytical ultracentrifugation, gel filtration, FRET, and ELISAL or the KinExA ^™ 3000 assay (available from Sapidyne Instruments (Boise, ID) obtained) to measure the affinity between the polypeptide and its bound receptor. Exemplary assays are detailed below.

i) Cell-free assay

Several in vitro cell-free assays for examining effector function (eg, FcR binding affinity) have been described in the art. Preferably, cell-based assays enable the evaluation of binding of altered antibodies to soluble Fc receptor forms, such as monomeric Fc receptors or multimeric Fc receptors of the invention. Automated HTS technology can be used for this screening procedure. Screening may use labels that allow detection (eg isotopic labels, chromophores, fluorophores, lumiphore or epitopes). A label can be attached to the multimeric Fc receptor or Fc-containing polypeptide to be analyzed.

Exemplary cell-free assays include, but are not limited to, FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer), Alphascreen (Amplied Luminescent Proximity Homogeneous) based assays, scintillation proximity assays, ELISA (enzyme-linked Immunosorbent assay), SPR (surface plasmon resonance, eg BIACORE(R), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis, analytical ultracentrifugation, and chromatography, including gel filtration chromatography.

ii) Cell-based assays

Several cell-based in vitro assays for examining effector function (eg, FcR binding affinity) of altered polypeptides have been described in the art. Preferably, the cell-based assay is capable of assessing the binding of altered antibodies to surface forms of Fc receptors. Exemplary cell-based assays include bridging assays and flow cytometry.

In an exemplary embodiment, the FcR binding affinity of the altered polypeptide can be measured using an FcR bridging assay. FcR (eg, FcRn or FcγR) binding affinity can be measured using assays based on the ability of an antibody to form a "bridge" between an antigen and an FcR-bearing cell.

ii) Model animal tests

For veterinary purposes or as animal models of human diseases (such as the above-mentioned immune diseases or disorders), the modified polypeptides of the present invention can also be administered to model animals in order to examine the modified polypeptides of the present invention by, for example, examining the half-life or biodistribution of antibodies potential for treatment. With regard to the latter, this animal model can be used to evaluate the efficacy of the antibodies of the invention (eg, to examine effector function, dosage, and schedule of administration).

In one embodiment, altered polypeptides of the invention can be examined for improvements in circulating half-life. An altered polypeptide having an enhanced half-life when administered to a model animal can or is expected to be more effective than a corresponding polypeptide without the mutation (e.g., an antibody of the same type (e.g., IgG such as IgG1) without mutations at positions where the altered polypeptide comprises mutations) ) remain in the loop longer. For example, since 21 days is a typical beta-phase half-life of human antibodies, altered antibodies of the invention may be those antibodies that have a circulation time longer than 21 days. In another embodiment, the altered polypeptide has a reduced half-life relative to the target antibody. For example, an altered antibody can be selected that has a reduced half-life (eg, less than 21 days) relative to the target antibody.

Examples of animal models that can be used to evaluate the efficacy of the altered polypeptides of the present invention in preventing or treating tumor formation include tumor xenograft models.

Examples of animal models that can be used to evaluate the efficacy of the altered polypeptides of the present invention in preventing or treating rheumatoid arthritis (RA) include adjuvant-induced RA, collagen-induced RA, and collagen mAb-induced RA (Holmdahl et al., Immunol. Rev. .184: 184, 2001; Holmdahl et al., Aging Res. Rev. 1: 135, 2002; Van den Berg, Curr. Rheumatol. Rep. 4: 232, 2002).

Examples of animal models that can be used to evaluate the efficacy of the altered polypeptides of the present invention in preventing or treating inflammatory bowel disease (IBD) include TNBS-induced IBD, DSS-induced IBD (Padol et al., Eur.J.Gastrolenterol.Hepatol.12:257 , 2000; Murthy et al., Dig. Dis. Sci. 38: 1722, 1993).

Examples of animal models that can be used to evaluate the efficacy of the altered polypeptides of the present invention in preventing or treating glomerulonephritis include anti-GBM-induced glomerulonephritis (Wada et al., Kidney Int. 49:761-767, 1996) and anti-thy1-induced glomerulonephritis (Schneider et al., Kidney Int. 56:135-144, 1999).

Examples of animal models that can be used to evaluate the efficacy of the antibody or antigen-binding fragment of the present invention in preventing or treating multiple sclerosis include experimental autoimmune encephalomyelitis (EAE) (Link and Xiao, Immunol. Rev. 184: 117-128, 2001).

VII. FcRn fusion protein

In another aspect, the invention provides novel reagents, eg, for purification, for determining the binding affinity of the altered Fc-containing polypeptides of the invention, and for panning for other Fc mutations that affect binding to FcRn.

An agent of the invention may be a multimeric Fc-binding protein comprising a first polypeptide and at least one other polypeptide, wherein said first and other polypeptides each comprise at least one Fc region operably linked to at least one monomeric Fc-binding domain or parts thereof.

In one embodiment, the Fc region or portion thereof and the Fc binding domain or portion thereof of each polypeptide are genetically fused together using techniques readily available in the art, respectively. In certain embodiments, multimers are formed by non-covalent association of said first and other polypeptides. In another embodiment, the multimer is formed by covalent association (eg, disulfide bonding) between said first and other polypeptides. Preferably, the first and second polypeptides are covalently bonded (eg via a disulfide bond) in their respective Fc regions to form a "dimeric" (or bidendate) Fc binding domain. In one embodiment, the Fc region may be altered (eg, mutated) so that its effector functions (especially binding to Fc receptors) are abolished or inactivated. The Fc region and Fc binding domain may be derived from any organism expressing the corresponding domains. Preferred species include human, monkey, mouse, rat and rabbit. A multimeric Fc receptor may be chimeric, eg the Fc region may be derived from a rodent (eg mouse or rat) and the Fc binding domain derived from a primate (eg human or monkey).

In certain preferred embodiments, agents of the invention may comprise an Fc binding domain derived from an Fc receptor (eg, Fc neonatal receptor) or an extracellular portion thereof. In one embodiment, the FcRn:Fc molecule of the present invention is a dimeric molecule; each monomer constituting the dimer comprises an ectodomain of the neonatal receptor α chain (for example, the α1 and α2 domains of the receptor) and an Fc The first polypeptide and the second β microglobulin (β ₂ m) polypeptide of the domain or portion thereof. In one embodiment, the Fc region is derived from IgG1. The DNA sequence of a preferred human FcRnα-Fc fusion is shown in SEQ ID NO:9 and the predicted amino acid sequence is shown in SEQ ID NO:10. The Fc region preferably contains at least one mutation that reduces its binding to monomeric or multimeric Fc receptors. For example, mutations can be made at amino acid positions 310, 311, 433 and 434 (EU numbering), such as described in Example 4.

Surprisingly, multimeric Fc receptors can be expressed at much higher levels than monomeric Fc receptors. For example, up to 100 mg/L can be obtained after stable transfection of mammalian cell lines (e.g. CHO) with expression vectors encoding FcRn:Fc fusion protein and _β2m (either together on the same vector or on different vectors). Yield.

The reagents of the present invention offer a number of advantages over existing reagents. The inventors have surprisingly discovered that the multimeric Fc receptors of the invention have greatly enhanced binding interactions with Fc-containing polypeptides. The binding affinity of hFcRnFc for immobilized hIgG was measured by Biacore, showing a binding affinity of 25 nanomolar compared to 2,000 nanomolar for monomeric hFcRn. For example, a reagent of the invention is capable of binding at least two separate FcR binding sites on an Fc-containing polypeptide (eg, an antibody Fc region). In particular, reagents of the invention are capable of binding to FcR binding sites on both heavy chains of the Fc region of an Fc polypeptide (eg, an antibody). These properties allow for enhanced binding interactions with Fc-containing polypeptides. In particular, reagents of the invention are capable of binding Fc-containing polypeptides with enhanced stability and/or affinity. Existing reagents typically comprise monomeric soluble Fc receptors or fragments thereof, which suffer from various deficiencies. For example, monomeric Fc receptors, especially human monomeric FcRn, cannot be used in many high-throughput assays due to their poor affinity and instability when coated, labeled, or immobilized on solid (e.g., plastic) surfaces. Screening (HTS) assay formats (eg HTS ELISA). Furthermore, Fc-polypeptides bind relatively weakly to monomeric Fc receptors, which makes it difficult to measure the binding signal. Compared to monomeric Fc receptors, multimeric Fc receptors of the invention provide more robust assays due to enhanced stability and improved (eg >100-fold) binding affinity for specific Fc-containing polypeptides .

Thus, the multimeric Fc receptors of the invention can be used in a variety of screening methods to examine changes in FcR binding affinity of any Fc-containing polypeptide. The multimeric Fc receptors of the invention can be used in any cell-free binding assay known in the art capable of assessing the binding of an Fc-containing polypeptide (eg, an antibody) to a multimeric Fc receptor. Depending on the requirements of the assay, the multimeric Fc receptors of the invention can be immobilized on a solid matrix or the multimeric Fc receptors of the invention can float freely in solution. Suitable cell-free binding assays are described herein. In one embodiment, multimeric Fc receptors of the invention can be used to examine changes in effector function of altered polypeptides of the invention.

In another embodiment, the multimeric Fc receptors of the invention can be used to purify Fc-containing polypeptides. For example, a multimeric Fc receptor of the invention can be immobilized on a column so that an Fc-containing polypeptide can be purified from a mixture based on its greater affinity for the multimeric Fc receptor. These methods offer certain advantages over existing affinity purification methods that require tags to facilitate purification, such as with His or epitope tags. Such tags may interfere with the intended use of the purified protein (eg, administration as a therapeutic agent), and if such tags are removed, methods of removing them require extensive purification steps, resulting in substantial losses in yield.

In an exemplary embodiment, a multimeric neonatal Fc receptor of the invention can facilitate binding of an Fc-containing polypeptide to a column at a first pH (e.g., acidic pH) and from a column at a second pH (e.g., neutral pH). Eluted on the column. This purification method has additional advantages over existing methods for purifying Fc-containing polypeptides. For example, Protein A affinity columns are commonly used to purify antibodies and other Fc-containing polypeptides. However, elution of the polypeptide from the column requires relatively harsh treatments (eg, low pH) that may destroy the polypeptide, resulting in a substantial loss of yield. In contrast, elution of Fc-containing polypeptides from multimeric neonatal Fc receptor affinity requires only a small increase from a first pH (eg, pH 5.8-6.5) to a second pH (eg, pH 7.0-7.5).

In another embodiment, the multimeric Fc receptors of the present invention (e.g., reagents of the present invention can be used to examine the safety of any Fc-containing protein-based therapy, especially monoclonal antibody therapy. For example, when administered to In patients, certain Fc-containing polypeptides (e.g., oxidized Fc-containing polypeptides) may exhibit undesired transcytosis into vulnerable tissues (e.g., placental transfer from mother to fetus). The Fc-containing polypeptides are expected to Polymeric Fc receptors bind with lower affinity and will elute before non-oxidized polypeptides. Methionine (e.g., EU positions 253 and 429) residues are particularly susceptible to oxidation. Therefore , Fc polypeptides can be screened based on binding to multimeric Fc receptors, and if the binding affinity to multimeric Fc receptors is at an acceptable threshold, Fc polypeptides can be selected based on therapeutic applications. For example, in one embodiment, Binding to multimeric Fc receptors is used to determine lot-to-lot consistency between polypeptide preparations or to perform quality control checks.

Multimeric Fc receptors of the invention can be produced using any of the methods described herein.

VIII. Further Modification of Altered Fc-Containing Polypeptides

Altered Fc-containing polypeptides can be further modified to provide the desired effect. For example, in certain embodiments, additional moieties, i.e., functional moieties, such as PEGylation moieties, blocking moieties, detectable moieties, diagnostic moieties and/or Or a therapeutic moiety is conjugated (ie, physically linked) that modifies the altered polypeptide. Chemical conjugation can be achieved randomly or by site-specific modification of specific residues within the altered polypeptide. Exemplary functional moieties are first described below, followed by useful chemistries and materials for linking such functional moieties to various amino acid side chain chemistries of altered polypeptides.

a) Functional part

Examples of useful functional moieties include, but are not limited to, PEGylation moieties, blocking moieties, detectable moieties, diagnostic moieties, and therapeutic moieties.

Exemplary PEGylation moieties include poly(alkylene) glycol moieties, such as PEG moieties, preferably PEG-maleimide moieties. Preferred PEGylated moieties (or related polymers) may be, for example, polyethylene glycol ("PEG"), polypropylene glycol ("PPG"), polyoxyethylated glycerol ("POG") and other polyoxyethylated Polyols, polyvinyl alcohol ("PVA") and other polyalkylene oxides, polyoxyethylated sorbitol or polyoxyethylated glucose. The polymers may be homopolymers, random or block copolymers, terpolymers based on the aforementioned monomers, linear or branched, substituted or unsubstituted, provided they have at least one active sulfone moiety. Polymeric moieties may be of any length or molecular weight, but these characteristics may affect biological properties. Particularly useful polymers having an average molecular weight of 2,000 to 35,000 Daltons are useful for reducing clearance rates in pharmaceutical applications. Furthermore, if two groups are attached to the polymer head-to-head, the length of the polymer may affect the effective distance and other spatial relationships between the two groups. Thus, one skilled in the art can vary the length of the polymer to optimize or confer the desired biological activity. PEG can be used in biological applications for several reasons. PEGs are typically clear, colorless, odorless, soluble in water, thermally stable, inert to many chemical agents, non-hydrolyzing, and non-toxic.

Preferably, the PEGylated moiety is attached to an altered Fc-containing polypeptide of the invention having an increased lifetime. PEGylated moieties can be used to further increase the half-life of the altered polypeptide by increasing the apparent molecular weight of the altered polypeptide molecule. Increased apparent molecular weight can reduce the rate of clearance from the body following subcutaneous or systemic administration. In many cases, PEGylation can also act to reduce antigenicity and immunogenicity. In addition, PEGylation can increase the solubility of the altered polypeptide.

