阅读说明：本技术 具有改变的糖基化和降低的效应物功能的包含fc的多肽 (FC-containing polypeptides with altered glycosylation and reduced effector function ) 是由 C·潘 H·邱 于 2013-09-12 设计创作，主要内容包括：本发明涉及具有改变的糖基化和降低的效应物功能的包含FC的多肽,具体提供了结合多肽(例如,抗体)及其药物偶联物,其包含具有改变的糖基化概貌和降低的效应物功能的Fc结构域。在具体的实施方案中,所述Fc结构域包含：根据EU编号在氨基酸位点298的天冬酰胺残基；和根据EU编号在氨基酸位点300的丝氨酸或苏氨酸残基。还提供了编码抗原结合多肽的核酸、用于制造这种抗原-结合多肽的重组表达载体和宿主细胞。还提供了使用本文公开的抗原-结合多肽来治疗疾病的方法。(The present invention relates to FC-containing polypeptides with altered glycosylation and reduced effector function, and specifically provides binding polypeptides (e.g., antibodies) and drug conjugates thereof comprising an FC domain with altered glycosylation profile and reduced effector function. In particular embodiments, the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine residue at amino acid position 300 according to EU numbering. Nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides are also provided. Also provided are methods of treating diseases using the antigen-binding polypeptides disclosed herein.)

1. An isolated binding polypeptide comprising an Fc domain having altered glycosylation, wherein the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine at amino acid position 300 according to EU numbering, and wherein the binding polypeptide exhibits reduced effector function due to the altered glycosylation.

2. The binding polypeptide of claim 1, further comprising an alanine residue at amino acid position 299 according to EU numbering and/or a glutamine residue at amino acid position 297 according to EU numbering.

3. An isolated binding polypeptide comprising an Fc domain, wherein the Fc domain comprises: a free asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

4. An isolated binding polypeptide comprising an Fc domain, wherein the Fc domain comprises: a modified asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

5. A composition comprising the binding polypeptide of any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

6. A method of treating a patient comprising administering an effective amount of the composition of claim 5.

7. An isolated polynucleotide encoding the binding polypeptide of any one of claims 1-4.

8.A vector comprising the polynucleotide of claim 7.

9. A host cell comprising the polynucleotide of claim 7 or the vector of claim 8.

10. A method of making a binding polypeptide comprising expressing the polynucleotide of claim 7 or the vector of claim 8 in a cell.

Background

Antibodies with reduced or eliminated Fc glycosylation have been used in the treatment of inflammatory and autoimmune diseases or disorders to reduce side effects or toxicity associated with unwanted effector functions (see, e.g., Chan and Carter, nat. reviews Immunology, 2010). However, antibody Fc domain glycosylation is critical to antibody structure, stability and function and glycosylation can result in antibodies with poor biophysical properties. Accordingly, there is a need in the art for engineered binding proteins with reduced effector function, but which retain the desirable glycosylated Fc domain characteristics.

Summary of The Invention

The present disclosure improves upon the prior art by providing binding polypeptides (e.g., antibodies or fusions) comprising an Fc domain with altered glycosylation and reduced effector function, and optionally drug conjugates thereof. In particular embodiments, the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine residue at amino acid position 300 according to EU numbering. The disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Also provided are methods of treating diseases using the antigen-binding polypeptides disclosed herein.

The inventors have also surprisingly found that the binding polypeptides (e.g., antibodies) of the present disclosure exhibit an altered glycosylation profile, which is advantageous in that binding of the binding polypeptide to Fc γ receptors is eliminated, thereby altering the effector function of the binding polypeptide while retaining the desirable biophysical properties conferred by glycosylation. In addition, the engineered N-linked glycosylation site at amino acid position 298 can also be used as a site for effector moiety conjugation (e.g., cytotoxic drugs).

Accordingly, in one aspect the invention provides an isolated binding polypeptide comprising an Fc domain having altered glycosylation, wherein the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine at amino acid position 300 according to EU numbering, and wherein the binding polypeptide exhibits reduced effector function due to the altered glycosylation. In one embodiment, the binding polypeptide further comprises an alanine residue at amino acid position 299 according to EU numbering. In another embodiment, the binding polypeptide further comprises a glutamine residue at amino acid position 297 according to EU numbering. In one embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is human.

In one embodiment, the side chain of the asparagine residue is linked to the glycan through a β -glycosylamide linker. In another embodiment, the glycan is a biantennary glycan. In another embodiment, the saccharide is a naturally occurring mammalian glycoform.

In another embodiment, the binding polypeptide has a lower affinity for an fey receptor than a binding polypeptide having a native Fc domain. In one embodiment, the Fc γ receptor is Fc γ RI and/or Fc γ RIIIa. In another embodiment, the binding polypeptide has a similar affinity for the FcRn receptor as a binding polypeptide having a native Fc domain.

In another embodiment, the glycan comprises a reactive aldehyde group. In another embodiment, the glycan comprises an oxidized saccharide residue comprising an aldehyde group with reactivity. In another embodiment, the oxidized saccharide residue is a terminal sialic acid or galactose.

In another embodiment, the glycan is linked to an effector moiety. In another embodiment, the effector moiety is a cytotoxin. In another embodiment, the cytotoxin is selected from the group of cytotoxins listed in table 1. In another embodiment, the effector moiety is a detection agent. In another embodiment, the effector moiety is linked to a saccharide residue of the glycan through an oxime or hydrazone linkage. In another embodiment, the saccharide residue is a terminal sialic acid or galactose residue of the glycan. In another embodiment, the effector moiety comprises a pH-sensitive linker, disulfide linker, enzyme-sensitive linker, or other cleavable linker moiety. In another embodiment, the effector moiety comprises a linker moiety selected from the group of linker moieties described in table 2 or 14.

In some embodiments, the binding polypeptide is an antibody or immunoadhesin.

In another aspect, the invention provides an isolated binding polypeptide comprising an Fc domain, wherein the Fc domain comprises: a free asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

In another aspect, the present invention provides an isolated binding polypeptide comprising an Fc structure, wherein the Fc domain comprises: a modified asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

In another embodiment, the effector moiety is linked to the saccharide residue of the glycan through a side chain of a modified asparagine residue. In one embodiment, the saccharide is a terminal sialic acid or galactose residue of the glycan. In one embodiment, the effector moiety is linked to a saccharide residue of the glycan through an oxime or hydrazone linkage. In one embodiment, the saccharide is a terminal sialic acid or galactose residue of the glycan. In another embodiment, the modified asparagine residue is linked to a drug effector moiety to form an Antibody Drug Conjugate (ADC).

In another embodiment, a composition comprises a binding polypeptide of any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

In another embodiment, the present invention provides a method of treating a patient comprising administering an effective amount of a composition of the present invention.

In another aspect, the invention provides an isolated polynucleotide encoding a binding polypeptide of the invention. In another aspect, the invention provides a vector comprising the polynucleotide or a host cell comprising the polynucleotide or vector.

In yet another aspect, the invention provides a method of making a binding polypeptide comprising expressing the polynucleotide or the vector in a cell.

In particular, the invention relates to the following:

1. an isolated binding polypeptide comprising an Fc domain having altered glycosylation, wherein the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine at amino acid position 300 according to EU numbering, and wherein the binding polypeptide exhibits reduced effector function due to the altered glycosylation.

2. The binding polypeptide of item 1, further comprising an alanine residue at amino acid position 299 according to EU numbering.

3. The binding polypeptide of item 1, further comprising a glutamine residue at amino acid position 297 according to EU numbering.

4. The binding polypeptide of any one of the preceding claims, wherein the Fc domain is an IgG1 Fc domain.

5. The binding polypeptide of any one of the preceding claims, wherein the Fc domain is human.

6. The binding polypeptide of any one of the preceding claims, wherein the side chain of the asparagine residue is linked to the glycan through a β -glycosylamide linker.

7. The binding polypeptide of item 4, wherein the glycan is a biantennary glycan.

8. The binding polypeptide of item 4 or 5, wherein the glycan is a naturally occurring mammalian glycoform.

9. The binding polypeptide of any one of the preceding claims, which has a lower affinity for an fey receptor than a binding polypeptide having a native Fc domain.

10. The binding polypeptide, wherein the Fc γ receptor is Fc γ RI and/or Fc γ RIIIa.

11. The binding polypeptide of any one of the preceding claims, which has a similar affinity for the FcRn receptor as a binding polypeptide having a native Fc domain.

12. The binding polypeptide of any one of claims 6-11, wherein the glycan comprises a reactive aldehyde group.

13. The binding polypeptide of any one of claims 6-11, wherein the glycan comprises an oxidized carbohydrate residue comprising a reactive aldehyde group.

14. The binding polypeptide of item 13, wherein the oxidized carbohydrate residue is a terminal sialic acid or galactose.

15. The binding polypeptide of any one of claims 6-11, wherein the glycan is linked to an effector moiety.

16. The binding polypeptide of item 15, wherein the effector moiety is a cytotoxin.

17. The binding polypeptide of item 16, wherein the cytotoxin is selected from the group of cytotoxins listed in Table 1.

18. The binding polypeptide of item 17, wherein the effector moiety is a detection agent.

19. The binding polypeptide of any one of claims 15-18, wherein the effector moiety is linked to a saccharide residue of the glycan through an oxime or hydrazone linkage.

20. The binding polypeptide of item 19, wherein the carbohydrate residue is a terminal sialic acid or galactose residue of the glycan.

21. The binding polypeptide of any one of claims 15-20, wherein the effector moiety comprises a pH-sensitive linker, disulfide linker, enzyme-sensitive linker, or other cleavable linker moiety.

22. The binding polypeptide of any one of claims 15-20, wherein the effector moiety comprises a linker moiety selected from the group of linker moieties described in table 2 or 14.

23. The binding polypeptide of any one of the preceding claims, which is an antibody or immunoadhesin.

24. An isolated binding polypeptide comprising an Fc domain, wherein the Fc domain comprises: a free asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

25. An isolated binding polypeptide comprising an Fc domain, wherein the Fc domain comprises: a modified asparagine residue at amino acid position 298 according to EU numbering; and a free serine or threonine residue at amino acid position 300 according to EU numbering.

26. The binding polypeptide of item 25, wherein the effector moiety is attached to the carbohydrate residue of the glycan through the side chain of the modified asparagine residue.

27. The binding polypeptide of item 25, wherein the carbohydrate is a terminal sialic acid or galactose residue of the glycan.

28. The binding polypeptide of item 25, wherein the effector moiety is linked to a saccharide residue of the glycan through an oxime or hydrazone linkage.

29. The binding polypeptide of item 25, wherein the carbohydrate is a terminal sialic acid or galactose residue of the glycan.

30. The binding polypeptide of item 25, wherein the modified asparagine residue is linked to a drug effector moiety to form an Antibody Drug Conjugate (ADC).

31. A composition comprising the binding polypeptide of any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

32. A method of treating a patient comprising administering an effective amount of the composition of item 31.

33. An isolated polynucleotide encoding the binding polypeptide of any one of claims 1-30.

34. A vector comprising the polynucleotide of item 33.

35. A host cell comprising the polynucleotide of item 33 or the vector of item 34.

36. A method of making a binding polypeptide comprising expressing the polynucleotide of item 33 or the vector of item 34 in a cell.

Brief Description of Drawings

FIG. 1 is a simplified illustration of the synthesis of antibody drug conjugates in which a toxin moiety is attached to an oxidized sialic acid residue of an antibody glycan using an oxime linker.

FIG. 2 is a Coomassie blue stained gel showing expression and purification of glycosylated mutants.

Fig. 3 depicts the results of a surface plasmon resonance experiment to evaluate the binding of α β TCR HEBE1 IgG antibody mutants to recombinant human Fc γ RIIIa (V158& F158).

Fig. 4 depicts the results of a surface plasmon resonance experiment to evaluate the binding of α β TCR HEBE1 IgG antibody mutants to recombinant human fcyri.

Figure 5 depicts the cytokine release profile for TNFa, GM-CSF, IFNy and IL10 from PBMC in the presence of mutant anti- α β TCR antibody (day 2).

Figure 6 depicts cytokine release profiles from PBMCs for IL6, IL4, and IL2 in the presence of mutant anti- α β TCR antibodies (day 2).

Figure 7 depicts cytokine release profiles for TNFa, GM-CSF, IFNy and IL10 from PBMC in the presence of mutant anti- α β TCR antibody (day 4).

Figure 8 depicts cytokine release profiles for IL6, IL4, and IL2 from PBMCs in the presence of mutant anti- α β TCR antibodies (day 4).

Fig. 9 depicts the results of experiments investigating the expression level of 2C3 mutant by immunoblot analysis and surface plasmon resonance.

Figure 10 depicts the results of experiments investigating 2C3 mutant glycosylation before and after PNGase F treatment.

FIG. 11 depicts the results of SDS-PAGE experiments investigating glycosylation sites on 2C3 mutants isolated from cell culture.

Figure 12 depicts the results of a surface plasmon resonance experiment to evaluate the binding of modified anti-CD 52 to recombinant human Fc γ RIIIa (V158). anti-CD 52, which contains the S298N/Y300S mutation in the Fc domain, was used to evaluate the effector functions of the modified molecules in binding to CD52 peptide (a), binding to Fc γ RIIIa (V158, B), and control binding to mouse fcrn (c).

Fig. 13 depicts the results of a surface plasmon resonance experiment investigating Fc binding properties of the 2C3 mutant.

FIG. 14 depicts the results of a surface plasmon resonance experiment investigating the binding of modified anti-CD 52 to Fc γ RIIIa (Val158) (supra) and Fc γ RIIIa (Phe 158). anti-CD 52 antibody comprising the S298N/Y300S mutation in the Fc domain was used to evaluate the effector function of the modified molecules in binding to Fc γ RIIIa (Val158, fig. 14A) and Fc γ RIIIa (Phe58, fig. 14B).

FIG. 15 depicts the analysis of C1q binding in the S298N/Y300S mutant and wild type 2C3 control (A), and the results of Eliza analysis confirming equivalent coating of wells.

Figure 16 depicts the results of a surface plasmon resonance experiment measuring the binding kinetics of the 2C3 mutant to CD-52 peptide 741.

FIG. 17 depicts the results of a surface plasmon resonance experiment comparing the antigen binding affinities of wild-type anti-CD 522C3 and A114N highly glycosylated mutants.

Figure 18 depicts the results of isoelectric focusing and mass spectrometry charge standard experiments to determine glycan content of 2C3 mutants.

FIG. 19 depicts the concentration (octants) and results of a plasmon resonance experiment comparing the antigen binding affinity of wild-type anti-CD 522C3 and the mutant.

FIG. 20 depicts the results of an SDS-PAGE experiment to determine dextran content of the anti-TME 1A 114N mutant.

FIG. 21 depicts the results of SDS-PAGE and hydrophobic interaction chromatography analysis of A114N anti-Her 2 mutant.

FIG. 22 depicts the results of SDS-PAGE experiments demonstrating coupling of PEG to the 2C3A 114N mutant via an aminooxy linker.

FIG. 23 depicts the results of an LC-MS experiment to determine the glucan content of highly glycosylated mutants against TEM 1A 114N.

Figure 24 depicts the results of an LC-MS experiment to determine dextran content of wild-type HER2 antibody and a114N anti HER-2 hyperglycosylated mutant.

Figure 25 depicts a typical method of performing site-specific conjugation of antibodies according to the methods of the invention.

Fig. 26 depicts the synthesis of the following typical effector moieties of the present invention: aminooxy-Cys-MC-VC-PABC-MMAE and aminooxy-Cys-MC-VC-PABC-PEG 8-Dol 10.

Figure 27 depicts characterization information for sialylated HER2 antibody.

Figure 28 depicts characterization information for oxidized sialylated anti-HER 2 antibodies.

Figure 29 depicts hydrophobic interaction chromatography of glycoconjugates prepared with three different sialylated antibodies with two different aminooxy groups.