Exemplary blocking moieties include cysteine adducts, cystine, mixed disulfide adducts, or others having sufficient steric bulk and/or charge to reduce antigen-dependent effectors Compounds that function (eg, by inhibiting the ability of the Fc region to bind to Fc receptors or complement proteins). Preferably, said blocking moiety is conjugated to an altered polypeptide of the invention having reduced effector function to further reduce effector function.

Exemplary detectable moieties that may be used for conjugation to the altered polypeptides of the invention include fluorescent moieties, radioisotopic moieties, radiopaque moieties, and the like, such as detectable labels such as biotin, fluorophores, chromophores, self- spin resonance probes, or radioactive labels. Exemplary fluorophores include fluorescent dyes (eg, fluorescein, rhodamine, etc.) and other light-emitting molecules (eg, luminal). A fluorophore may be context sensitive such that its fluorescence will change if it is positioned in close proximity to one or more residues in the modified protein that undergo a structural change upon binding a substrate (eg, a dansyl probe). Exemplary radiolabels include atoms containing one or more hyposensitive nuclei ( ¹³ C, ¹⁵ N, ² H ^{, 125} I, 123 I, ⁹⁹ Tc, ⁴³ K, ⁵² Fe, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In etc.) small molecules. Other useful moieties are known in the art.

Examples of diagnostic moieties that may be used in conjugation to the altered polypeptides of the invention include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for the determination of the presence, absence or level of a molecule (eg, a target peptide, protein or proteins) associated with a disease or condition. This diagnosis is also suitable for prognosing and/or diagnosing a disease or condition and its course.

Exemplary therapeutic moieties that can be used in conjugation to the altered polypeptides of the invention include, for example, anti-inflammatory, anti-cancer, anti-neurodegenerative, and anti-infective agents. The functional portion may also have one or more of the functions described above.

Exemplary therapeutic agents include radionuclides with high energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA and thus useful for inducing cellular (eg, cancerous) death. Exemplary high energy radionuclides include: ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re and ¹⁸⁸ Re. These isotopes typically produce energetic alpha or beta particles with short path lengths. These radionuclides kill cells in their vicinity, such as neoplastic cells to which the conjugate is attached or have entered. They have little or no effect on non-targeted cells and are essentially non-immunogenic.

Exemplary therapeutic agents also include cytotoxic agents, such as cytostatic agents (e.g., alkylating agents, DNA synthesis inhibitors, DNA intercalators or crosslinkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, Cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenic agents, etc.

Exemplary therapeutic agents also include alkylating agents, such as drugs of the anthracycline family (e.g., adriamycin, carminomycin, cyclosporin-A, chloroquine (chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, porfiromycin, anthracenediones, and aziridines) . In another embodiment, the chemotherapeutic moiety is a cytostatic agent, such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine-1,4-dioxide (3-amino-1,2,4-benzotriazine 1,4-dioxide), aminopterin (aminopterin), cytosine β-D-arabinofuranoside (cytosineβ-D-arabinofuranoside), 5-fluoro-5' -Deoxyuridine (5-fluoro-5'-deoxyuridine), 5-fluorouracil (5-fluorouracil), 9-[1,3-dihydroxy-2-propoxymethyl]guanine (ganciclovir), hydroxyurea ( hydroxyurea), actinomycin-D (actinomycin-D) and mitomycin C (mitomycin C). Exemplary DNA intercalators or crosslinkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide ( cyclophosphamide), cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and Oxaliplatin.

Exemplary therapeutic agents also include transcriptional regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin. Other exemplary cytostatics compatible with the present invention include Ansamycin, benzoquinones, quinoid derivatives (e.g., quinolones, genistein, bactacyclin) , busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl - diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, methylcycline nitrosourea (semustine)).

Exemplary therapeutic agents also include cytotoxic nucleosides such as adenosylarabinoside, cytarabine, cytosinearabinoside, 5-fluorouracil, fludarabine, 5-fluorodechlorouridine ( floxuridine), ftrafur, and 6-mercaptopurine; tubulin-binding agents such as Taxoids (eg, Paclitaxel, Docetaxel, taxane), Norco Nocodazole, Rhizoxin, dolastatins (for example, dolastatin-10, -11 or -15), colchicine and colchicinoids (for example ZD6126), combretastatins ( such as combretastatin A-4, AVE-6032), and vinca alkaloids (such as vinblastine, vincristine, vindesine, and vinorelbine ) (navelbine)); anti-angiogenic compounds such as angiostatin K1-3, DL-alpha-difluoromethyl-ornithine, endostatin, fumagillin , genistein, minocycline, staurosporine, and (±) thalidomide.

Exemplary therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylethylstilbestrol) (diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminogluthetimide), 17- (Allylamino)-17-demethoxygeldanamycin (Geldanamycin), 4-amino-1,8-naphthalimide, apigenin, brefeldin A ), cimetidine, dichloromethylene-diphosphate, leuprolide (Leuprorelin), luteinizing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin protein and thapsigargin.

Exemplary therapeutic agents also include enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzo-imidazole 1-β-D-ribofuranoside, etoposide, Formestane, fostriecin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), Mo Mevinolin, trichostatin A, tyrphostin AG34 and tyrphostin AG879.

Exemplary therapeutic agents also include gene modulators such as 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen , melatonin, Mifepristone, Raloxifene, trans-retinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis- Retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

Exemplary therapeutic agents also include cytotoxic agents, such as drugs of the pteridine family, diyenes, and podophyllotoxins. Particularly useful members of these classes include, for example, methylpterin, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vinblastine, Xin, epoxy vinblastine (leurosine) and so on.

Still other cytotoxins compatible with the teachings herein include auristatins (e.g. auristatin E and monomethylauristan E), calicheamicin, gramicidin D, maytansanoids (e.g. maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxy anthracindione , mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogs or homologues .

Other types of functional moieties are known in the art and can be readily employed in the methods and compositions of the invention in light of the teachings herein.

b) The chemistry used to link the functional moiety to the amino acid side chain

The chemistry used to link the aforementioned functional moieties (which may be small molecules, nucleic acids, polymers, peptides, proteins, chemotherapeutics, or other types of molecules) to specific amino acid side chains is known in the art (details for specific linkers For a review, see eg, Hermanson, G.T., Bioconjugate Techniques, Academic Press (1996)).

Examples of linking groups known in the art for sulfhydryl moieties (e.g., cysteine, or thiol side chain chemistry) include, but are not limited to, activated acyl groups (e.g., α-haloacetate, chloroacetic acid or chloroacetamide), activated alkyl groups, Michael acceptors such as maleimide or acrylic groups, groups that react with sulfhydryl moieties by redox reactions, and activated disulfide groups. The mercapto moiety can also be obtained by reacting with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)-propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro -2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloromercury benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2 -Oxa-1,3-oxadiazole.

In a preferred embodiment, cysteine or other amino acid with sulfhydryl side chain chemistry is attached during or after the preparation of the Fc-containing polypeptide. For example, when cell culture is used to prepare the modified Fc-containing polypeptide, conditions are provided such that free cysteines in solution are able to form cysteine adducts with the sulfhydryl side chains of the Fc-containing polypeptide. The adducts thus formed can be used to inhibit glycosylation and/or effector functions, or, alternatively, be subsequently subjected to reducing conditions to remove the adducts and thereby allow the use of one of the sulfhydryl chemistries described above.

Examples of linking groups known in the art for hydroxyl moieties (e.g., serine, threonine, or tyrosine side chain chemistries) include those described above for sulfhydryl moieties, including activated acyl groups, activated Alkyl groups and Michael acceptors.

Examples of linking groups known in the art for amino moieties (e.g., asparagine or arginine side chain chemistries) include, but are not limited to, N-succinimidyl, N-sulfosuccinimidyl, N- -phthalimidyl, N-sulfophthalimide, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3- Sulfonyl-4-nitrophenyl, 3-carboxy-4-nitrophenyl, imidate (eg, methyl picolimidate), pyridoxal phosphate, pyridoxal, hydrogen chloride Chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, and 2,4-pentanedione.

Examples of linking groups known in the art for acidic moieties (eg, aspartate or glutamate side chain chemistry) include activated esters and activated carbonyls. The acidic moiety can also optionally be synthesized by reacting with a carbodiimide (R'N-C-N-R') such as 1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or 1- Ethyl-3-(4-aza-4,4-dimethylpentyl)carbodiimide.

When the desired functional moiety is a PEGylated moiety, PEGylation reactions well known in the art can be used. For example, in one approach, PEGylation is achieved by acylation or alkylation with reactive polyethylene glycol molecules (or similar reactive water-soluble polymers). A water-soluble polymer for PEGylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG). In another embodiment, the polymer used for PEGylation is polyethylene glycol-maleimide (ie, PEG-maleimide).

The method of preparing a PEGylated antibody or antibody fragment of the present invention generally comprises the steps of: a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, in a state that can cause binding of the antibody or antibody fragment to a or multiple PEG groups, and b) obtaining a reaction product. It will be apparent to those skilled in the art how to select optimal reaction conditions or acylation reactions based on known parameters and desired results. In one embodiment, specific amino acid residues can be targeted, e.g., altering a first amino acid residue in order to inhibit glycosylation of a second amino acid residue (preferably where the first amino acid is cysteine and has sulfhydryl chemistry) .

IX. Methods of prevention, diagnosis and treatment

The invention has general use when an altered polypeptide (eg, antibody or fusion protein) of the invention binds to a cell surface antigen and this binding elicits the requisite effector response. An example of an effector-mediated response is reduction of the underlying cause of a disorder (eg, elimination of tumor cells or antigen-bearing cells involved in an immune or inflammatory response). In another embodiment, one or more symptoms of a disorder may be reduced. In another embodiment, the compositions described herein can be used in diagnostic reagents (eg, antibodies for tumor imaging) to alter effector-mediated responses. The methods described herein can be used in subjects who are at risk of developing a disease or who are currently exhibiting a disease condition.

A. Antitumor therapy

Thus, in certain embodiments, the altered polypeptides of the invention are useful in the prevention or treatment of cancer. In one embodiment, the altered polypeptide blocks autocrine or paracrine growth (eg, by binding to a receptor without transducing a signal, or by binding to a growth factor). In preferred embodiments, the altered polypeptide is capable of binding a tumor-associated antigen.

In one embodiment, the altered polypeptide reduces tumor volume, inhibits tumor growth, and/or prolongs survival of a tumor-bearing animal. In general, the present invention can be used to prevent or treat any neoplasm that contains an antigenic marker that allows the modified antibody to target cancer cells. Exemplary cancers or neoplasia that may be prevented or treated include, but are not limited to, bladder cancer, breast cancer, head and neck cancer, prostate cancer, colon-rectal cancer, melanoma, or skin cancer, breast cancer, ovarian cancer, cervical cancer, intrauterine Cancers of the membrane, kidney, lung (eg, small cell and non-squamous cell carcinoma), pancreatic cancer, and multiple myeloma. More particularly, the modified antibodies of the invention can be used in the treatment of Kaposi's sarcoma, CNS neoplasia (capillary hemangioblastoma, meningioma and brain metastases), melanoma, gastrointestinal and renal sarcomas, striated muscle Sarcoma, glioblastoma (preferably glioblastoma multiforme), leiomyosarcoma, retinoblastoma, papillary cystadenocarcinoma of the ovary, Wilm's tumor or small cell lung cancer. It will be appreciated that, in light of the present disclosure, appropriate starting polypeptides can be derived without undue experimentation for tumor-associated antigens associated with the aforementioned neoplasia.

Examples of blood cancers suitable for treatment using the present invention include Hodgkin's and non-Hodgkin's lymphomas and leukemias, including ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia ( CLL) and monocytic leukemia. It will be appreciated that the altered polypeptides and methods of the present invention are particularly effective in the treatment of various B-cell lymphomas, including low-grade/follicular non-Hodgkin's lymphoma (NHL), cellular lymphoma (FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small lymphocytic NHL Schizocytic NHL, bulky disease NHL, and Waldenstrom's macroglobulinemia. Those skilled in the art will appreciate that these lymphomas often have different names due to changing classification systems, and that patients with lymphomas classified under different names may also benefit from the combined treatment regimens of the present invention. In addition to the neoplastic conditions described above, it will be appreciated that the present invention may also be advantageously used in the treatment of other malignancies bearing compatible tumor-associated antigens.

B. Immune Disease Treatment

In addition to neoplastic conditions, the altered polypeptides of the invention are also particularly effective in treating autoimmune diseases or abnormal immune responses. In this regard, it will be appreciated that the altered polypeptides of the invention may be used to control, suppress, modulate or eliminate undesired immune responses to external and self-antigens. For example, in one embodiment, the antigen is an autoantigen. In another embodiment, the antigen is an allergen. In other embodiments, the antigen is an alloantigen or a xenoantigen. The use of the modified polypeptides disclosed herein in reducing the immune response to alloantigens and xenoantigens is particularly useful in transplantation, for example to inhibit the transplant recipient's reaction to a donor graft, such as a tissue or organ graft or a bone marrow graft. repel. In addition, suppression or elimination of donor T cells in bone marrow transplants can also be used to suppress graft-versus-host disease.

In other embodiments, the altered polypeptides of the invention can be used to treat immune disorders, including, but not limited to, allergic bronchopulmonary aspergillosis; allergic rhinitis; autoimmune hemolytic anemia; acanthosis; allergic contact Dermatitis; Addison's disease; atopic dermatitis; alopecia areata; alopecia universalis; amyloidosis; anaphylactoid purpura; anaphylactoid reaction; aplastic aplastic anemia; hereditary angioedema; idiopathic angioedema; ankylosing spondylitis; cranial arteritis; giant cell arteritis; Takyayasu's arteritis; temporal arteritis; asthma; ataxia-telangiectasia; autoimmune oophoritis; autoimmune orchitis; autoimmune autoimmune polyendocrine failure; Behcet's disease; Berger's disease; Buerger's disease; bronchitis; bullous pemphigus; chronic mucocutaneous candidiasis; Caplan's syndrome syndrome; post-myocardial infarction syndrome; post-pericardiotomy syndrome; carditis; celiac sprue; Chagas' disease; Chediak- Higashi syndrome; Churg-Strauss disease; Cirrhosis; Cogan's syndrome; cold agglutinin disease; CREST syndrome; Crohn's disease; cryoglobulinemia; cryptogenic Cryptogenic Fibrosing Alveolitis, dermatitisherpetifomis; dermatomyositis; diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; discoid lupus erythematosus; eosinophilia Eosinophilic fasciitis; episcleritis; drythema elevatum diutinum; erythema marginatum; erythema multiforme; erythema nodosum ; Familial Mediterranean fever; Felty's syndrome; Pulmonary fibrosis; Allergic glomerulonephritis (anaphylactoid); Glomerulonephritis (post-streptococcalglomerulonephritis); post-transplantation glomerulonephritis; membranous glomerulopathy (glomerulopathy, membranous); Goodpasture's syndrome; Granuloma annulare; Allergic granulomatosis (allergic); Granulomatous myositis; Grave's disease; Hashimoto's Thyroiditis; Hemolytic disease of the newborn; Henoch-Schoenlein purpura; chronic active and chronic progressive hepatitis; histiocytosis X; hypereosinophilic syndrome; idiopathic platelets idiopathicthrombocytopenic purpura; Job's syndrome; juvenile dermatomyositis; juvenile rheumatoid arthritis (juvenile chronic arthritis); Kawasaki's disease; keratitis keratoconjunctivitis sicca; Landry-Guilain-Barre-Strohl syndrome; Leprosy, lepromatous; Loeffler's syndrome; lupus; lupus nephritis; Lyell's syndrome; Lyme Lyme disease; lymphomatoid granulomatosis; systemic mastocytosis (systemic); mixed connective tissue disease; mononeuritis multiplex ( Mononeuritis multiplex; Muckle-Wells syndrome; mucocutaneous lymph node syndrome; mucocutaneous lymph node syndrome; multicentric reticulohistiocytosis; multiple sclerosis; myasthenia gravis; mycosis fungoides; necrotizing vasculitis, systemic; nephrotic syndrome; overlap syndrome; panniculitis ( panniculitis; paroxysmal cold hemoglobinuria; paroxysmal nocturnal hemoglobinuria; pemphigoid; pemphigus; pemphigus erythematosus Pemphigus erythematosus; Pemphigus foliaceus; Pemphigus vulgaris; Pigeonbreeder's disease; Hypersensitivity Pneumonitis; Polyarterial nodosa polyarteritis nodosa; Polymyalgia rheumatica; polymyositis; polyneuritis, idiopathic; portuguese familial polyneuropathy; aura Pre-eclampsia/eclampsia; Primary biliary cirrhosis; Progressive systemic sclerosis (scleroderma); Psoriasis; Psoriatic arthritis; Alveolar Pulmonary alveolar proteinosis; pulmonary fibrosis, Raynaud's phenomenon/syndrome; Reidel's thyroiditis; Reiter's syndrome, relapsing polychondritis; rheumatic fever; Rheumatoid arthritis; Sarcoidosis; scleritis; sclerosing cholangitis; scleroderma, serum sickness; Sezary syndrome; Sjogren's syndrome; Stevens-Johnson syndrome; Still's disease; subacute sclerosing panencephalitis; sympathetic ophthalmia; systemic lupus erythematosus; transplant rejection; ulcerative colitis ); undifferentiated connective tissue disease; chronic urticaria (urticaria, chronic); cold-induced urticaria (urticaria, cold); uveitis (uveitis); vitiligo (vitiligo); Weber- Christian disease; Wegener's granulomatosis; and Wiskott-Aldrich syndrome.

C anti-inflammatory treatment

In yet other embodiments, the altered polypeptides of the invention can be used to treat inflammatory conditions caused (at least in part) or exacerbated by inflammation, such as increased blood flow, edema, activation of immune cells (e.g., hyperplasia, cytokine production or enhanced phagocytosis). Exemplary inflammatory disorders include those in which inflammatory factors (e.g., matrix metalloproteinases (MMP), nitric oxide (NO), TNF, interleukins, plasma proteins; cellular defense systems, cytokines, lipid metabolites, proteases, toxic Free radicals, mitochondria, apoptosis, adhesion molecules, etc.) or inflammatory factors are present in a given area or tissue in abnormal amounts (eg, amounts that can be beneficially altered, eg, to benefit the subject). The inflammatory process is the response of living tissue to injury. The cause of inflammation can be due to physical damage, chemical substances, microorganisms, tissue necrosis, cancer or other factors. Acute inflammation is short-lived, lasting only a few days. However, if it persists for a longer period of time, it may be referred to as chronic inflammation.

Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and relapsing inflammatory disorders. Acute inflammatory conditions are generally of relatively short duration and last from about a few minutes to about 1 to 2 days, although they can also last several weeks. Cardinal features of acute inflammatory conditions include increased blood flow, extravasation of body fluids and plasma proteins (edema), and extravasation of white blood cells such as neutrophils. Chronic inflammatory conditions are generally of longer duration, eg weeks to months to years or even longer, and are histologically associated with the presence of lymphocytes and macrophages and proliferation of blood vessels and connective tissue. A relapsing inflammatory disorder includes a disorder that recurs after a period of time or recurs periodically. Examples of relapsing inflammatory disorders include asthma and multiple sclerosis. Some conditions can fall into one or more categories.

Inflammatory disorders are generally characterized by heat, flushing, swelling, pain, and loss of function. Exemplary causes of inflammatory conditions include, but are not limited to, microbial infections (such as bacterial, viral, and fungal infections), physical factors (such as burns, radiation, and trauma), chemical factors (such as toxins and corrosive substances), tissue necrosis, and Various types of immune responses. Examples of inflammatory conditions include but are not limited to Alzheimer's disease; severe asthma, atherosclerosis, cachexia, CHF-ischemia and coronary restenosis; osteoarthritis, rheumatoid arthritis, fibrosis / Radiation-induced or juvenile arthritis; acute and chronic infections (bacterial, viral and fungal); acute and chronic bronchitis, sinusitis and other respiratory infections, including colds; acute and chronic gastroenteritis and colitis and Crohn's disease; acute and chronic cystitis and urethritis; acute respiratory distress syndrome; cystic fibrosis; acute and chronic dermatitis; psoriasis; acute and chronic conjunctivitis; acute and chronic serositis (pericarditis, peritonitis uremic pericarditis; acute and chronic cholecystitis; acute and chronic vaginitis; stroke, trauma-induced inflammation of the brain or central nervous system, and Ulcerative colitis; acute and chronic uveitis; drug reactions; diabetic nephropathy, and burns (thermal, chemical, and electrical). Other inflammatory disorders or conditions that may be treated with the antibodies or antigen-binding fragments of the invention include inflammation due to corneal transplantation, chronic obstructive pulmonary disease, hepatitis C, lymphoma, multiple myeloma, and osteoarthritis.

In another embodiment, the polypeptides of the invention can be used to prevent or treat neurodegenerative diseases, including, but not limited to, Alzheimer's disease, stroke, and traumatic brain or central nervous system injury. Other neurodegenerative diseases include ALS/motor neuron disease, diabetic peripheral neuropathy, diabetic retinopathy, Huntington's disease, macular degeneration, and Parkinson's disease. In a preferred embodiment, altered polypeptides with reduced binding affinity for FcRn are used in the treatment of neurological disorders because they cannot cross the blood-brain barrier as efficiently as polypeptides with a higher binding affinity for FcRn. For example, in one embodiment, an altered polypeptide of the invention is injected into the cerebrospinal fluid to treat neurodegenerative diseases.

In prophylactic applications, a pharmaceutical composition or medicament comprising a polypeptide of the invention is administered to a subject at risk of (or suffering from but asymptomatic) a disease for which an Fc region-containing Diseases treated by the polypeptides, such as disorders of the immune system, wherein the administration is in an amount sufficient to eliminate or reduce the risk, reduce the severity of the disease, or delay the onset of the disease (wherein the disease includes the biochemistry, tissue clinical and/or behavioral symptoms, their complications, and intermediate pathological phenotypes that develop during disease progression).

In therapeutic applications, a composition or medicament is administered to a subject already suffering from such a disease in an amount sufficient to treat or at least partially arrest the symptoms (biochemical, histological, and/or behavioral), including its complications and intermediate pathological phenotypes that arise during disease development. Polypeptides of the invention are particularly useful for modulating the biological activity of cell surface antigens present in blood where the disease being treated or prevented is at least in part due to abnormally high or low biological activity of said antigen.

In some methods, administering an agent reduces or eliminates an immune disorder such as inflammation. An amount sufficient to accomplish treatment or prophylaxis is defined as a therapeutically or prophylactically effective dose. In both prophylactic and therapeutic regimens, typically the agent is administered in several doses until an adequate immune response is achieved.

It will be appreciated that, based on the selection of target molecules to which the modified polypeptides of the invention bind, the polypeptides of the invention may be used in the treatment of many conditions not explicitly mentioned herein. It will also be appreciated that any antibody or fusion protein known in the art may be modified in accordance with the methods of the invention and used to treat the condition for which it is indicated.

D. Application method

The altered polypeptides of the invention may be used for prophylaxis and/or therapy by startingeral, topical, intravenous, oral, intraarterial, intracranial, intraperitoneal or intranasal means. The term startingeral is used herein to include intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The most typical routes of protein drug administration are intravascular, subcutaneous or intramuscular, but other routes may also be effective. In some methods, the agent is injected directly into specific tissues where deposits have accumulated, such as intracranial injections. In some methods, the antibody is administered in a sustained release composition or device (eg, a Medipad ^™ device). The protein drug can also be administered through the respiratory tract, for example using a dry powder inhalation device.

For the treatment of the above-mentioned conditions, the effective dose of the composition of the present invention will vary with many different factors, including the mode of administration, the target site, the physiological condition of the subject, whether the subject is human or animal, other substances administered. Medications, and whether treatments are preventive or curative. Typically, the subject is a human, but non-human mammals including transgenic mammals can also be treated.

For passive immunization with antibodies, dosages range from about 0.0001 to 100 mg/kg, more usually 0.01 to 20 mg/kg of host body weight. For example, dosages may be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. The dose may be administered to the subject daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary therapy entails long-term, eg, at least 6 months, administration of multiple doses. Other exemplary treatment regimens entail biweekly administration or monthly administration or once every 3 to 6 months. Exemplary dosage regimens include 1-10 mg/kg or 15 mg/kg continuously daily, 30 mg/kg on alternate days, or 60 mg/kg weekly. In some methods, two polypeptides with different binding specificities are administered simultaneously, in which case the doses of each polypeptide administered are within the indicated ranges respectively.

Typically the polypeptide is administered multiple times. The interval between two single doses can be weekly, monthly or yearly. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml, and in some methods to achieve a plasma antibody concentration of 25-300 μg/ml. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary with the half-life of the polypeptide in the subject. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. As discussed herein, half-life also depends on the specific mutations present in the altered polypeptide.

Dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a composition containing an antibody of the invention or a cocktail thereof is administered to a subject not already in a disease state in order to enhance the subject's resistance. This amount is defined as a "prophylactically effective dose". In this application, the exact dose again depends on the subject's health and general immune status, but is usually 0.1 to 25 mg/dose, especially 0.5 to 2.5 mg/dose. Relatively low doses can be administered chronically at relatively dilute intervals. Some subjects can continue to receive treatment for the remainder of their lives.

In therapeutic applications, it is sometimes desirable to administer relatively high doses (e.g., about 1 to 200 mg antibody/dose, more typically 5-25 mg doses) at relatively short intervals until disease progression is slowed or terminated, preferably until the subject Subjects showed partial or complete improvement in disease symptoms. Thereafter, the patient may receive a prophylactic administration regimen.

Dosages of nucleic acid encoding antibodies range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg of DNA per subject. Doses of infectious viral vectors can vary from 10 to 100, or more, virions per dose.

Those skilled in the art can determine by routine experimentation the effective non-toxic amount of the altered polypeptide for the purpose of treating the disease. For example, a therapeutically effective amount of a modified polypeptide can vary according to factors such as the subject's stage of disease (eg, stage I versus stage IV tumors), age, sex, medical complications (eg, immunosuppressive conditions or diseases), and body weight. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

E. Monitoring of Therapy

Treatment of a subject with a disease or condition can be monitored using standard methods. Some methods require determining a baseline value, such as a baseline value of an antibody level or antibody profile in a subject, prior to administration of a dose of the agent, and comparing this to the profile or level after treatment. A significant increase in the level or profile (i.e., above the typical experimental error range for repeated measurements of the same sample (expressed as one standard deviation from the mean of these measurements) indicates a positive therapeutic outcome (i.e., administration of the agent has achieved expected response.) A negative treatment outcome is indicated if the value for the immune response does not change significantly or decreases.

In other methods, control level values or spectral values (ie, mean plus standard deviation) are determined for a control population. Typically, individuals in the control population have not received prior treatment. The mean of the subject's level or profile after administration of the therapeutic agent is then compared to the control value. A significant increase (eg, above one standard deviation from the mean) relative to a control value indicates a positive or adequate treatment outcome. The absence of a significant increase or the occurrence of a decrease indicates a negative or inadequate treatment outcome. Administration of the agent is generally continued while the level is increasing relative to control values. As before, the arrival of a plateau relative to a control value indicates that treatment administration may be discontinued or the dose and/or frequency of treatment administration may be reduced.

In other methods, control level or profile values (eg, mean plus standard deviation) are determined from a control population consisting of individuals who have undergone treatment with a therapeutic agent and whose levels or profiles have plateaued in response to treatment. The level or profile measured in the subject is compared to the control value. Treatment may be discontinued if the measured value in the subject is not significantly different (eg, greater than 1 standard deviation) from the control value. If the subject's level is significantly lower than the control value, this provides justification for continued administration of the agent. If a subject's levels are persistently below control levels, a change in therapy may be required.