Figure 30 shows HIC chromatograms of anti-Her 2a114 glycosylation mutant conjugates with AO-MMAE prepared using GAM (+) chemistry.

Figure 31 depicts a comparison of the efficacy of anti-HER 2 glycoconjugates and sulfhydryl conjugates in vitro.

Figure 32 depicts a comparison of potency of anti-FAP B11 glycoconjugates and sulfhydryl conjugates in vitro.

Figure 33 depicts a comparison of in vivo efficacy of anti-HER 2 glycoconjugates and sulfhydryl conjugates in a HER2+ tumor cell xenograft model.

Fig. 34 depicts the results of an LC-MS experiment to determine the glucan content of mutant anti- α β TCR antibodies comprising the S298N/Y300S mutation.

Fig. 35 depicts the results of a circular dichroism experiment to determine the relative thermostability of a wild-type anti- α β TCR antibody and a mutant anti- α β TCR antibody comprising the S298N/Y300S mutation.

Figure 36 depicts the results of cell proliferation experiments for ADCs prepared with anti-HER antibody with a114N hyperglycosylation mutation and AO-MMAE.

Detailed Description

The present disclosure provides binding polypeptides (e.g., antibodies) and drug conjugates thereof, comprising an Fc domain, wherein the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine residue at amino acid position 300 according to EU numbering. The disclosure also provides nucleic acids encoding antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. And methods of treating diseases using the antigen binding polypeptides disclosed herein are provided.

I. Definition of

The term "binding polypeptide" or "binding polypeptide" as used herein refers to a polypeptide (e.g., an antibody) that comprises at least one binding site responsible for selective binding to a target antigen of interest (e.g., a human antigen). Typical binding sites include antibody variable domains, the ligand binding site of a receptor, or the receptor binding site of a ligand. In certain aspects, a binding polypeptide of the invention comprises a plurality (e.g., two, three, four, or more) binding sites.

The term "native residue" as used herein refers to an amino acid residue that is naturally occurring at a particular amino acid site of a binding polypeptide (e.g., an antibody or fragment thereof) and that has not been modified, introduced, or altered by the hand of man. The term "altered binding polypeptide" or "altered binding polypeptide" as used herein includes binding polypeptides (e.g., antibodies or fragments thereof) comprising at least one non-natural mutated amino acid residue.

The term "specifically binds" as used herein refers to antibodies or antigen-binding fragments thereof that bind to an antigen at up to about 1x 10^-6M、1x 10^-7 M、1x 10^-8 M、1x 10^-9 M、1x 10^-10 M、1x 10^-11 M、1x 10^-12M or less dissociation constant (Kd), and/or an ability to bind to an antigen with at least two-fold affinity compared to its affinity for non-specific antigen binding.

The term "antibody" as used herein refers to such assemblies (e.g., intact antibody molecules, antibody fragments, or variants thereof) that have significant known specific immunoreactive activity for an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins comprise light and heavy chains with or without an interchain covalent linkage between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood.

As will be described in further detail below, the genetic term "antibody" includes five different antibody types that are biochemically distinguishable. All five antibody types are clearly within the scope of the present disclosure, and the following discussion will be directed primarily to immunoglobulin molecules of the IgG class. For IgG, the immunoglobulin comprises two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of 53,000 and 70,000. The four chains are joined in a "Y" configuration by disulfide bonds, wherein the light chain cradles the chain starting at the opening of the "Y" and continues through the variable region.

Immunoglobulin light chains are classified as either kappa or lambda (kappa, lambda). Each type of heavy chain may be associated with a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent linkers when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence continues from the N-terminus at the forked end of the Y-configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses (e.g., gamma l-gamma 4). The nature of this chain determines the "class" of antibody to be IgG, IgM, IgA, IgG or IgE respectively. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been well characterized and are known to confer functional properties. Modified versions of each of these types and isoforms may be readily discerned by those skilled in the art in view of the present disclosure, and are therefore also within the scope of the present disclosure.

Both the light and heavy chains are divided into regions with structural and functional homology. The term "region" refers to a portion or portion of an immunoglobulin or antibody chain and includes constant or variable regions, as well as discrete portions or portions of more such regions. For example, the light chain variable region comprises "complementarity determining regions" or "CDRs" as defined herein dispersed in a "framework region" or "FR".

A region in an immunoglobulin heavy or light chain can be defined as a "constant" (C) region or a "variable" (V) region, depending on whether the multiple class member relatively lacks sequence variation within the region in the case of the "constant region" or significant variation within the region in the case of the "variable region". The terms "constant region" and "variable region" may also be used functionally. In this context, it is understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. In turn, the constant regions of immunoglobulins or antibodies confer important effector functions such as secretion, placental mobility, Fc receptor binding, complement fixation, and the like. The subunit structures and three-dimensional configurations of constant regions of various immunoglobulin classes are well known.

The constant and variable regions of immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a globular region of a heavy or light chain that includes peptide loops (e.g., including 3 to 4 peptide loops) that are stabilized by, for example, β -sheet sheets and/or intrachain disulfide bonds. The constant region domain on an immunoglobulin light chain may be referred to interchangeably as a "light chain constant region domain", "CL region", or "CL domain". The constant region on a heavy chain (e.g., the hinge, CH1, CH2, or CH3 domain) may be referred to interchangeably as a "heavy chain constant region domain," a "CH" region domain, or a "CH domain. The variable region on the light chain may be referred to interchangeably as a "light chain variable region domain", "VL region domain" or "VL domain". The variable domain on the heavy chain may be referred to interchangeably as a "heavy chain variable region domain", "VH region domain" or "VH domain".

By convention, the numbering of the variable constant region domains increases as they are further and further from the antigen binding site or amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-termini of the heavy and light chains, respectively. Accordingly, the domains of the light chain immunoglobulin are aligned in the VL-CL orientation, while the domains of the heavy chain are aligned in the VH-CH 1-hinge-CH 2-CH3 orientation.

Amino acid positions in the heavy chain constant region, including amino acid positions in the CH1, hinge, CH2, CH3 and CL domains, can be numbered according to the Kabat index numbering system (see Kabat et al, in "Sequences of proteins of Immunological Interest", u.s.dept.health and Human Services, 5 th edition, 1991). Alternatively, antibody amino acid positions may be numbered according to the EU index numbering system (see Kabat et al, supra).

As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

The term "CH 1 domain" as used herein includes the first (amino-terminal most) constant region domain of an immunoglobulin heavy chain, which extends, for example, from position 114-. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule and does not form part of the Fc region of the immunoglobulin heavy chain.

The term "hinge region" as used herein includes the portion of the heavy chain molecule that connects the CH1 domain with the CH2 domain. The hinge region comprises about 25 residues and is flexible, thus allowing independent movement of the two N-terminal antigen-binding regions. The hinge region can be further divided into three distinct domains: upper, middle and lower hinge domains (Roux et al J.Immunol.1998, 161: 4083).

The term "CH 2 domain" as used herein includes the portion of a heavy chain immunoglobulin molecule that extends from, for example, positions 244-360 (EU positions 231-340) in the Kabat index numbering system. The CH2 domain is unique in that it is not closely paired with another domain. In contrast, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. In one embodiment, a binding polypeptide of the present disclosure includes a CH2 domain from an IgG1 molecule (e.g., a human IgG1 molecule).

The term "CH 3 domain" as used herein includes the heavy chain portion of an immunoglobulin molecule that extends about 110 residues from the N-terminus of the CH2 domain, e.g., from about 361-476 position (EU position 341-445) in the Kabat index numbering system. The CH3 domain typically forms the C-terminal portion of an antibody. In some immunoglobulins, however, an additional domain may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain of the μ chain of IgM and the e chain of IgE). In one embodiment, a binding polypeptide of the present disclosure includes a CH3 domain from an IgG1 molecule (e.g., a human IgG1 molecule).

The term "CL domain" as used herein includes the constant region domain of an immunoglobulin light chain, which extends, for example, from about positions 107A-216 Kabat. The CL domain is adjacent to the VL domain. In one embodiment, a binding polypeptide of the disclosure includes a CL domain from a kappa light chain (e.g., a human kappa light chain).