In other approaches, the polypeptide levels or profiles of subjects not currently receiving treatment but who have had a prior course of treatment are monitored to determine if treatment needs to be restarted. The levels or profiles measured in a subject can be compared to values previously achieved by the subject after a prior course of treatment. A significant decrease (ie, above the typical margin of error for repeated measurements of the same sample) relative to the previous measurement indicates that treatment can be resumed. Alternatively, the value measured in the subject can be compared to a control value (mean plus standard deviation) determined in a population of subjects who have undergone the course of treatment. Alternatively, values measured in subjects can be compared to those measured in a population consisting of prophylactically treated subjects (who remained asymptomatic of disease) or curatively treated subjects (who exhibited improvement in disease characteristics). ) for comparison with the control values determined in the population composed of . In all these cases, a significant decrease (ie, greater than the standard deviation) relative to the control value indicates that the subject should restart treatment.

The polypeptide profile after administration typically exhibits an immediate peak in antibody concentration followed by an exponential decay. With no further administration, the decay will approach pre-treatment levels over days to months, depending on the half-life of the antibody used. For example, the half-life of some human antibodies is about 20 days.

In some methods, a baseline measurement of a polypeptide for a given antigen is taken in a subject prior to administration, a second measurement is taken shortly after administration to determine peak polypeptide levels, and one or more additional measurements are taken periodically to monitor polypeptide levels attenuation. A further dose of the polypeptide is administered when the level of the polypeptide falls to baseline or a predetermined percentage (eg, 50%, 25%, or 10%) of the post-baseline peak. In some methods, the background-subtracted peak or subsequent measured level is compared to a reference level previously determined in other subjects to form a beneficial prophylactic or therapeutic regimen. If the measured level of polypeptide is significantly lower than the reference level (eg, below the mean minus one standard deviation of the reference value in a population of subjects benefiting from treatment), administration of a further dose of the polypeptide is indicated.

Other methods include monitoring any art-recognized physiological symptom (eg, physical or mental symptom) that is routinely used by a researcher or physician to diagnose or monitor disease during the course of treatment.

F. Combination therapy

The altered polypeptides of the invention may optionally be administered in combination with other agents (including any of the agents of Section VIII above) known or determined to be effective in treating a condition or disease in need of treatment (eg, prophylactic or therapeutic). In addition, polypeptides of the invention may also be conjugated to moieties that add functionality to the polypeptides, such as PEG, tags, drugs, or markers.

It is also understood that the altered polypeptides of the present invention may be combined or used in combination (eg, to provide a combination therapy regimen) with any chemotherapeutic agent(s) capable of digesting, reducing, inhibiting or controlling the growth of neoplastic cells in vivo. Exemplary chemotherapeutic agents compatible with the present invention include alkylating agents, vinca alkaloids (eg, vincristine, vinblastine), procarbazine, aminopterin, and prednisone. The four-drug combination MOPP (mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone) is very effective in the treatment of various types of lymphoma and comprises a preferred embodiment of the present invention. In MOPP-resistant patients, ABVD (eg, doxorubicin, bleomycin, vincristine, and dacarbazine), ChlVPP (chlorambucil, vincristine, procarbazine and prednisone), CABS (cyclohexylnitrosourea, doxorubicin, bleomycin, and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin , bleomycin, and vincristine) or BCVPP (nitrosourea mustard, cyclophosphamide, vincristine, procarbazine, and prednisone) in combination. For standard dosing and schedules, see Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, "Harrison's Principles of Internal Medicine" 1774-1788 (Kurt J. Isselbacher et al., eds., 13th ed.1994) and V.T. DeVita et al., (1997) and references cited therein. These therapies may be used unchanged or modified as needed for a particular patient in combination with one or more of the modified polypeptides of the invention described herein.

Other treatment regimens that can be used in the present invention include the use of alkylating agents such as cyclophosphamide or chlorambucil alone, or drug combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and albino C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus ammonia Methotrexate, bleomycin, and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, and leucovorin Folic acid plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and formyl tetrahydrofolate) and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vinblastine, fixed-dose prednisone, bleomycin, and leucovorin). Standard dosages and schedules for each of these regimens can be readily determined by those skilled in the art. CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside, and etoposide. Other compatible chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2&apos;-deoxycoformycin and fludarabine.

Salvage treatment is used for patients with intermediate and advanced NHL who cannot achieve disease remission or relapse. Rescue therapy uses drugs such as cytosine arabinoside, carboplatin, cisplatin, etoposide, and ifosfamide (given alone or in combination). In certain neoplastic conditions that are recurrent or aggressive, the following regimens are often used: IMVP-16 (ifosfamide, methotrexate, and etoposide), MIME (methyl-gag, ifosfamide , methotrexate, and etoposide), DHAP (dexamethasone, high-dose cytarabine, and cisplatin), ESHAP (etoposide, methylpredisolone) , HD cytarabine, cisplatin), CEPP (B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin) and CAMP (cyclohexylnitrosourea, mitogen anthraquinone, cytarabine, and prednisone) (each with a known rate and schedule of administration).

The amount of chemotherapeutic agent that can be used in combination with the modified polypeptide of the invention can vary from subject to subject, or can be administered according to methods well known in the art. See, eg, Bruce A Chabner et al., Antineoplastic Agents, "Goodman & Gilman's The Pharmacological Basis of Therapeutics" 1233-1287 ((Joel G. Hardman et al., eds., 9th ed.1996).

Although modified polypeptides may be administered as described herein, it must be emphasized that in other embodiments, modified polypeptides may be administered to healthy patients as first-line therapy. In this embodiment, the modified polypeptides of the invention may be administered to patients with normal or average red bone marrow reserve and/or to patients who have not, currently have or are losing normal or average red bone marrow reserve. As used herein, administration of a modified polypeptide in association or combination with adjuvant therapy means sequential, simultaneous, coextensive, concurrent, concomitant , or contemporaneous administration or application of the therapy and the antibody disclosed herein. It will be apparent to those skilled in the art that the administration or timing of the individual components of a combination therapy regimen can be scheduled to enhance the overall efficacy of the treatment. For example, a chemotherapeutic agent may be administered on a standard, well-known course of treatment, followed by administration of the radioimmunoconjugate of the invention over several weeks. Conversely, cytotoxins conjugated to modified polypeptides can be administered intravenously, followed by external beam-targeted radiation to the tumor. In still other embodiments, the modified polypeptide can be administered concurrently with one or more selected therapeutic agents in one outpatient setting. Those skilled in the art (eg, experienced oncologists) will readily be able to identify effective combination treatment regimens without undue experimentation based on the selected adjuvant therapy and the teachings of this specification.

In this regard, it is understood that the combination of the modified polypeptide and the chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and the modified polypeptide can be administered in any order or simultaneously. In selected embodiments, modified polypeptides of the invention are administered to patients who have previously undergone chemotherapy. In other embodiments, the modified polypeptide and chemotherapy are administered substantially simultaneously or concurrently. For example, modified antibodies can be administered to a patient undergoing a course of chemotherapy. In a preferred embodiment, the modified antibody is administered within 1 year of any chemotherapeutic agent or treatment. In other preferred embodiments, the modified polypeptide is administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment. In yet other preferred embodiments, the modified polypeptide is administered within 4, 3, 2, or 1 week of any chemotherapeutic agent or treatment. In still other preferred embodiments, the modified polypeptide is administered within 5, 4, 3, 2, or 1 day of the selected chemotherapeutic agent or treatment. It is also understood that the two agents or treatments may be administered within about hours or minutes (ie, substantially simultaneously).

IX. Pharmaceutical Compositions

The pharmaceutical composition of the present invention includes at least one Fc-containing modified polypeptide prepared by the methods described herein in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" refers to at least one ingredient in a pharmaceutical product normally used for the administration of an active ingredient. Likewise, the carrier may contain any pharmaceutical excipient used in the art and any form of excipient for administration. Compositions can be, for example, injection solutions, aqueous suspensions or solutions, non-aqueous suspensions or solutions, solid and liquid oral formulations, salves, gels, ointments, intradermal patches, Creams, lotions, tablets, capsules, sustained-release preparations, etc. Other excipients may include, for example, coloring agents, taste-masking agents, dissolution aids, suspending agents, compressing agents, enteric coatings, sustained release aids, and the like.

The agents of the invention are often administered in the form of pharmaceutical compositions comprising the active therapeutic agent and various other pharmaceutically acceptable ingredients. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania (1980)). The preferred form depends on the desired mode of administration and therapeutic use. The composition may also, depending on the desired formulation, include a pharmaceutically acceptable non-toxic carrier or diluent (defined as an excipient commonly used in formulating pharmaceutical compositions for administration to animals or humans). Diluents can be chosen so as not to interfere with the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or preparation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

Polypeptides may be administered in the form of depost injection or implant formulations which may be formulated in such a way as to allow for slow release of the active ingredient. An exemplary composition comprises 5 mg/mL of polypeptide formulated in an aqueous buffer (consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl). An exemplary general formulation buffer is 20 mM sodium citrate, pH 6.0, 10% sucrose, 0.1% Tween 80.

Typically, compositions are prepared for injection as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Formulations can also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymers for enhanced adjuvant effect (see discussion above) (see Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97, 1997).

The invention is further illustrated by some examples which should not be construed as limiting. The contents of all references, patents and published patent applications used throughout this application, as well as the Figures and Sequence Listing, are hereby incorporated by reference.

Example

Example 1 - Preparation of Altered Antibodies with Chimeric Fc Regions

To evaluate the binding of the human rabbit IgG chimeric constructs, amino acids likely to affect binding were first determined to be within 10 A of the interaction interface between the two interacting proteins. Based on the crystal structures of rat IgG2a and rat FcRn, amino acids on rat IgG2a Fc that were within 10 A of the interface of these two molecules were determined. Homology alignment of the rabbit IgG1 Fc and rat IgG1 Fc regions then allowed determination of which amino acids on the rabbit Fc region were likely to be within 10 Å of the interaction interface. The counterparts of the rat and human Fc regions were then determined. Chimeric molecules were then constructed, including all mutants within the 10 A interface. Each single amino acid was then individually substituted into hFc and analyzed to determine the contribution of each individual constituent amino acid to binding. Combinations of amino acid mutants were then constructed using molecules that showed enhanced hFcRn binding. In this way, only bound positive contributors were identified.

Each mutant was analyzed for affinity using Biacore, and molecules showing an increase in affinity of 25% or better were scored as positive results. The test was carried out at pH6.

Mutant antibodies are transiently expressed in 293 cells by standard techniques known to those of ordinary skill in the art. The expressed protein contains λ-type human F(ab), which can be easily purified using protein L. This antibody is reactive with epitopes not expressed in animals (pK to be determined). Detection of this mAb in blood was achieved by ELISA using antigens reactive with the variable regions and detection with anti-human H&L HRP (horseradish peroxidase) secondary antibody. These assays and animal models can be readily established by those of ordinary skill in the art.

The Kd of rabbit IgG1 for soluble monomeric hFcRn was measured on Biacore. This value was compared to the Kd determined for human IgG1 on the same chip. In these experiments, IgG was immobilized on different quadrants of the chip and direct comparisons of Kd values could be determined. Purchase protein and verify protein purity by SDS PAGE and analytical gel filtration prior to use.

A sequence alignment of rabbit IgG1 and human IgG1 has been completed and a sequence determined from the crystal structure of rat IgG2a/rFcRn located within 10 Å of the interface of human and rabbit IgG has been identified.

Identification of specific amino acid substitutions in the IgG constant region of human antibodies and IgG fusion proteins that increase antibody half-life. The mutations of the present invention are specifically derived from a chimeric protein construct between the rabbit IgG1 constant region (rFc) and hIgG1 constant region (hFc).

The effect of these changes was then determined by measuring the binding affinity of hFcRn to the mutated hFc region and compared with native hFc, the h/rFc chimera showed greater affinity for hFcRn. The in vivo half-lives of these molecules are then determined in monkeys or other appropriate models such as knock-in or transgenic hFcRn mice to demonstrate that the chimeric proteins have increased half-lives relative to native hFc.

Results: Biacore data have been obtained where hFcRn shows a Kd value for binding of rabbit IgG to hFcRn that is two times tighter than hIgG. Examples of residues for substitution include: EU positions 280, 281, 282, 283, 285, 286, 288, 289, 290, 305, 307, 308, 309, 315, 340, 344, and 378. More particularly, a polypeptide of the invention may contain at least one amino acid mutation selected from the group consisting of: Asp280Asn (wherein D represents the amino acid position to be mutated (by substitution) at said EU position (278), N represents the polypeptide to effect the change待通过替代进入该位置的氨基酸)，Gly281Glu，Val282Glu，Glu283Gln，His285Arg，Asn286Thr，Lys288Arg，Thr289Pro，Lys290Pro，Val305Thr，Thr307Pro，Val308Ile，Leu309Thr，Asn3153Arg，Lys340Arg，Arg344Leu，Ala378Ser，Ser383Lys，Glu386Lys，Pro387Ala，和Asn389Asp (according to the EU numbering system).

Example 2 - Identification of Candidate Residues by Electrostatic Optimization

In this example, methods for modifying the affinity of antibody constant regions for human FcRn are described. To obtain mutants with altered binding affinity of the Fc region for FcRn at acidic and neutral pH, we applied electrostatic charge optimization techniques to a homologue model of human Fc that binds to human FcRn. Models of the human Fc/FcRn complex at acidic (6.0) and neutral (7.4) pH were obtained from the crystal structure of the rat Fc/FcRn complex (PDB ) using the MODELLER program (Accelrys, Inc., San Diego, CA). code: 1I1A) and were energy minimized in CHARMM (Accelrys, Inc., San Diego, CA). During computational optimization, we used electrostatic charge optimization to determine the positions of Fc residues that could modulate the binding of Fc and FcRn at acidic and neutral pH (Lee and Tidor, J. Chem. Phys. 106:8681-8690 , 1997; Kangas and Tidor, J. Chem. Phys. 109:7522-7545, 1998). These calculations were done at two pH values, acidic (6.0) pH and neutral (7.4) pH, since Fc is known to bind FcRn in the pinocytic vacuole at acidic pH and release in the extracellular space at neutral pH. Enhanced binding of Fc to FcRn at neutral pH has the potential to impair Fc half-life. A reduction in half-life would be an expected consequence if IgG cannot be released from cell-associated FcRn in typical IgG clearance mechanisms. Consequently, Fc-containing proteins with increased affinity for FcRn at higher pH will be cleared from the blood, resulting in a protein with a shorter half-life.