The term "Fc region" as used herein is defined as the portion of the heavy chain constant region that begins at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, 114 from the first residue of the weight variable region) and terminates at the C-terminus of the antibody. Accordingly, a complete Fc region comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

The term "native Fc" as used herein refers to a molecule comprising a non-antigen-binding fragment sequence resulting from digestion or otherwise produced by an antibody, whether in monomeric or multimeric form, and may comprise a hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and the immunoglobulin may be any immunoglobulin, although IgG1 and IgG2 are preferred. Native Fc molecules consist of monomeric polypeptides, bound by covalent (i.e., disulfide bonds) and non-covalent into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the type (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA 2). An example of a native Fc is a disulfide-bonded dimer produced by papain-digested IgG. The term "native Fc" as used herein is universally applicable to monomeric, dimeric and multimeric forms.

The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor FcRn (neonatal Fc receptor). Typical Fc variants and their interaction with rescue receptors (slope receptors) are well known in the art. Thus, the term "Fc variant" may comprise a molecule or sequence that is humanized from a non-human native Fc. In addition, native Fc includes regions that can be removed, as these regions provide structural features or biological activity that are not required for the antibody-like binding polypeptides of the invention. Thus, the term "Fc variant" encompasses a molecule or sequence that lacks one or more native Fc sites or residues, or a molecule or sequence in which one or more Fc sites or residues have been modified, which affect or participate in: (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity when expressed in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors rather than rescue receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

The term "Fc domain" as used herein encompasses native Fc and Fc variants as well as the sequences defined above. Like Fc variants and native Fc molecules, the term "Fc domain" includes molecules in monomeric or multimeric form, whether resulting from digestion of an intact antibody or otherwise produced.

The variable regions of the antibody allow it to selectively recognize and specifically bind to epitopes on the antigen, as indicated above. That is, the VL and VH domains of an antibody combine to form a variable region (Fv) that defines an antigen-binding site in three dimensions. The tetrad antibody structure forms an antigen binding site that is present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three Complementarity Determining Regions (CDRs) on each of the heavy and light chain variable regions. The term "antigen binding site" as used herein includes sites (e.g., cell surface or soluble antibodies) that specifically bind to (immunoreact with) an antigen. The antigen binding site includes immunoglobulin heavy and light chain variable regions and the binding site formed by these variable regions determines the specificity of the antibody. The antigen binding site is formed by one antibody and another, different variable region. The altered antibodies of the present disclosure include at least one antigen binding site.

In some embodiments, a binding polypeptide of the present disclosure includes at least two antigen binding domains that provide for binding of the binding polypeptide to a selected antigen. The antigen binding domains need not be from the same immunoglobulin molecule. In this regard, the variable region may be or be from any type of animal that can be induced to produce a humoral response against a desired antigen and produce immunoglobulins. Thus, the variable region of a binding polypeptide may be of mammalian origin, for example, it may be human, murine, rat, goat, sheep, non-human primate (e.g., cynomolgus monkey, macaque etc.), lupin or camelid (e.g., from camels, llamas and related species).

In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, discrete amino acid sequences that are specifically positioned to form an antigen binding site as the antibody assumes a three-dimensional conformation in an aqueous environment. The remainder of the heavy and light variable domains exhibit less intramolecular variability in amino acid sequence and are referred to as framework regions. The framework regions predominantly adopt a β -sheet configuration while the CDRs form loops that connect to and, in some cases, form part of the β -sheet structure. Thus, these framework regions act to form a scaffold, providing six CDRs with positioning in the correct orientation through inter-chain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on the immunoreactive antigen. The complementary surface promotes non-covalent binding between the antibody and the immunoreactive epitope.

Exemplary binding polypeptides of the invention include antibody variants. The term "antibody variant" as used herein includes antibodies in altered synthetic and engineered forms, and thus not naturally occurring, e.g., antibodies comprising portions of at least two heavy chains rather than two complete heavy chains (e.g., domain deleted antibodies or miniantibodies); multiple specific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or to different epitopes on a single antigen); heavy chain molecules that bind to scFv molecules, and the like. Furthermore, the term "antibody variant" includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind three, four, or more copies of the same antigen).

The term "valency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to a target molecule or a specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site can specifically bind to the same or different molecules (e.g., can bind to different ligands or different antigens, or different epitopes on the same antigen). The subject binding polypeptides preferably have at least one binding site specific for a human antigen molecule.

The term "specificity" refers to the ability to specifically bind to (e.g., immunoreact with) a given target antigen (e.g., a human target antigen). The binding polypeptide may be monospecific and comprise one or more binding sites that specifically bind to the target, or the polypeptide may be multispecific and comprise two or more binding sites that specifically bind to the same or different targets. In some embodiments, a binding polypeptide of the invention is specifically directed against two different (e.g., non-overlapping) portions of the same target. In certain embodiments, the binding polypeptides of the invention are specifically directed against more than one target. Typical binding polypeptides (e.g., antibodies) comprising an antigen binding site that binds to an antigen expressed on tumor cells are well known in the art and one or more CDRs from such an antibody may be included in an antibody of the invention.

The term "linking moiety" includes moieties capable of linking an effector moiety and a binding polypeptide disclosed herein. The linker moiety may be selected so as to be cleavable (e.g., enzymatically cleavable or pH sensitive) or non-cleavable. Typical linking moieties are set forth herein in table 2.

The term "effector moiety" as used herein includes diagnostic and therapeutic agents (e.g., proteins, nucleic acids, lipids, drug moieties, and fragments thereof) having biological or other functional activity. For example, a modified binding polypeptide comprising an effector moiety conjugated to a binding polypeptide has at least one additional function or property as compared to a non-conjugated antibody. For example, conjugation of a cytotoxic drug (e.g., effector moiety) to a binding polypeptide results in the formation of a binding polypeptide (i.e., other than antigen binding) that has drug cytotoxicity as a secondary function. In another example, conjugation of the second binding polypeptide to the binding polypeptide can confer additional binding properties. In certain embodiments, when the effector moiety is a gene-encoded therapeutic or diagnostic protein or nucleic acid, the effector moiety may be synthesized or expressed by peptide synthesis or recombinant DNA methods well known in the art. On the other hand, when the effector is a non-gene encoded peptide or drug moiety, the effector moiety may be artificially synthesized or purified from a natural source. The term "drug moiety" as used herein includes anti-inflammatory, anti-cancer, anti-infection (e.g., antifungal, antibacterial, antiparasitic, antiviral, etc.) and anesthetic therapeutic agents. In another embodiment, the drug moiety is an anti-cancer or cytotoxic agent. Compatible drug moieties may also include prodrugs. Typical effector moieties are set forth in table 1 herein.

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active agent that is less active, reactive, or susceptible to side effects than the parent drug and that is capable of being activated by an enzyme or otherwise converted in vivo to a more active form. Prodrugs compatible with the compositions of the present disclosure include, but are not limited to, phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphoric acid-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouracil prodrugs convertible to more active, non-cytotoxic drugs. One skilled in the art can chemically modify the contemplated drug moiety or prodrug thereof to further facilitate reaction of the compound to prepare modified binding polypeptides of the disclosure. The drug moiety also includes derivatives, pharmaceutically acceptable salts, esters, amides, and ethers of the drug moieties described herein. Derivatives include modifications to the drugs identified herein that may improve or not significantly reduce the intended therapeutic activity of the particular drug.

The term "anti-cancer agent" as used herein includes agents that are not conducive to neoplastic or tumor cell growth and/or proliferation, and may act to reduce, inhibit or destroy malignancy. Examples of such agents include, but are not limited to, cytostatics, alkylating agents, antibiotics, cytotoxic nucleosides, tubulin binding agents, hormones, hormone antagonists, cytotoxic agents, and the like. Cytotoxic agents include tomaymycin derivatives (tomaymycin derivatives), maytansine derivatives (maytansinoids), cryptophycin derivatives, anthracycline derivatives, diphosphonic acid derivatives, leptomycin derivatives (leptin derivatives), streptonigrin derivatives (streptonigrin derivatives), auristatin derivatives, and duocarmycin derivatives. Any agent that acts to prevent or slow the growth of immunoreactive cells or malignant cells is within the scope of the present disclosure.

The term "antigen" or "target antigen" as used herein refers to a molecule or portion of a molecule that is capable of being bound by a binding site of a binding polypeptide. The target antigen may have one or more epitopes.

Binding polypeptides

In one aspect, the present disclosure provides binding polypeptides (e.g., antibodies, antibody fragments, antibody variants, and fusion proteins) comprising an Fc domain, wherein the Fc domain comprises: an asparagine residue at amino acid position 298 according to EU numbering; and a serine or threonine residue at amino acid position 300 according to EU numbering.

Fc domains from any immunoglobulin (e.g., IgM, IgG, IgD, IgA, and IgE) type and species can be used for the binding polypeptides disclosed herein. Chimeric Fc domains comprising portions of Fc domains from different species or Ig classes may also be employed. In some embodiments, the Fc domain is a human IgG1 Fc domain. In the case of the human IgG1 Fc domain, mutations to asparagine at Kabat position 298, the wild-type amino acid, and serine or threonine at Kabat position 300, resulted in the formation of a consensus site for N-linked glycosylation (i.e., an N-X-T/S sequence, where X is any amino acid except proline). However, in the case of Fc domains of other species and/or Ig classes or isotypes, it will be understood by those skilled in the art that if proline is present, it may be necessary to mutate the Kabat 299 position of the Fc domain to regenerate the N-X-T/S sequence.

The binding polypeptides disclosed herein encompass any binding polypeptide comprising an Fc domain having an N-linked glycosylation site at position 298 according to Kabat numbering. In certain embodiments, the binding polypeptide is an antibody, or a fragment or derivative thereof. Any antibody from any source or variety can be used for the binding polypeptides disclosed herein. Suitable antibodies include, but are not limited to, human antibodies, humanized antibodies, or chimeric antibodies.

In some embodiments, a binding polypeptide of the present disclosure can comprise an antigen-binding fragment of an antibody. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to or competes for antigen binding (i.e., specific binding) with an intact antibody (i.e., the intact antibody from which it was derived). Antigen-binding fragments may be produced by recombinant or biochemical methods well known in the art. Typical antigen binding fragments include Fv, Fab 'and (Fab') 2. In a preferred embodiment, the antigen binding fragments of the present disclosure are altered antigen binding fragments that include at least one engineered glycosylation site. In a typical embodiment, the altered antigen-binding fragment of the present disclosure comprises an altered VH domain as described above. In another typical embodiment, the altered antigen-binding fragment of the present disclosure comprises an altered CH1 domain as described above.

In typical embodiments, the binding polypeptide comprises a single chain variable region sequence (ScFv). The single chain variable region sequence comprises a single polypeptide having one or more antigen binding sites, for example, a VL domain linked to a VH domain by a flexible linker. ScFv molecules can be constructed in either the VH-linker-VL orientation or the VL-linker-VH orientation. The flexible hinge connecting the VL and VH domains that make up the antigen binding site preferably comprises from about 10 to about 50 amino acid residues. Linking peptides are known in the art. A binding polypeptide of the invention may comprise at least one scFv and/or at least one constant region. In one embodiment, a binding polypeptide of the present disclosure may include at least one scFv linked to or fused to an antibody or a fragment comprising a CH1 domain (e.g., a CH1 domain comprising an asparagine residue at Kabat position 114) and/or a CH2 domain (e.g., a CH2 domain comprising an asparagine residue at EU 298 and a serine or threonine residue at EU300 position).

In some typical embodiments, the binding polypeptides of the present disclosure are multivalent (e.g., tetravalent) antibodies produced by fusing a DNA sequence encoding the antibody to an ScFv molecule (e.g., an altered ScFv molecule). For example, in one embodiment, these sequences are combined, thereby linking the ScFv molecule (e.g., an altered ScFv molecule) at its N-terminus or C-terminus to the Fc fragment of an antibody via a flexible linker (e.g., gly/ser linker). In another embodiment, the tetravalent antibodies of the present disclosure may be generated by fusing an ScFv molecule to a linker peptide, which is fused to a CH1 domain (e.g., a CH1 domain comprising an asparagine residue at Kabat position 114) to construct an ScFv-Fab tetravalent molecule.

In another embodiment, a binding polypeptide of the disclosure is an altered miniantibody. The altered miniantibodies of the present disclosure are dimeric molecules consisting of two polypeptide chains each comprising an ScFv molecule fused to a CH3 domain or portion thereof via a linker peptide (e.g., an altered ScFv molecule comprising an altered VH domain as described above). Miniantibodies can be generated by constructing the ScFv component and linking the peptide-CH 3 component using methods described in the art (see, e.g., us patent 5,837,821 or WO 94/09817 Al). In another embodiment, tetravalent miniantibodies can be constructed. The tetravalent miniantibody may be constructed in the same manner as the miniantibody except that the two ScFV molecules are linked using a flexible linker. The linked scFv-scFv construct was then linked to the CH3 domain.

In another embodiment, a binding polypeptide of the present disclosure comprises a bispecific antibody. Bispecific antibodies are dimeric tetravalent molecules, each molecule having a polypeptide similar to an scFv molecule, but typically having a short (less than 10 and preferably 1-5) amino acid residue linker connecting the two variable domains, such that the VL and VH domains cannot interact on the same polypeptide chain. Instead, the VL and VH domains on one polypeptide chain interact with the VH and VL domains (respectively) on a second polypeptide chain (see, for example, WO 02/02781). The bispecific antibodies of the present disclosure comprise scFv molecules fused to a CH3 domain.

In other embodiments, the binding polypeptides of the invention comprise multispecific or multivalent antibodies comprising one or more series variable domains, e.g., Tandem Variable Domain (TVD) polypeptides, on the same polypeptide chain. Typical TVD polypeptides include the "double-headed" or "double-Fv" configuration described in U.S. Pat. No.5,989,830. In a double Fv configuration, the variable domains of two different antibodies are expressed in a tandem orientation on two separate chains (one heavy and one light chain), with one polypeptide chain having two-fold VH in tandem separated by a peptide linker (VH 1-linker-VH 2), and the other polypeptide chain consisting of a complementary VL domain linked in tandem by a peptide linker (VL 2-linker-VL 1). In the cross-double head configuration, the variable domains of two different antibodies are expressed in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), one of which has two VH domains in series separated by a peptide linker (VH 1-linker-VH 2), and the other of which consists of complementary VL domains connected in series in opposite directions by a peptide linker (VL 2-linker-VL 1). Other antibody variants based on the "double-Fv" format include the double-variable-domain IgG (DVD-IgG) bispecific antibody (see US patent No.7,612,181) and the TBTI format (see US 2010/0226923 a 1). The addition of constant domains to each chain of a bi-Fv (CH1-Fc to the heavy chain and kappa or lambda constant domains to the light chain) resulted in a functional bispecific antibody without any additional modifications (i.e., significant addition of constant domains to enhance stability).

In another typical embodiment, the binding polypeptide comprises a crossed double variable domain IgG (CODV-IgG) bispecific antibody based on a "double-headed" configuration (see US20120251541 a1, which is incorporated herein by reference in its entirety). CODV-IgG antibody variants have one polypeptide chain with a VL domain linked in series with a CL domain (VL1-L1-VL2-L2-CL) and a second polypeptide chain with a complementary VH domain linked in series with a CH1 domain in the opposite direction (VH2-L3-VH1-L4-CH1), wherein the polypeptide chains form a crossed light-heavy chain pair. In some embodiments, the second polypeptide may be further linked to an Fc domain (VH2-L3-VH1-L4-CH 1-Fc). In certain embodiments, linker L3 is at least twice the length of linker L1 and/or linker L4 is at least twice the length of linker L2. For example, L1 and L2 may be 1-3 amino acid residues in length, L3 may be 2 to 6 amino acid residues in length, and L4 may be 4 to 7 amino acid residues in length. Examples of suitable linkers include single glycine (Gly) residues; bisglycine peptide (Gly-Gly); tripeptide (Gly-Gly); a peptide having four glycine residues (Gly-Gly); a peptide having five glycine residues (Gly-Gly); a peptide having six glycine residues (Gly-Gly); a peptide having seven glycine residues (Gly-Gly); a peptide having eight glycine residues (Gly-Gly). Other combinations of amino acid residues may be used, such as the peptides Gly-Gly-Gly-Gly-Ser and the peptides Gly-Gly-Gly-Gly-Gly-Ser.