These mutation predictions can be divided into those involving: (1) mutations at residues involved in the interaction interface that become partially hidden upon binding (improving the interaction by forming hydrogen bonds across the interface); Mutations of polar residues that become secluded upon binding and thus pay the price for desolvation but do not form any direct electrostatic interactions with FcRn (usually by mutating to wild-type residues have similar shape to hydrophobic residues or by adding residues that can cause favorable electrostatic interactions to obtain improvements); and (3) mutations of surface residues on the Fc that are present in In the region of non-complementary potential. These mutations are thought to improve the long-range electrostatic interactions between Fc and FcRn without disturbing the packing interactions at the binding interface, and include both mutations that preserve and alter molecular charge.

When performing charge optimization, various constraints are set to represent the natural side chain characteristics. For example, for net side chain charges of -1, 0, and +1, optimize with the additional constraint that no atom has a charge exceeding an absolute value of 0.85 electron charge units. Models were prepared using the standard protocol for hydrogen addition with the program CHARMM (Accelrys, Inc. San Diego, CA). In addition to the N-terminus of the β2-microglobulin chain, N-acetamide and N-formamide patches were added to the N-terminus and C-terminus, respectively. Models were prepared separately for acidic and neutral pH values to account for the different protonation and rotamer states of histidine residues and the rotamer states of asparagine and glutamine. Using the continnum electrostatics model, we performed electrostatic charge optimization for each side chain of amino acids (candidate residues) in Fc located within 15 Å of the FcRn interface at acidic and neutral pH values. Based on the results of charge optimization, mutations were identified for additional computational analysis. In the process, we visually inspected the optimal charge distribution and designed mutations that were closer to the optimal situation than the current residues. Charge optimization gives charges at atomic centers, but does not produce real mutations. A round of charge optimization was performed with the addition of various constraints to represent the characteristics of native side chains. For example, optimization was performed for net side chain charges of -1, 0, and +1 with the additional constraint that no atoms have charges exceeding 0.85 electron charge units in absolute value. Just select the position where substitution at the ideal charge distribution can produce a difference in human FcRn binding of not less than 0.3 kcal/mole between neutral pH and acidic pH. analyzed the ideal charge distribution on existing side chains and proposed a set of substitutions from 20 natural amino acids.

The following example shows the optimization results obtained for Valine 284, Histidine 285, Asparagine 286 and Lysine 290 (examples of selected residues) of the Fc molecule. The Mut (mutation energy) column corresponds to the difference in free energy of binding from the native residue to a completely uncharged side chain isostere (i.e., a residue with the same shape but no or partial charge on the atom) ( kcal/mol). Negative numbers indicate predicted increases in binding affinity. The Opt-1 column corresponds to the binding free energy difference that can be obtained with an optimal charge distribution in the side chains and a net side chain charge of -1. Columns Opt0 and Opt1 correspond to the binding free energy difference with the optimal charge at net charges of 0 and +1, respectively.

For acidic pH, the following results were obtained:

Residue Mut Opt-1 Opt0 Opt1

V284 0.0 -0.9 -0.3 0.5

H285 -0.6 -2.0 -1.6 -1.1

N286 0.0 -1.1 -0.2 1.1

K290 -0.7 -0.8 -0.9 -0.8

For neutral pH, the following results were obtained:

Residue Mut Opt-1 Opt0 Opt1

V284 0.0 -0.2 -0.3 -0.3

H285 0.1 -0.2 -0.1 0.2

N286 0.1 -0.3 -0.3 -0.3

K290 -0.2 0.1 -0.3 -0.3

Based on these results and a visual analysis of the model, an improved transition to glutamate that can take advantage of these free energies of binding is proposed. In fact, for all positions, a human FcRn binding difference of ΔΔG greater than 0.3 kcal/mol between neutral and acidic pH could be obtained by mutating the wild-type amino acid to glutamic acid.

Mutant side chains were built by performing a rotamer dihedral scan in CHARMM, using 30 or 60 degree dihedral angle increments, to determine the optimal position for each side chain. Binding energies for wild-type and mutant complexes were then calculated using Poisson-Boltzmann electrostatic energies and additional terms for van der Waals energies and covert surface area.

Calculations are performed at acidic and neutral pH using the amino acid charge settings of PARSE or CHARMM. Using a consensus scoring scheme means that the results obtained using both charge settings should have consistent predictors in order to produce meaningful predictions.

The following example illustrates the use of this method on valine 284, histidine 285, asparagine 286, and lysine 288 of an Fc molecule:

Residue No. Mutation G _6.0parse G _7.4parse G _6.0charmm G _7.4charmm

V 284 E -1.7 0 .7 -1.4 -0.3

H 285 E -2.5 0.4 -2.3 -1.2

N 286 E -0.4 1 .2 -1.5 -0.7

K 290 E -0.9 -0.4 -1.8 0.7

Binding of human Fc and human FcRn relative to wild-type Fc and FcRn at acidic pH with ΔΔG below -0.3 kcal/mol for both charge settings, indicating increased affinity relative to wild-type human Fc . At neutral pH, ΔΔG has different predictors under the two charge settings, suggesting that the mutants are unlikely to have higher human FcRn binding affinity than wild-type human Fc at neutral pH (in the context of the consensus scoring scheme ). Therefore, the V284E, H285E, N286E and K290E mutants (examples of selected mutants) are expected to have a longer FcRn-mediated half-life in the organism than the wild-type molecule.

In summary, we performed the computational approach described above to predict mutations that could differentially modulate the FcRn-binding affinity of Fc molecules at neutral and acidic pH values. We propose that amino acid substitutions in human Fc that can lead to increased FcRn affinity at acidic pH, but do not affect or reduce interaction affinity at neutral pH, will lead to Fc molecules with longer in vivo half-life. Amino acid substitutions that result in increased Fc/FcRn binding at neutral pH and/or reduced Fc/FcRn binding at acidic pH will appear to result in reduced half-life in vivo.

Example 4: Construction of altered Fc polypeptides

The changes predicted by the method of the present invention were introduced into the starting polypeptide encoding the heavy chain of the humanized IgG1 monoclonal antibody huCBE11. Figures 1A and 1B show the nucleotide sequence (SEQ ID NO: 3) and predicted amino acid sequence (SEQ ID NO: 4) of the heavy chain, respectively. Mutations were introduced into the huCBE11 heavy chain carried on an expression vector designated pEAG1787 by site-directed mutagenesis using standard recombinant DNA techniques. The antibody variable region is residues 1-120, and the human IgG1 constant region is residues 121-449. The C-terminal lysine residue of huIgG1 was genetically removed. For reference: the N-linked glycosylation site (EU residue number N297) is residue 300 in the above sequence. Figure 2 shows the amino acid sequence of the Fc region of huCBE11 according to the EU numbering index.

HuCBE11 monoclonal antibody is a humanized IgG1, κ recombinant antibody that recognizes human lymphotoxin β receptor. Cloning, chimerization and humanization of mAb CBE11 are described in US Patent Application 2004/0058394. The HuCBE11 light chain is carried in an expression vector called pEAG1754. The variable region is residues 1-107, and the human kappa constant region is residues 108-214. Figures 3A and 3B show the nucleotide sequence (SEQ ID NO: 5) and predicted amino acid sequence (SEQ ID NO: 6) of the light chain, respectively.

Wild-type and altered polypeptides were expressed by transient co-transfection of the human embryonic kidney cell line (HEK293E) with the heavy chain vector (pEAG1787) and light chain vector (pEAG1754).

Embodiment 5: Construction of FcRn-Fc fusion protein agent

Dimeric Fc-binding proteins were constructed by genetically fusing the extracellular domain of a neonatal Fc receptor with the Fc region of an IgG1 antibody.

Briefly, human β2 microglobulin cDNA was cloned from human placental polyA+ RNA by RT-PCR. The nucleotide sequence (SEQ ID NO: 7) and predicted amino acid sequence (SEQ ID NO: 8) of human β2 microglobulin cDNA are shown in Figures 4A and 4B, respectively. The cDNA corresponding to the alpha chain of human FcRn was cloned from human placental polyA+ RNA by RT-PCR and sequenced. The extracellular domain (ECD) of the human FcRnα chain (residues 1-297) was subcloned for fusion with a human Fc region derived from an IgGl antibody.

The Fc region (residues 398-535) was derived from a previously described human IgGl antibody (see US Patent No. 5,928,643). The IgG1 Fc region contains a truncated hinge, but retains the CH2 and CH3 domains of the antibody, and the C-terminal lysine residue of the CH3 domain is genetically removed. Figures 1A and 1B show the nucleotide sequence (SEQ ID NO: 3) and predicted amino acid sequence (SEQ ID NO: 4) of IgG1 antibody heavy chain cDNA.

To eliminate the possibility of the fusion protein binding FcRn, the Fc region was modified from its wild-type at four positions (shown as underlined residues 388, 389, 511 and 512 in the sequence shown in Figure 5B) by site-directed mutagenesis. Sequences are mutated to produce FcRn binding affinity and serum half-life that have previously been shown to significantly reduce FcRn (Kim et al., 1994, Eur.J.Immunol.24:542-548; and Popov et al. 1996, Mol.Immunol.33 Human equivalent ("huM4Fc") of the H310A/Q311N/H433A/N434Q (EU numbering) mutant (EU numbering: 521-530).

Using standard recombinant DNA techniques, this Fc region was fused to the C-terminus of the FcRn ECD to form the FcRn-Fc fusion protein cDNA. The β2 microglobulin and FcRn-Fc fusion protein cDNAs were inserted into an expression vector (pEAG1761) carrying both cDNAs in a tandem transcription unit driven by the cytomegalovirus immediate early promoter. CHO cells were transfected with pEAG1761 to generate a stable cell line secreting a soluble dimer (heterotetrameric FcRn-Fc fusion protein consisting of two β2-microglobulin and two FcRn α-Fc fusion chains). Figures 5A and 5B show the nucleotide sequence (SEQ ID NO: 9) and predicted amino acid sequence (SEQ ID NO: 10) of the human FcRnα-Fc fusion cDNA encoded by pEAG1761, respectively.

Conditioned medium (from CHO or 293 cells) containing secreted FcRn-huM4Fc (α chain and β2-microglobulin) fusion protein was cultured in an Amicon stirred cell unit using a 10,000 MWCO polyethersulfone membrane. Concentrate about 6 times to 150ml. The concentrate was dialyzed overnight against 40 volumes of 20 mM MES (pH 5.8), 150 mM NaCl, followed by centrifugation at 2060 x g for 10 min at 4°C to pellet cell debris. This was applied to a 6 ml human IgG-Sepharose 6 Fast Flow (Amersham Biosciences) column pre-equilibrated in 20 mM MES (pH 5.8), 150 mM NaCl. Bound FcRnFc was eluted with a single-step buffer change (pH8 in 20 mM Tris, 150 mM NaCl). hIgG-Sepharose columns were successfully used to capture rat, mouse and human FcRn-huM4Fc proteins.

Eluted fractions were analyzed by non-reducing SDS-PAG (4%-20% gradient gel), and fractions containing FcRn-huM4Fc were pooled for further purification by anion exchange chromatography. Protein concentration was determined spectrophotometrically using a 1 mg/ml human or rat protein solution (A ₂₈₀ value 1.80), and a 1 mg/ml mouse protein solution (A ₂₈₀ value 1.84). Approximately 70 mg of hFcRn-M4Fc was eluted from the IgG-Sepharose column. This pool was dialyzed against 20 mM Tris pH 8 and then applied to a 5 ml DEAE column. Bound protein was eluted from the DEAE column using a linear gradient of increasing salt up to a final concentration of 0.5M NaCl (20 column volumes). FcRn-huM4Fc protein eluted at approximately 150 mM-175 mM NaCl (30%-35% 0.5M NaCl buffer). The purity of the DEAE fractions was analyzed by SDS-PAGE as above, and the fractions containing FcRn-huM4Fc were pooled to give 48 mg of a homogeneous FcRnFc pool.

This DEAE pool was concentrated and centrifuged through a Vivaspin concentration unit (10,000 MWCO) to buffer exchange with 20 mM MES (pH 5.8), 150 mM NaCl buffer for -80°C storage, or for size exclusion chromatography (Superdex200 16 /60, Amersham Biosciences)—the final purification step—to remove minor aggregates.

Example 6: Analysis of FcRn binding affinity of altered polypeptides

The altered human monoclonal antibodies of the invention were identified by their ability to bind biotinylated FcRn using various cell-free assays.

a) Purification of the altered Fc polypeptide

Using the FcRn-huM4Fc fusion protein of Example 4, a human monoclonal antibody containing an altered Fc region was purified. To facilitate purification, the FcRn-huM4Fc fusion protein was immobilized on Sepharose 4 FastFlow Media. A 2 ml hFcRnFc column was prepared by coupling approximately 20 mg of hFcRn to NHS-activated Sepharose 4 Fast Flowmedia (Amersham Biosciences) in PBS (pH 2.2) overnight at 4°C with shaking. The column was washed with PBS prior to purification. 1 mg per FcRnFc column.