In some embodiments, the binding polypeptides include immunoadhesin molecules comprising a non-antibody binding domain (e.g., a receptor, ligand or cell adhesion molecule) fused to an antibody constant domain (see, e.g., Ashkenazi et al, Methods,19958(2), 104-115, which is incorporated herein by reference in its entirety).

In certain embodiments, the binding polypeptide comprises an immunoglobulin-like domain. Suitable immunoglobulin-like domains include, but are not limited to, fibronectin domains (see, e.g., Koide et al) (2007), methods mol. biol.352: 95-109, which is incorporated herein by reference in its entirety), DARPin (see, for example, Stumpp et al (2008) Drug discov. today 13 (15-16): 695-: 2668-76, incorporated herein by reference in its entirety), Lipocalins (see, e.g., Skerra et al (2008) FEBS j.275 (11): 2677-83, which is incorporated herein by reference in its entirety), Affilins (see, for example, Ebersbach et al (2007) j.mol.biol.372 (1): 172-85, which is incorporated herein by reference in its entirety), affitns (see, e.g., Krehenbrink et al (2008). 1058-68, which is incorporated herein by reference in its entirety), Avimers (see, e.g., Silverman et al (2005) nat. biotechnol.23 (12): 1556-61, which is incorporated herein by reference in its entirety), Fynomers (see, for example, grabulivski et al (2007) J BiolChem 282 (5): 3196-3204, which is incorporated herein by reference in its entirety) and a Kunitz domain peptide (see, for example, Nixon et al (2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which is incorporated herein by reference in its entirety).

N-linked glycans

In some embodiments, the Fc domain of a binding polypeptide disclosed herein is glycosylated at an engineered arginine at position 298 (N298) according to EU numbering. N-linked glycans are typically attached to the nitrogen group of the N298 side chain through a β -glycosylamide linker. However, other suitable art-recognized linkers may be employed.

Any type of naturally occurring or synthetic (i.e., non-natural) N-linked glycan can be linked to N114. For example, the glycan can be a natural glycan or an engineered glycan comprising a non-natural linker. In certain embodiments, the glycan comprises a sugar that can be oxidized (e.g., by periodate treatment) to produce a group suitable for coupling to an effector moiety (e.g., a reactive aldehyde group). Suitable sugars that can be oxidized include, but are not limited to, galactose and sialic acid (e.g., N-acetylneuraminic acid). In some embodiments, the glycan is a biantennary glycan. In some embodiments, the glycan is a naturally occurring mammalian glycoform.

Glycosylation can be achieved by any means known in the art. In some embodiments, the glycosylation is obtained by expressing the binding polypeptide in a cell capable of N-linked glycosylation. Any natural or engineered cell (e.g., prokaryotic or eukaryotic) can be used. In general, mammalian cells are used to affect glycosylation. N-glycans produced in mammalian cells are generally referred to as complex N-glycans (see, e.g., Drickamer K, Taylor ME (2006). Introduction to glycobiology, 2 nd edition, which is incorporated herein by reference in its entirety). These complex N-glycans have typically two to six external branches with an internal core structure Man₃GlcNAc₂A linked sialylsialylsialyllactosamine (sialyllactosamine) sequence. Complex N-glycans have at least one branch, and preferably at least two alternating GlcNAc and galactose (Gal) residues to terminate oligosaccharides, such as, for example: NeuNAc-; NeuAc alpha 2,6 GalNAc alpha 1-; NeuAc alpha 2,3 Gal beta 1,3 GalNAc alpha 1-; and NeuAc α 2,3/6 Gal β 1,4 GlcNAc β 1; in addition, sulfate esters may be present on galactose, GalNAc, and GlcNAc residues, while phosphate esters may be present on mannose residues. The NeuAc may be O-acetylated or substituted by NeuGl (N-glycolylneuraminic acid). Complex N-glycans may also have intrachain substitutions that bisect GlcNAc and core fucose (Fuc).

Additionally or alternatively, glycosylation can be achieved or modified in vitro by enzymatic means. For example, one or more glycosyltransferases may be employed to add specific sugar residues to N298, and one or more glycosidases may be employed to remove unwanted sugars from N-linked glycans. Such enzymatic methods are known in the art (see, e.g., WO/2007/005786, which is incorporated herein by reference in its entirety).

Immune effector function and Fc modification

In certain embodiments, a binding polypeptide of the invention can comprise an antibody constant region (e.g., an IgG constant region, e.g., a human IgG1 or IgG4 constant region) that mediates one or more effector functions. For example, binding of the C1 component of complement to the constant region of an antibody activates the complement system. Complement activation is critical in opsonization and lysis of cellular pathogens. Activation of complement also stimulates an inflammatory response and may be involved in autoimmune allergies. In addition, antibodies bind to receptors on a variety of cells via the Fc region, where the Fc receptor binding site on the antibody Fc region binds to an Fc receptor (FcR) on the cell. There are a variety of Fc receptors specific for different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Binding of antibodies to Fc receptors on cell surfaces can trigger a number of important and diverse biological responses including phagocytosis and destruction of antibody-coated microparticles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production. In a preferred embodiment, a binding polypeptide (e.g., an antibody or antigen-binding fragment thereof) of the invention binds to an Fc-gamma receptor. In alternative embodiments, a binding polypeptide of the invention may comprise a constant region that lacks one or more effector functions (e.g., ADCC activity) and/or is incapable of binding to an Fc γ receptor.

Certain embodiments of the invention include antibodies in which at least one amino acid in one or more constant region domains is deleted or otherwise altered to provide a desired biochemical property, such as reduced or enhanced effector function, ability to non-covalently dimerize, increased ability to localize to a tumor site, reduced serum half-life, or increased serum half-life when compared to an intact, unaltered antibody having about the same immunogenicity. For example, certain antibodies described herein for use in diagnostic and therapeutic methods are domain deleted antibodies comprising a polypeptide chain similar to an immunoglobulin heavy chain, but lacking at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, the binding polypeptide comprises constant regions derived from different antibody isotypes (e.g., constant regions from two or more human IgG1, IgG2, IgG3, or IgG 4). In other embodiments, the binding polypeptide comprises a chimeric hinge (i.e., a hinge comprising a hinged portion derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and a middle hinge domain of IgG 1). In one embodiment, the binding polypeptide includes an Fc region from a human IgG4 molecule or portion thereof and a Ser228Pro mutation (Eu numbering) at the core hinge region of the molecule.

In certain embodiments, the Fc portion can be mutated to increase or decrease effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (by point mutation or otherwise) can decrease Fc receptor binding of the modified antibody in circulation, thereby increasing tumor localization. In other cases, constant region modifications consistent with the present invention may moderate complement binding and thus reduce serum half-life and nonspecific binding of conjugated cytotoxins. While other modifications to the constant region may be used to modify disulfide bonds or oligosaccharide moieties, which allow for improved localization due to increased antigen specificity and flexibility. The physiological profile, bioavailability, and other biochemical effects of the modification, such as tumor localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

In certain embodiments, the Fc domain employed in the antibodies of the invention is an Fc variant. The term "Fc variant" as used herein refers to an Fc domain having at least one amino acid substitution relative to the wild-type Fc domain from which the Fc domain is derived. For example, wherein the Fc domain is derived from a human IgG1 antibody, the Fc variant of the human IgG1 Fc domain comprises at least one amino acid substitution relative to the Fc domain.

The amino acid substitutions of the Fc variants can be located at any position within the Fc domain (i.e., at any amino acid position under EU convention). In one embodiment, the Fc variant comprises a substitution at an amino acid position located in the hinge domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH2 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH3 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution at an amino acid position located in the CH4 domain or portion thereof.

Binding polypeptides of the invention can employ any art-recognized Fc variant known to confer improvement (e.g., reduction or enhancement) in effector function and/or FcR binding. Such Fc variants may include, for example, International PCT publications WO88/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO 2/029207A 2, WO 2/2A 2/2A 2, WO 2A 2/2A 2/2, WO 2A 2/2A 2; 5,739,277; 5,834,250; 5,869,046; 6,096,871, respectively; 6,121,022; 6,194,551; 6,242,195, respectively; 6,277,375; 6,528,624, respectively; 6,538,124, respectively; 6,737,056; 6,821,505, respectively; 6,998,253, respectively; and 7,083,784, each of which is incorporated herein by reference. In a typical embodiment, a binding polypeptide of the invention may comprise an Fc variant comprising an amino acid substitution at EU 268 (e.g., H268D or H268E). In another typical embodiment, a binding polypeptide of the invention may comprise an amino acid substitution at EU 239 site (e.g., S239D or S239E) and/or EU 332 site (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide of the invention may comprise an Fc variant comprising an amino acid substitution that alters the antigen-dependent effector function of an antibody, particularly the circulating half-life of the binding polypeptide. Such binding polypeptides exhibit increased or decreased binding to FcRn when compared to binding polypeptides lacking these substitutions, and thus have increased or decreased half-lives in serum, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and these molecules have useful application in methods of therapeutically treating mammals in which it is desirable for the administered antibody to have a long half-life, e.g., for treating chronic diseases or disorders. Conversely, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules may also be useful, for example, for administration to mammals where reduced circulation time may be advantageous, e.g., for in vivo diagnostic imaging or where the initial antibody has toxic side effects when present in circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful for treating diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be expected include those intended to be localized to the brain, kidney and/or liver. In a typical embodiment, the altered binding polypeptides (e.g., antibodies or antigen-binding fragments thereof) of the invention exhibit reduced transport from the vascular system across the epithelium of the glomerulus. In another embodiment, the altered binding polypeptides (e.g., antibodies or antigen-binding fragments thereof) of the invention exhibit reduced transport from the brain across the Blood Brain Barrier (BBB) into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. The FcRn binding loop comprises amino acid residues 280-299 (numbering according to EU). Typical amino acid substitutions that alter FcRn binding activity are disclosed in international PCT publication No. wo05/047327, which is incorporated herein by reference. In some typical embodiments, a binding polypeptide (e.g., an antibody or antigen-binding fragment thereof) of the invention comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering). In yet other exemplary embodiments, the binding molecules of the invention comprise a human Fc domain with the double mutation H433K/N434F (see, e.g., U.S. patent No.8,163,881).

In other embodiments, the binding polypeptides described herein for use in the diagnostic and therapeutic methods have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, that is altered to reduce or eliminate glycosylation. For example, a binding polypeptide (e.g., an antibody or antigen-binding fragment thereof) of the invention can also comprise an Fc variant comprising an amino acid substitution that alters Fc glycosylation of the antibody. For example, the Fc variants can have reduced glycosylation (e.g., N-or O-linked glycosylation). In typical embodiments, the Fc variant comprises reduced glycosylation of N-linked glycans normally present at amino acid position 297(EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In a specific embodiment, the antibody comprises an Fc variant having an amino acid substitution at amino acid position 228 or 229(EU numbering). In more specific embodiments, the antibody comprises an IgG1 or IgG4 constant region comprising the S228P and T299A mutations (EU numbering).

Effector moiety

In certain embodiments, a binding polypeptide of the present disclosure comprises an effector moiety. In general, these effector moieties are coupled (directly or via a linker moiety) to an N-linked glycan on the binding polypeptide (e.g., an N-linked glycan linked to N298 of the CH2 domain (EU numbering) and/or N114 of the CH1 domain (Kabat numbering)). In certain embodiments, the binding polypeptide is a full length antibody comprising two CH1 domains having a glycan at position 114 Kabat, wherein both glycans are conjugated to one or more effector moieties.

Any effector moiety can be added to the binding polypeptides disclosed herein. The effector moiety preferably adds a non-native function to the altered antibody or fragment thereof without significantly altering the intrinsic activity of the binding polypeptide. The effector moiety may be, for example, but not limited to, a therapeutic agent or a diagnostic agent. Modified binding polypeptides (e.g., antibodies) of the present disclosure can include one or more effector moieties, which can be the same or different.

In one embodiment, the effector moiety may be of formula (I):

H₂N-Q-CON-X

a compound of the formula (I),

wherein:

A) q is NH or O; and is

B) CON is a connecting part; and is

C) X is a therapeutic agent as defined herein.

Linker moiety for linking therapeutic agent to H₂N-Q-. The linker moiety may comprise at least one of any suitable component known to those skilled in the art, including, for example, an alkylene component, a polyethylene glycol component, a poly (glycine) component, a poly (oxazoline) component, a carbonyl component, a component derived from a cysteine amide, a component derived from valine coupled to citrulline, and a component derived from 4-aminobenzyl carbamate, or any combination thereof.

In another embodiment, the effector moiety of formula (I) may be of formula (Ia):

H₂N-Q-CH₂-C(O)-Z-X

formula (Ia)

Wherein:

A) q is NH or O; and is

B) Z is Cys- (MC)_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_f，

Wherein

Cys is a component derived from cysteine amide;

mc is a maleimide-derived component;

vc is a component derived from valine coupled to citrulline;

PABC is a component derived from 4-aminobenzyl carbamate;

v.X is a therapeutic agent as defined herein;

a is 0 or 1;

b is 0 or 1;

c is 0 or 1; and is

ix.f is 0 or 1

"component derived from cysteine amide" is a compound of formula (I) and H₂N-Q-CH₂-c (o) -point of attachment. In one embodiment, the "A cysteine amide-derived component "may refer to one or more portions of an effector moiety having the structure:

in one embodiment, the "Cys" component of the effector moiety may comprise one such moiety. For example, the following structure indicates an effector moiety with one such moiety (where the "Cys" component is indicated by the dashed box):

in another embodiment, the "Cys" component of the effector moiety may comprise two or more such moieties. By way of example, the following portions include two such portions:

as can be seen from the structure, each "Cys" component carries (MC)_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_f-an X group.

In one embodiment, the phrase "maleimide-derived component" may refer to any moiety of an effector moiety having the structure:

wherein d is an integer from 2 to 5. Any Cys- (MC) included in the effector moiety_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_fThe number of MC components in the-X group is indicated by the subscript "a" and can be 0 or 1. In one embodiment, a is 1. In another embodiment, b is 0.

In one embodiment, the "Cys" component may be linked to the "MC" component via a sulfur atom in the "Cys" component, as shown by the dashed box in the following structure:

in one embodiment, the phrase "a component derived from valine coupled to citrulline" can refer to any portion of the effector moiety having the following results:

including any Cys- (MC) in the effector moiety_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_fThe number of VC components in the-X group is indicated by the subscript "b" and may be 0 or 1. In one embodiment, b is 1. In another embodiment, b is 0.

In one embodiment, the phrase "component derived from 4-aminobenzyl carbamate" may refer to any moiety of an effector moiety having the structure:

including any Cys- (MC) in the effector moiety_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_fThe number of PABC components in the-X group may be represented by the subscript "c" and may be 0 or 1. In one embodiment, c is 1. In another embodiment, c is 0.

In one embodiment, "C₁₆H₃₂O₈C₂H₄"refers to the following structure:

any Cys- (MC) included in the effector moiety_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈C₂H₄)_fC in the-X group₁₆H₃₂O₈The number of units can be represented by the subscript "f". In one embodiment, f is 1. In another embodiment, f is 0.