1 mg of each altered human IgG1 mAb was applied to the column in loading buffer 20 mM MES (pH 5.8), 150 mM NaCl. The column was then washed with 10 column volumes of loading buffer and the bound mAb was eluted with a pH step gradient (20 mM Tris, 150 mM NaCl) increasing the pH to 8.0.

b) Biotinylation of FcRn-huM4Fc (bhFcRnFc):

Biotin XX sulfosuccinimidyl ester (XX = two aminocaproyl chains) was used to biotin FcRn-huM4Fc for the competitive, bead-based, FRET and AlphaScreen assays described below. This reagent is available from Molecular Probes' Mini-Biotin-XX protein labeling kit. FcRnFc was dialyzed against PBS 150 mM Na/K phosphate, pH 7.0 and 150 mM NaCl. To 0.72 mg/ml hFcRnFcIgG (500 μl) was added 1/10 volume of 1 M Na bicarbonate (50 μL) carbonate (NaHCO3) stock solution. Then 10 μl of biotin-XX reagent (dissolved in water just before use as described in Molecular Probes) was added. Reactions were incubated for 1 hour at room temperature and then dialyzed extensively in PBS at 4°C. The biotinylated fusion protein (bhFcRnFc) was stable when frozen at -80°C.

c) Direct binding test:

Direct binding of unlabeled hFcRn-Fc to the altered Fc polypeptides of the invention was assessed using an ELISA format.

All washes and dilutions were performed in pH 6.0 PBS unless otherwise stated. Plates were coated at 100 μl per well overnight at 4°C with 5 μg/ml altered IgG antibody. Plates were washed 3 times and then coated with 1% BSA for 2 hours. Plates were then washed 3 times and incubated with 100 μL of the appropriate serially diluted FcRnFc for 90 minutes. Wash the plate again 3 times, and add 100 μL of the appropriate secondary mAb-HRP or streptavidin-HRP, incubate for 90 minutes, wash the plate again, and develop according to the appropriate method for the selected secondary mAb, and read Absorbance. Using a four-parameter fit of the binding data shown in Figure 10, the apparent dissociation constant (Kd) was determined.

d) Competitive binding assay:

The relative binding affinities of the variant antibodies to the labeled dimeric FcRn construct (bhFcRnFc) were determined using two proximity assays (FRET and AlphaScreen) in which changes in the fluorescently labeled control antibody (5C8) compared to Example 4 were evaluated Competitive binding between peptides.

i) FRET binding assay:

FRET assays were performed in black half-well ELISA plates with a total reaction volume of 30 μL. Each reaction mixture contained in pH 5.8 buffer with 100 mM NaCl and 0.005% PO detergent (Biacore, Tween-20): a) 1 nM control antibody (h5C8) labeled with europium; c) appropriate competitor mAb from Example 4 (0-5 μMolar; 0-0.75 mg/ml); and d) 200 nM bhFcRnFc. Finally bhFcRnFc was added to the reaction mixture.

Reactions were incubated at room temperature for 30 minutes before being read on an LJL Analyst (Molecular Devices) using an excitation wavelength of 615 and a fluorescence emission wavelength of 665. The settings for the LJL Analyst are 50 μs time delay and 400 μs readout. The signal ratio ([Delta]F) (665nm/615nm) of APC and Eu was plotted against the concentration of competitor mAb to determine IC50 ([mu]g/ml). Negative control reactions lacked europium-labeled hAb and competitor IgG, and positive controls contained no competitor IgG.

Relative _IC50 values (mutant IC50/wt control IC50) were determined for each altered antibody and plotted (Figure 6). Figure 6 shows that antibodies containing mutations at EU positions 285, 286, 290 and 304, especially antibodies containing mutations H285E, N286D, K290E and S304D, lead to enhanced relative binding affinities (relative IC ₅₀ ) to dimeric FcRn/Fc fusion proteins. <1). Conversely, many altered antibodies (i.e., those containing mutations at EU positions 252, 255, 279, 282, 284, 285, 287, 288, 290, 304, 306, 309, 376, 434, and 438 ) showed a reduced apparent binding affinity (relative _IC50 > 1) for dimeric FcRn/Fc fusion proteins. In particular, mutations at EU positions 282 (V282E), 290 (K290D), 438 (Q438E) and 434 (N434L) showed significantly reduced binding affinity to FcRn. For example, mutation N434L resulted in approximately 400-fold lower FcRn binding affinity than the control antibody.

ii) ALPHA Screen binding test:

AlphaScreen is a bead-based assay that uses donor and acceptor beads separately conjugated to a binding partner (ie, a dimeric Fc binding protein and an altered Fc polypeptide). Donor beads are coated with a photosensitizer that generates singlet oxygen upon laser excitation (eg, at 680 nm). The chemiluminescence-coated acceptor beads react with this singlet oxygen, releasing light at 520-620 nm. Due to the very short half-life of singlet oxygen in solution, the chemiluminescence of the acceptor bead is proportional to the proximity of the donor bead. Thus, the binding of donor and acceptor beads results in a greatly enhanced chemiluminescence signal.

To two-fold serial dilutions of the altered antibody of Example 4 was added a biotinylated Fc/FcRn fusion protein (bhFcRnFc) in 384-well white Costar plates (Corning, Acton, MA). After 30 min room temperature incubation, human IgG1-conjugated acceptor beads (conjugated by PerkinElmer Biosignal Inc., Montreal, Canada) and streptavidin-conjugated receptor beads were added at 20 μg/ml (final concentration) in a final reaction volume of 25 μl. Composite donor beads (PerkinElmer Biosignal Inc., Montreal, Canada). This arrangement results in a reaction mixture containing streptavidin-conjugated donor beads bound to the Fc/FcRn fusion protein and IgG1-conjugated acceptor beads bound to the altered antibody.

The reaction mixture was incubated at room temperature for 1 hour and read on a Fusion-Aplha reader (PerkinElmer Biosignal Inc., Montreal, Canada). PBS, 0.1% bovine serum albumin and 0.01 Tween-20, pH 6.0 were used as assay buffers. Human IgG1 was used as a positive control. Assay buffer at pH 7.0 was used as a negative control to verify that human IgGl does not bind to human FcRnFc. Results were analyzed using GraphPad Prism software.

The _IC50 values (μg/ml) determined for each of the altered antibodies are summarized in FIG. 7 . Figure 7 illustrates that antibodies containing mutations at EU positions 284, 285, 286 and 288, especially mutations V284E, H285E, N286D, K290E, lead to enhanced apparent binding affinity (IC ₅₀ <1). Conversely, many altered antibodies (i.e., those containing mutations at EU positions 252, 255, 279, 282, 284, 285, 286, 290, 304, 306, 309, 314, 313, 345, 376, 434 and 438 Altered antibodies) exhibit reduced apparent binding affinity (IC ₅₀ >1) for dimeric FcRn/Fc fusion proteins relative to control antibodies. In particular, EU positions 252 (M252S and M252T), 434 (N434L), and 438 (Q438E) showed significantly reduced binding affinity to FcRn. For example, mutation Q438E resulted in approximately 1500-fold lower FcRn binding affinity than the control antibody.

equivalent scheme

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are intended to be covered by the following claims.

sequence listing

SEQ ID NO: 1

231 APELLGG

238 PSVFLFPPKP

248 KDTLMISRTP

258 EVTCVVVDVS

268 HEDPEVKFNW

278 YVDGVEVHNA

288 KTKPREEQYN

298 STYRVVSVLT

308 VLHQDWLNGK

318 EYKCKVSNKA

328 LPAPIEKTIS

338 KAK

SEQ ID NO: 2

341 GQPREPQ

348 VYTLPPSRDE

358 LTKNQVSLTC

368 LVKGFYPSDI

378 AVEWESNGQP

388 ENNYKTTPPV

398 LDSDGSFFLY

408 SKLTVDKSRW

418 QQGNVFSCSV

428 MHEALHNHYT

438 QKSLSLSPG

The mature huCBE11 heavy chain DNA sequence (SEQ ID NO.3) encoded by pEAG1787

1 GAGGTACAAC TGGTGGAGTC TGGGGGAGGC TTAGTGAAGC CTGGAGGGTC

51 CCTGAGGCTC TCCTGTGCAG CCTCTGGATT CACTTTCAGT GACTATTACA

101 TGTATTGGTT TCGCCAGGCC CCGGGAAAGG GGCTGGAGTG GGTCGCAACC

151 ATTAGTGATG GTGGTAGTTA CACCTACTAT CCAGACAGTG TGAAGGGGCG

201 ATTCACCATC TCCAGAGACA ATGCCAAGAA CAGCCTCTAC CTGCAGATGA

251 GCAGCCTGAG GGCTGAGGAC ACAGCTGTGT ATTACTGCGC AAGAGAGGAG

301 AATGGTAACT TTTACTACTT TGACTACTGG GGCCAAGGGA CCACGGTCAC

351 CGTCTCCTCA GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT

401 CCTCCAAGAG CACCTCTGGG GGCACAGCGG CCCTGGGCTG CCTGGTCAAG

451 GACTACTTCC CCGAACCGGT GACGGTGTCG TGGAACTCAG GCGCCCTGAC

501 CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA GGACTCTACT

551 CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC

601 TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA

651 AGTTGAGCCC AAATCTTGTG ACAAGACTCA CACATGCCCA CCGTGCCCAG

701 CACCTGAACT CCTGGGGGGA CCGTCAGTCT TCCTCTTCCC CCCAAAACCC

751 AAGGACACCC TCATGATCTC CCGGACCCCT GAGGTCACAT GCGTGGTGGT

801 GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG TACGTGGACG

851 GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC

901 AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT

951 GAATGGCAAG GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC

1001 CCATCGAGAA AACCATCTCC AAAGCCAAAG GGCAGCCCCG AGAACCACAG

1051 GTGTACACCC TGCCCCCATC CCGGGATGAG CTGACCAAGA ACCAGGTCAG

1101 CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC GCCGTGGAGT

1151 GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG

1201 TTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA

1251 GAGCAGGTGG CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG

1301 CTCTGCACAA CCACTACACG CAGAAGAGCC TCTCCCTGTC TCCCGGTTGA

Predicted amino acid sequence of mature huCBE11 heavy chain (SEQ ID NO.4)

1 EVQLVESGGG LVKPGGSLRL SCAASGFFTS DYYMYWFRQA PGKGLEWVAT

51 ISDGGSYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRAED TAVYYCAREE

101 NGNFYYFDYW GQGTTVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

151 DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSLGTQT

201 YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP

251 KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN

301 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

351 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

401 LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG*

DNA sequence (SEQ ID NO.5) of the mature huCBE11 light chain encoded by pEAG1754

1 GATATCCAGA TGACCCAGTC TCCATCATCC TTGTCTGCAT CGGTGGGAGA

51 CAGGGTCACT ATCACTTGCA AGGCGGGTCA GGACATTAAA AGCTATTTAA

101 GCTGGTACCA GCAGAAACCA GGGAAAGCGC CTAAGCTTCT GATCTATTAT

151 GCAACAAGGT TGGCAGATGG GGTCCCATCA AGATTCAGTG GCAGTGGATC

201 TGGTACAGAT TATACTCTAA CCATCAGCAG CCTGCAGCCT GAGGATTTCG

251 CAACTTATTA CTGTCTACAG CATGGTGAGA GCCCGTGGAC GTTCGGTGGA

301 GGCACCAAGC TGGAGATCAA ACGAACTGTG GCTGCACCAT CTGTCTTCAT

351 CTTCCCGCCA TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT

401 GCCTGCTGAA TAACTTCTAT CCCAGAGAGG CCAAAGTACA GTGGAAGGTG

451 GATAACGCCC TCCAATCGGG TAACTCCCAG GAGAGTGTCA CAGAGCAGGA

501 CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG CTGAGCAAAG

551 CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC

601 CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG

Predicted amino acid sequence of mature huCBE11 light chain (SEQ ID NO.6)

1 DIQMTQSPSS LSASVGDRVT ITCKAGQDIK SYLSWYQQKP GKAPKLLIYY

51 ATRLADGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCLQ HGESPWTFGG

101 GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

151 DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

LSSPVTKSFN RGEC*

The DNA sequence (SEQ ID NO.7) of the human beta-2-microglobulin encoded by pEAG1761

1 ATGTCTCGCT CCGTGGCCTT AGCTGTGCTC GCGCTACTCT CTCTTTCTGG

51 CCTGGAGGCT ATCCAGCGTA CTCCAAAGAT TCAGGTTTAC TCACGTCATC

101 CAGCAGAGAA TGGAAAGTCA AATTTCCTGA ATTGCTATGT GTCTGGGTTT

151 CATCCATCCG ACATTGAAGT TGACTTACTG AAGAATGGAG AGAGAATTGA

201 AAAAGTGGAG CATTCAGACT TGTCTTTCAG CAAGGACTGG TCTTTCTATC

251 TCTTGTACTA CACTGAATTC ACCCCCACTG AAAAAGATGA GTATGCCTGC

301 CGTGTGAACC ATGTGACTTT GTCACAGCCC AAGATAGTTA AGTGGGATCG

351 AGACATGTAA

The predicted amino acid sequence of human β-2-microglobulin (SEQ ID NO.8)

1 MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKS NFLNCYVSGF

51 HPSDIEVDLL KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC

101 RVNHVTLSQP KIVKWDRDM*

DNA sequence (SEQ ID NO.9) of the human FcRnα-Fc fusion protein encoded by pEAG1761

1 ATGGGGGTCC CGCGGCCTCA GCCCTGGGCG CTGGGGCTCC TGCTCTTTCT

51 CCTTCCTGGG AGCCTGGGCG CAGAAAGCCA CCTCTCCCTC CTGTACCACC

101 TTACCGCGGT GTCCTCGCCT GCCCCGGGGA CTCCTGCCTT CTGGGTGTCC

151 GGCTGGCTGG GCCCGCAGCA GTACCTGAGC TACAATAGCC TGCGGGGCGA

201 GGCGGAGCCC TGTGGAGCTT GGGTCTGGGA AAACCAGGTG TCCTGGTATT

251 GGGAGAAAGA GACCACAGAT CTGAGGATCA AGGAGAAGCT CTTTCTGGAA

301 GCTTTCAAAG CTTTGGGGGG AAAAGGTCCC TACACTCTGC AGGGCCTGCT

351 GGGCTGTGAA CTGGGCCCTG ACAACACCTC GGTGCCCACC GCCAAGTTCG

401 CCCTGAACGG CGAGGAGTTC ATGAATTTCG ACCTCAAGCA GGGCACCTGG

451 GGTGGGGACT GGCCCGAGGC CCTGGCTATC AGTCAGCGGT GGCAGCAGCA

501 GGACAAGGCG GCCAACAAGG AGCTCACCTT CCTGCTATTC TCCTGCCCGC

551 ACCGCCTGCG GGAGCACCTG GAGAGGGGCC GCGGAAACCT GGAGTGGAAG

601 GAGCCCCCCT CCATGCGCCT GAAGGCCCGA CCCAGCAGCC CTGGCTTTTC

651 CGTGCTTACC TGCAGCGCCT TCTCCTTCTA CCCTCCGGAG CTGCAACTTC

701 GGTTCCTGCG GAATGGGCTG GCCGCTGGCA CCGGCCAGGG TGACTTCGGC

751 CCCAACAGTG ACGGATCCTT CCACGCCTCG TCGTCACTAA CAGTCAAAAAG

801 TGGCGATGAG CACCACTACT GCTGCATTGT GCAGCACGCG GGGCTGGCGC

851 AGCCCCTCAG GGTGGAGCTG GAATCTCCAG CCAAGTCCTC CGTCGACAAA

901 ACTCACACAT GCCCACCGTG CCCAGCACCT GAACTCCTGG GGGGACCGTC

951 AGTCTTCCTC TTCCCCCCAA AACCCCAAGGA CACCCTCATG ATCTCCCGGA

1001 CCCCTGAGGT CACATGCGTG GTGGTGGACG TGAGCCACGA AGACCCTGAG

1051 GTCAAGTTCA ACTGGTACGT GGACGGCGTG GAGGTGCATA ATGCCAAGAC

1101 AAAGCCGCGG GAGGAGCAGT ACAACAGCAC GTACCGTGTG GTCAGCGTCC

1151 TCACCGTCCT GGCTAACGAC TGGCTGAATG GCAAGGAGTA CAAGTGCAAG

1201 GTTCCCAACA AAGCCCCTCCC AGCCCCCCATC GAGAAAACCA TCTCCAAAGC

1251 CAAAGGGCAG CCCCGAGAAC CACAGGTGTA CACCCTGCCC CCATCCCGGG

1301 ATGAGCTGAC CAAGAACCAG GTCAGCCTGA CCTGCCTGGT CAAAGGCTTC

1351 TATCCCAGCG ACATCGCCGT GGAGTGGGAG AGCAATGGGC AGCCGGAGAA

1401 CAACTACAAG ACCACGCCTC CCGTGTTGGA CTCCGACGGC TCCTTCTTCC

1451 TCTACAGCAA GCTCACCGTG GACAAGAGCA GGTGGCAGCA GGGGAACGTC

1501 TTCTCATGCT CCGTGATGCA TGAGGCTCTG GCGCAGCACT ACACGCAGAA

1551 GAGCCTCTCC CTGTCTCCGG GTTGA

The predicted amino acid sequence of human FcRnα-Fc fusion protein (SEQ ID NO.10)

1 MGVPRPQPWA LGLLLLFLLPG SLGAESHLSL LYHLTAVSSP APGTPAFWVS

51 GWLGPQQYLS YNSLRGEAEP CGAWVWENQV SWYWEKETTD LRIKEKLFLE

101 AFKALGGKGP YTLQGLLGCE LGPDNTSVPT AKFALNGEEF MNFDLKQGTW

151 GGDWPEALAI SQRWQQQDKA ANKELTFLLF SCPHRLREHL ERGRGNLEWK

201 EPPSMRLKAR PSSPGFSVLT CSAFSFYPPE LQLRFLRNGL AAGTGQGDFG

251 PNSDGSFHAS SSLTVKSGDE HHYCCIVQHA GLAQPLRVEL ESPAKSSVDK

301 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE

351 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVL AN D WLNGKEYKCK

401 VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF

451 YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV

501 FSCSVMHEAL AQ HYTQKSLS LSPG*

Claims (70)

1. comprise the polypeptide of the change of the FcRn bound fraction in Fc district at least, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

EU amino acid position 248 is replaced by charged amino acid;

EU amino acid position 249 is replaced by positively charged amino acid;

EU amino acid position 251 is replaced by polare Aminosaeren or Methionin;

EU amino acid position 252 is replaced by polare Aminosaeren;

EU amino acid position 255 is replaced by polare Aminosaeren;

EU amino acid position 256 is replaced by Methionin;

EU amino acid position 257 is replaced by charged amino acid;

EU amino acid position 258 is replaced by polare Aminosaeren or charged amino acid;

Substituting of EU amino acid position 277;

EU amino acid position 279 is replaced by charged amino acid;

EU amino acid position 280 is replaced by charged amino acid;

EU amino acid position 281 is replaced by charged amino acid or glutamine;

EU amino acid position 282 is replaced by charged amino acid;

EU amino acid position 284 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 285 is replaced by positively charged amino acid, polare Aminosaeren, or aspartic acid;

EU amino acid position 286 is replaced by L-glutamic acid, Threonine, or methionine(Met);

EU amino acid position 287 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 288 is replaced by charged amino acid;

Substituting of EU amino acid position 289;

EU amino acid position 304 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 305 is replaced by polare Aminosaeren or charged amino acid;

Substituting of EU amino acid position 306;

EU amino acid position 307 is replaced by polarity or charged amino acid;

EU amino acid position 308 is replaced by charged amino acid;

EU amino acid position 309 is replaced by charged amino acid;

EU amino acid position 310 is replaced by charged amino acid or polare Aminosaeren;

EU amino acid position 311 is replaced by positively charged amino acid;

EU amino acid position 312 is replaced by positively charged amino acid or polare Aminosaeren;

EU amino acid position 313 is replaced by charged amino acid;

EU amino acid position 315 is replaced by charged amino acid;

EU amino acid position 316 is replaced by positively charged amino acid;

EU amino acid position 317 is replaced by charged amino acid or polare Aminosaeren;

EU amino acid position 340 is replaced by charged amino acid;

EU amino acid position 343 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 344 is replaced by leucine;

EU amino acid position 345 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 376 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 378 is replaced by Serine;

EU amino acid position 383 is replaced by charged amino acid;

EU amino acid position 385 is replaced by charged amino acid;

EU amino acid position 389 is replaced by electronegative amino acid;

EU amino acid position 424 is replaced by charged amino acid;

EU amino acid position 426 is replaced by charged amino acid;

EU amino acid position 430 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 431 is replaced by charged amino acid;

EU amino acid position 432 is replaced by polare Aminosaeren;

EU amino acid position 434 is replaced by Methionin, arginine, or leucine;

EU amino acid position 436 is replaced by electronegative amino acid; With

EU amino acid position 438 is replaced by charged amino acid.

2. comprise the polypeptide of the change of the FcRn bound fraction in Fc district at least, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

The Methionin of EU amino acid position 248 is replaced by charged amino acid;

The aspartic acid of EU amino acid position 249 is replaced by positively charged amino acid;

The leucine of EU amino acid position 251 is replaced by polare Aminosaeren or Methionin;

Substitute the methionine(Met) of EU amino acid position 252;

The arginine of EU amino acid position 255 is replaced by polare Aminosaeren;

The Threonine of EU amino acid position 256 is replaced by Methionin;

The proline(Pro) of EU amino acid position 257 is replaced by charged amino acid;

The L-glutamic acid of EU amino acid position 258 is replaced by polare Aminosaeren or charged amino acid;

Substitute the tryptophane of EU amino acid position 277;

The Xie Ansuan of EU amino acid position 279 is replaced by charged amino acid;

The aspartic acid of EU amino acid position 280 is replaced by charged amino acid;

The glycine of EU amino acid position 281 is replaced by charged amino acid or glutamine;

The Xie Ansuan of EU amino acid position 282 is replaced by charged amino acid;

The Xie Ansuan of EU amino acid position 284 is replaced by polare Aminosaeren or charged amino acid;

The Histidine or the L-Ala of EU amino acid position 285 are replaced by charged amino acid or polare Aminosaeren;

The Histidine or the L-Ala of EU amino acid position 285 are replaced by positively charged amino acid, polare Aminosaeren, or aspartic acid;

The l-asparagine or the Methionin of EU amino acid position 286 are replaced by L-glutamic acid, Threonine, or methionine(Met);

The L-Ala of EU amino acid position 287 is replaced by polare Aminosaeren or charged amino acid;

The Methionin of EU amino acid position 288 is replaced by charged amino acid;

Substitute the Threonine of EU amino acid position 289;

The Serine of EU amino acid position 304 is replaced by polare Aminosaeren or charged amino acid;

The Xie Ansuan of EU amino acid position 305 is replaced by polare Aminosaeren or charged amino acid;

Substitute the leucine or the Xie Ansuan of EU amino acid position 306;

The Threonine or the Xie Ansuan of EU amino acid position 307 are replaced by polarity or charged amino acid;

The Xie Ansuan of EU amino acid position 308 is replaced by charged amino acid;

The leucine of EU amino acid position 309 is replaced by charged amino acid;

The Histidine of EU amino acid position 310 is replaced by charged amino acid or polare Aminosaeren;

The glutamine of EU amino acid position 311 is replaced by positively charged amino acid;

The aspartic acid or the leucine of EU amino acid position 312 are replaced by positively charged amino acid or polare Aminosaeren;

The l-asparagine of EU amino acid position 313 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 315 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 316 is replaced by positively charged amino acid;

The Methionin of EU amino acid position 317 is replaced by charged amino acid or polare Aminosaeren;

The Methionin of EU amino acid position 340 is replaced by charged amino acid;

The proline(Pro) of EU amino acid position 343 is replaced by polare Aminosaeren or charged amino acid;

The arginine of EU amino acid position 344 is replaced by leucine;

The L-glutamic acid of EU amino acid position 345 is replaced by polare Aminosaeren or charged amino acid;

The aspartic acid of EU amino acid position 376 is replaced by polare Aminosaeren or charged amino acid;

The L-Ala of EU amino acid position 378 is replaced by Serine;

The Serine of EU amino acid position 383 is replaced by charged amino acid;

The glycine of EU amino acid position 385 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 389 is replaced by electronegative amino acid;

The Serine of EU amino acid position 424 is replaced by charged amino acid;

The Serine of EU amino acid position 426 is replaced by charged amino acid;

The L-glutamic acid of EU amino acid position 430 is replaced by polare Aminosaeren or charged amino acid;

The leucine of EU amino acid position 431 is replaced by charged amino acid;

The Histidine of EU amino acid position 432 is replaced by polare Aminosaeren;

The l-asparagine of EU amino acid position 434 is replaced by Methionin, arginine, or leucine;

The tyrosine of EU amino acid position 436 is replaced by electronegative amino acid; With

The glutamine of EU amino acid position 438 is replaced by charged amino acid.

3. the polypeptide of claim 1 or 2 change wherein is positioned at EU amino acid position 277,289,306,344, or at least one the locational amino acid in 378 is by charged amino acid, polare Aminosaeren, or nonpolar amino acid substitutes.

4. the polypeptide of the change of claim 3, wherein said charged amino acid is electronegative amino acid.

5. the polypeptide of the change of claim 4, wherein said electronegative amino acid is selected from aspartic acid and L-glutamic acid.

6. the polypeptide of the change of claim 3, wherein said charged amino acid is positively charged amino acid.

7. the polypeptide of the change of claim 6, wherein said positively charged amino acid is selected from arginine, Histidine, and Methionin.

8. the polypeptide of the change of claim 7, wherein said positively charged amino acid is Methionin.

9. the polypeptide of the change of claim 3, wherein polare Aminosaeren is selected from methionine(Met), phenylalanine, tryptophane, Serine, Threonine, tyrosine, l-asparagine, glutamine and halfcystine.

10. the polypeptide of the change of claim 3, wherein nonpolar amino acid is selected from L-Ala, leucine, Isoleucine, Xie Ansuan, glycine and proline(Pro).

11. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change are antibody or its fragment.

12. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change is a fusion rotein.

13. the polypeptide of the change of claim 1 or 2, wherein Fc district or its FcRn bound fraction derive from people's antibody.

14. the polypeptide of the change of claim 13, it comprises complete Fc district.

15. the polypeptide of the change of claim 14, wherein initial polypeptide comprises the aminoacid sequence of SEQ ID NO:2.

16. each the polypeptide of change of claim 11-13, wherein Fc district or its Fc bound fraction are the IgG isotypes.

17. the polypeptide of the change of claim 16, wherein said IgG isotype is the IgG1 subclass.

18. each the polypeptide of change of claim 11-13, wherein said polypeptide comprises one or more inhuman amino-acid residues at the complementary determining region (CDR) of VL or VH.

19. the polypeptide of the change of claim 11 or 13, wherein said polypeptide can be in conjunction with (a) antigen and (b) FcR.

20. the polypeptide of the change of claim 19, wherein said antigen is tumor associated antigen.

21. the polypeptide of the change of claim 12, wherein said polypeptide can be in conjunction with (a) part and (b) FcR.

22. the polypeptide of the change of claim 19 or 21, wherein FcR is FcRn.

23. the polypeptide of the change of claim 19 or 21, wherein said polypeptide and the initial polypeptide that does not contain described sudden change with different binding affinities in conjunction with FcR.

24. the polypeptide of the change of claim 23, the binding affinity of the polypeptide of wherein said change is compared with high about 1.5 times to about 100 times of the beginning polypeptide.

25. the polypeptide of the change of claim 34, the binding affinity of the polypeptide of wherein said change is compared with low about 1.5 times to about 100 times of the beginning polypeptide.

26. the polypeptide of the change of claim 19 or 21, the polypeptide of wherein said change demonstrates a kind of binding affinity to FcR when a pH, and when the 2nd pH FcR is demonstrated different binding affinities.

27. the polypeptide of the change of claim 26, the binding affinity of the polypeptide of wherein said change when a pH than at the 2nd pH the time high about 1.5 times to about 100 times.

28. the polypeptide of the change of claim 27, the binding affinity of the polypeptide of wherein said change when a pH than at the 2nd pH the time low about 1.5 times to about 100 times.

29. each the polypeptide of change of claim 11-13, the polypeptide of wherein said change when being administered to the patient, demonstrates and the different circulating half-life of initial polypeptide that does not contain sudden change.

30. the polypeptide of the change of claim 29, the transformation period of the polypeptide of wherein said change is grown up about 1 hour to about 1 week than the initial polypeptide that does not contain sudden change.

31. the polypeptide of the change of claim 30, the transformation period of the polypeptide of wherein said change is lacked about 1 hour to about 1 week than the initial polypeptide that does not contain sudden change.

32. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change can combine with albumin A or G.

33. comprise the pharmaceutical composition of polypeptide of the change of claim 1 or 2.

34. comprise the nucleic acid molecule of the nucleotide sequence of coding claim 1 or 2 polypeptide.

35. the nucleic acid molecule of claim 34, it is an expression vector.

36. comprise the host cell of the expression vector of claim 35.

37. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

EU amino acid position 284 is replaced by L-glutamic acid;

EU amino acid position 285 is replaced by L-glutamic acid;

EU amino acid position 286 is replaced by aspartic acid;

EU amino acid position 288 is replaced by L-glutamic acid or aspartic acid;

EU amino acid position 290 is replaced by L-glutamic acid; With

EU amino acid position 304 is replaced by aspartic acid,

The polypeptide of wherein said change is compared with the initial polypeptide that does not contain sudden change and is demonstrated longer circulating half-life.

38. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

The Xie Ansuan of EU amino acid position 284 is replaced by L-glutamic acid;

The Histidine of EU amino acid position 285 is replaced by L-glutamic acid;

The l-asparagine of EU amino acid position 286 is replaced by aspartic acid;

The Methionin of EU amino acid position 288 is replaced by L-glutamic acid or aspartic acid;

The Methionin of EU amino acid position 290 is replaced by L-glutamic acid; With

The Serine of EU amino acid position 304 is replaced by aspartic acid,

The polypeptide of wherein said change is compared with the initial polypeptide that does not contain sudden change and is demonstrated longer circulating half-life.

39. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

EU amino acid position 248 is replaced by aspartic acid;

EU amino acid position 249 is replaced by arginine or Methionin;

EU amino acid position 250 is replaced by arginine or Methionin;

EU amino acid position 251 is replaced by arginine, Methionin, or l-asparagine;

EU amino acid position 252 is replaced by Serine or Threonine;

EU amino acid position 254 is replaced by Serine or Threonine;

EU amino acid position 256 is replaced by arginine, L-glutamic acid, or Methionin;

EU amino acid position 255 is replaced by leucine, aspartic acid or methionine(Met);

EU amino acid position 260 is replaced by Methionin;

EU amino acid position 257 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 277 is replaced by arginine, aspartic acid, glutamine, or Methionin;

EU amino acid position 279 is replaced by L-glutamic acid;

EU amino acid position 281 is replaced by glutamine;

EU amino acid position 282 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 287 is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro), or Threonine;

EU amino acid position 284 is replaced by aspartic acid;

EU amino acid position 285 is replaced by aspartic acid or phenylalanine;

EU amino acid position 286 is replaced by L-glutamic acid or methionine(Met);

EU amino acid position 288 is replaced by aspartic acid;

EU amino acid position 290 is replaced by aspartic acid;

EU amino acid position 304 is replaced by aspartic acid or L-glutamic acid;

EU amino acid position 305 is replaced by arginine;

EU amino acid position 306 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 307 is replaced by arginine, aspartic acid, or L-glutamic acid;

EU amino acid position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

EU amino acid position 310 is replaced by arginine, leucine, Methionin or l-asparagine;

EU amino acid position 312 is replaced by arginine, l-asparagine, or Methionin;

EU amino acid position 313 is replaced by aspartic acid, arginine, or Methionin;

EU amino acid position 315 is replaced by aspartic acid or L-glutamic acid;

EU amino acid position 343 is replaced by glutamine or Methionin;

EU amino acid position 345 is replaced by arginine or glutamine;

EU amino acid position 374 is replaced by arginine, Methionin, or leucine;

EU amino acid position 376 is replaced by l-asparagine;

EU amino acid position 426 is replaced by arginine, aspartic acid, or L-glutamic acid;

EU amino acid position 428 is replaced by arginine, glutamine, or Methionin;

EU amino acid position 430 is replaced by Methionin;

EU amino acid position 431 is replaced by proline(Pro);

EU amino acid position 432 is replaced by arginine;

EU amino acid position 434 is replaced by leucine or Methionin; With

EU amino acid position 438 is replaced by L-glutamic acid,

The polypeptide of wherein said change demonstrates short circulating half-life than the initial polypeptide that does not contain sudden change.

40. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that described polypeptide comprises at least one and is selected from following sudden change:

The Methionin of EU amino acid position 248 is replaced by aspartic acid;

The aspartic acid of EU amino acid position 249 is replaced by arginine or Methionin;

The Threonine of EU amino acid position 250 is replaced by arginine or Methionin;

The leucine of EU amino acid position 251 is replaced by arginine, Methionin, or l-asparagine;

The methionine(Met) of EU amino acid position 252 is replaced by Serine or Threonine;

The methionine(Met) of EU amino acid position 254 is replaced by Serine or Threonine;

The Threonine of EU amino acid position 256 is replaced by arginine, L-glutamic acid, or Methionin;

The arginine of EU amino acid position 255 is replaced by leucine, aspartic acid or methionine(Met);

The Threonine of EU amino acid position 260 is replaced by Methionin;

The proline(Pro) of EU amino acid position 257 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The tryptophane of EU amino acid position 277 is replaced by arginine, aspartic acid, glutamine, or Methionin;

The Xie Ansuan of EU amino acid position 279 is replaced by L-glutamic acid;

The glycine of EU amino acid position 281 is replaced by glutamine;

The Xie Ansuan of EU amino acid position 282 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The L-Ala of EU amino acid position 287 is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro), or Threonine;

The Xie Ansuan of EU amino acid position 284 is replaced by aspartic acid;

The Histidine of EU amino acid position 285 is replaced by aspartic acid or phenylalanine;

The l-asparagine of EU amino acid position 286 is replaced by L-glutamic acid or methionine(Met);

The Methionin of EU amino acid position 288 is replaced by aspartic acid;

The Methionin of EU amino acid position 290 is replaced by aspartic acid;

The Serine of EU amino acid position 304 is replaced by aspartic acid or L-glutamic acid;

The Xie Ansuan of EU amino acid position 305 is replaced by arginine;

The leucine of EU amino acid position 306 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The Threonine of EU amino acid position 307 is replaced by arginine, aspartic acid, or L-glutamic acid;

The leucine of EU amino acid position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

The Histidine of EU amino acid position 310 is replaced by arginine, leucine, Methionin or l-asparagine;

The aspartic acid of EU amino acid position 312 is replaced by arginine, l-asparagine, or Methionin;

The tryptophane of EU amino acid position 313 is replaced by aspartic acid, arginine, or Methionin;

The l-asparagine of EU amino acid position 315 is replaced by aspartic acid or L-glutamic acid;

The proline(Pro) of EU amino acid position 343 is replaced by glutamine or Methionin;

The L-glutamic acid of EU amino acid position 345 is replaced by arginine or glutamine;

The proline(Pro) of EU amino acid position 374 is replaced by arginine, Methionin, or leucine;

The aspartic acid of EU amino acid position 376 is replaced by l-asparagine;

The Serine of EU amino acid position 426 is replaced by arginine, aspartic acid, or L-glutamic acid;

The methionine(Met) of EU amino acid position 428 is replaced by arginine, glutamine, or Methionin;

The L-glutamic acid of EU amino acid position 430 is replaced by Methionin;

The L-Ala of EU amino acid position 431 is replaced by proline(Pro);

The leucine of EU amino acid position 432 is replaced by arginine;

The l-asparagine of EU amino acid position 434 is replaced by leucine or Methionin; With

The glutamine of EU amino acid position 438 is replaced by L-glutamic acid,

The polypeptide of wherein said change demonstrates short circulating half-life than the initial polypeptide that does not contain sudden change.

41. the method for the polypeptide of the change of preparation claim 1 or 2, described method comprises:

(a) with the nucleic acid molecule transfectional cell of nucleotide sequence of the polypeptide that comprises the described change of encoding; With

(b) from the polypeptide of described cell or the described change of cell conditioned medium liquid purifying.

42. the method for the antibody of preparation claim 11 or 13, described method comprises:

(a) provide and comprise encoding said antibody variable region of light chain (V _L) and constant region (C _L) first nucleic acid molecule of nucleotide sequence;

(b) provide and comprise encoding said antibody variable region of heavy chain (V _H) and constant region (CH ₁, CH ₂, and CH ₃) second nucleic acid molecule of nucleotide sequence;

(c) with first and second nucleic acid molecule transfectional cell under the condition of the antibody of change that allow to express the light chain that comprises described coding and heavy chain; With

(d) from described cell or the described antibody of cell conditioned medium liquid purifying.

43. the method for claim 42, wherein said cell are 293 cells.

44. it is conjugated protein to comprise the dimerization Fc of first and second polypeptide chains, wherein first and second polypeptide chains respectively comprise the Fc plot structure territory that at least one operationally is connected in conjunction with the territory with Fc.

45. the dimerization Fc of claim 44 is conjugated protein, described Fc structural domain the combining with reduction or elimination and FcRn of wherein suddenling change.

46. the dimerization Fc of claim 44 is conjugated protein, wherein said first and second polypeptide chains are covalently bound.

47. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains comprises the extracellular domain of FcRn.

48. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains is attached to β2Wei Qiudanbai in conjunction with last.

49. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains derives from people FcRn.

50. the dimerization Fc of claim 59 is conjugated protein, the wherein said conjugated protein aminoacid sequence shown in the SEQ IDNO:10 that comprises.

51. be used to measure the polypeptide that comprises Fc district FcRn bound fraction at least method at the binding affinity of FcR, this method comprises makes that the polypeptide that comprises the FcRn bound fraction in Fc district at least and the dimerization Fc of claim 44 are conjugated protein to be contacted and determine this interactional avidity.

52. be used in the library screening that comprises the polypeptide of the FcRn bound fraction in Fc district at least FcRn being had the method for those polypeptides of the binding affinity of change, wherein said method comprises:

(a) the library member is contacted with the dimerization Fc of claim 44 is conjugated protein; With

(b) measure described polypeptide to the protein-bonded binding affinity of dimerization Fc; With

(c) selection has the those polypeptides of the binding affinity of change to FcRn.

53. be used for containing the method for the polypeptide of the FcRn bound fraction in Fc district at least from the polypeptide mixture purifying, described method comprises the mixture application of sample to the protein-bonded affinity column of the dimerization Fc that contains claim 44, wash-out comprises the polypeptide of the FcRn bound fraction in Fc district at least, the described polypeptide of purifying thus.

54. the method for claim 53, wherein when a pH with described mixture application of sample to affinity column, and when the 2nd pH from the described polypeptide of affinity column wash-out.

55. the method for claim 54, wherein said polypeptide in the purifying engineering not by sex change.

56. be used to identify with initial polypeptide compare the method for polypeptide that FcRn has the binding affinity of change, described method comprises:

The associated change in conjunction with free energy of performance of the space of the best charge distribution of initial polypeptide amino acid and initial polypeptide when (a) determining in solvent in conjunction with FcRn;

Change at least one the candidate amino acid residue position in having of the initial polypeptide initial polypeptide to be finished when (b) being accredited as in conjunction with free energy in conjunction with FcRn; With

(c) identify the elected amino acid be positioned at this amino acid position, so that this sudden change mixing in initial polypeptide will cause FcRn is had the polypeptide of change of the binding affinity of change.

57. the method for claim 56 also comprises selected amino acid is mixed in the initial polypeptide.

58. the method for claim 56 comprises that also the polypeptide that calculate to change compares variation in conjunction with free energy with initial polypeptide in conjunction with FcRn the time in conjunction with FcRn the time..

59. the method for claim 58, wherein said calculation procedure comprise at first that on computers mould is built described sudden change, the variation of calculations incorporated free energy then in initial polypeptide.

60. the method for claim 58, wherein said calculation procedure is used the following at least a definite method that is selected from: use and determine the static bound energy based on the method for Poisson-Boltzmann formula; Determine the Fan Dewaershi bound energy; Determine bound energy with using based on the solvent-accessible surface Method for Area.

61. the method for claim 56, wherein said sudden change is an amino acid replacement.

62. the method for claim 61, wherein said amino acid replacement cause mixing the elected amino acid that has different electric charges with candidate amino acid.

63. the method for claim 56, wherein said sudden change increase the polypeptide that contains Fc that changes and FcRn in solvent in conjunction with the time between the two in conjunction with free energy, that reduces described change thus contains the binding affinity of Fc polypeptide to FcRn.

64. the method for claim 56, wherein said sudden change reduce the polypeptide that contains Fc that changes and FcRn in solvent in conjunction with the time between the two in conjunction with free energy, what increase described change thus contains the binding affinity of Fc polypeptide to FcRn.

65. identify the method for polypeptide of the change that contains Fc that FcRn is had the binding affinity of change two different pH levels, described method comprises:

(a) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when a pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(b) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when the 2nd pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(c) by the residue of charge distribution, the different charge distribution of the demonstration under the first and second pH levels relatively, identifying has at least one candidate amino acid residue position in conjunction with free energy when changing initial polypeptide and combine with FcRn to be finished in the initial polypeptide; With

(d) select to be positioned at the elected amino acid of described amino acid position, so as should elected amino acid to mix cause behind the initial polypeptide to FcRn have change in conjunction with polypeptide affinity, that contain Fc, that change.

66. the method for claim 65, wherein a pH is about 7.4.

67. the method for claim 65, the avidity of wherein said polypeptide when a pH than at the 2nd pH the time high about 1.5 times to about 100 times.

FcRn is demonstrated during at the 2nd pH the polypeptide of the change of different avidity 68. when a pH FcRn demonstrated a kind of avidity, wherein said polypeptide comprises the aminoacid sequence by the method prediction of claim 56.

69. comprise the pharmaceutical composition of the polypeptide of claim 68.

70. comprise the nucleic acid molecule of nucleotide sequence of the polypeptide of the claim 68 of encoding.

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