In one embodiment, a is 1, b is 1, c is 1, and f is 0.

a) Therapeutic effector moieties

In certain embodiments, the binding polypeptides of the present disclosure are conjugated to an effector moiety comprising a therapeutic agent, e.g., a drug moiety (or prodrug thereof) or a radiolabeled compound. In one embodiment, the therapeutic agent is a cytotoxin. Typical cytotoxic effectors are set forth in part in table 1 herein.

TABLE 1 typical cytotoxic Effector moiety

Further exemplary drug moieties include anti-inflammatory, anti-cancer, anti-infection (e.g., antifungal, antibacterial, antiparasitic, antiviral, etc.), and anesthetic therapeutics. In a further embodiment, the drug moiety is an anti-cancer agent. Typical anti-cancer agents include, but are not limited to, cytostatics, enzyme inhibitors, gene modulators, cytotoxic nucleosides, tubulin binding agents or tubulin inhibitors, proteasome inhibitors, hormones and hormone antagonists, anti-angiogenic agents, and the like. Typical cytostatic anticancer agents include alkylating agents such as the anthracycline family of drugs (e.g., doxorubicin, carminomycin, cyclosporin-a, chloroquine, methotrexate, mithramycin, methylmitomycin, streptonigrin, methylmitomycin, anthracenedione, and ethylenimine). Other cytostatic anticancer agents include DNA synthesis inhibitors (e.g., methotrexate and methotrexate dichloride, 3-amino-1, 2, 4-benzotriazine-1, 4-dioxide, aminopterin, cytosine β -D-arabinofuranoside, 5-fluoro-5' -deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C), DNA intercalators or crosslinkers (e.g., bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-dichlorodiammineplatinum (II) (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcriptional modulators (e.g., actinomycin D, daunorubicin, doxorubicin, homoharringtonite, and idarubicin). Other exemplary cytostatics compatible with the present disclosure include ansamycins, quinone derivatives (e.g., quinolones, genistein, bacacyclin), busulfan, ifosfamide, nitrogen mustard, dichloromethyl diethylamine, triethyleneimine benzoquinone, disazoquinone, carboxanone, indoloquinone (indoquinone) EO9, diazepinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

Typical cytotoxic nucleoside anticancer agents include, for example, vidarabine, cytarabine, 5-fluorouracil, fludarabine, floxuridine, furacil and 6-mercaptopurine. Typical anti-cancer tubulin binding agents include taxanes (taxoids) (e.g., paclitaxel, docetaxel, taxanes), nocodazole, rhizomycin, dolastatins (e.g., dolastatin-10, -11, or-15), colchicines and colchicinoids (e.g., ZD6126), combretastatins (e.g., combretastatin a-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)). Typical anti-cancer hormones and hormone antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroxyprogesterone acetate), estrogens (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), 17- (allylamino) -17-demethoxygeldanamycin, 4-amino-1, 8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene diphosphonic acid, leuprolide (leuprolide), luteinizing hormone releasing hormone, pimelane-A, rapamycin, sex hormone binding globulin, and thapsigargin. Typical anti-cancer, anti-angiogenic compounds include angiostatin Kl-3, DL-a-difluoromethyl-ornithine, endostatin, fumonisin, genistein, minocycline, staurosporine, and (. + -.) thalidomide.

Typical anti-cancer enzyme inhibitors include, but are not limited to, S (+) -camptothecin, curcumin, (-) -deguelin, 5, 6-dichlorobenzene-imidazole 1- β -D-ribofuranoside, etoposide, fulvestrant, forskocin, hispidin, 2-imino-1-imidazolidineacetic acid (cyclocreatine), meninine, trichostatin a, tyrphostin AG34, and tyrphostin AG 879.

Typical anticancer group modulators include 5-aza-2' -deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin a aldehyde), retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin a), tamoxifen, and troglitazone.

Other preferred classes of anti-cancer agents include, for example, pteridine family drugs, Diynenes, and podophyllotoxins. Particularly effective members of these classes include, for example, methotrexate, podophyllotoxin or podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vindesine, vinblastine and the like.

Still other anti-cancer agents compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristatin E), geldanamycin, calicheamicin, gramicin D, maytansinoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, teniposide, colchicine, dihydroanthridine, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs thereof, or homologs thereof.

Yet other anti-cancer agents compatible with the teachings herein include tomaymycin (tomaymycin) derivatives, maytansine derivatives, cryptophycin derivatives, anthracycline derivatives, diphosphonic acid derivatives, lepomycin derivatives, streptonigrin derivatives, auristatin derivatives, and duocarmycin (duocarmycin) derivatives.

Another class of compatible anti-cancer agents that can be used as drug moieties are radiosensitizing drugs that are effective against tumors or immune-reactive cells. This drug moiety enhances sensitivity to ionizing radiation, thereby increasing the efficacy of radiation therapy. Without being bound by theory, an antibody partially modified by a radiosensitizer and internalized by tumor cells can deliver the radiosensitizer to the vicinity of the nucleus where radiosensitization will be maximized. Antibodies that lose the radiosensitizing moiety are rapidly cleared from the blood, localizing residual radiosensitizer in the target tumor and providing minimal uptake in normal tissues. After removal from the blood, adjuvant radiotherapy can be administered by external beam radiation specific to the tumor, radioactivity implanted directly into the tumor, or systemic radioimmunotherapy with the same modified antibodies.

In one embodiment, the therapeutic agent comprises a radionuclide or radiolabel with high-energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA leading to cell death. Typical high-energy radionuclides include: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188 Re. These isotopes generally produce high energy alpha-or beta-particles with short path lengths. Such radionuclides kill neoplastic cells in close proximity thereto, for example, to which the conjugate attaches or enters. It has little or no effect on the non-localized cells and is essentially non-immunogenic. Alternatively, the high energy isotope may be generated by thermal radiation to other stable isotopes, such as boron neutron capture therapy (Guan et al, PNAS, 95: 13206-.

In one embodiment, the therapeutic agent is selected from MMAE, MMAF and PEG8-Do 110.

Typical therapeutic effector moieties include the following structures:

in one embodiment, the effector moiety is selected from:

in some embodiments, the effector moiety comprises more than one therapeutic agent. These multiple therapeutic agents may be the same or different.

i. Diagnostic effector moiety

In certain embodiments, a binding polypeptide of the present disclosure is conjugated to an effector moiety comprising a diagnostic agent. In one embodiment, the diagnostic agent is a detectable small molecule label such as biotin, a fluorophore, a chromophore, a spin resonance probe, or a radiolabel. Typical fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, and the like) and other luminescent molecules (e.g., luminol). The fluorophore may be environmentally sensitive such that its fluorescence changes if it is located near one or more residues in the modified binding polypeptide that undergo a structural change upon binding to a substrate (e.g., dansyl probe). Typical radiolabels comprise small molecules containing atoms with one or more poorly sensitive nuclei (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In and the like). Preferably, the radionuclide is a gamma, photon or positron emitting radionuclide having a half-life suitable to allow activity or detection after the time elapsed between administration and localization to the imaging site.

In one embodiment, the diagnostic agent is a polypeptide. Typical diagnostic polypeptides include enzymes having fluorescing or chromophoric activity, for example enzymes having the ability to cleave to form fluorophores or chromophores as substrates for products (i.e., reporter proteins such as luciferases). Other diagnostic proteins may have intrinsic fluorescent or chromophoric activity (e.g., green, red and yellow fluorescent bioluminescent jellyfish proteins (aequorin) from bioluminescent marine organisms) or they may comprise proteins containing one or more low energy radionuclides (13C, 15N, 2H, 125I, 124I, 123I, 99Tc, 43K, 52Fe, 64Cu, 68Ga, 111In, etc.).

With respect to the use of the radiolabeled conjugates of the present disclosure, the binding polypeptides of the present disclosure may be directly labeled (e.g., by iodination) or may be indirectly labeled through the use of a chelator. The phrases "indirect labeling" and "indirect labeling means" as used herein both mean the covalent attachment of a chelator to a binding polypeptide and the binding of at least one radionuclide to the chelator. Such chelating agents are generally referred to as bifunctional chelating agents because they bind both polypeptides and radioisotopes. Typical chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetetramine pentaacetic acid ("MX-DTPA") and cyclohexyldiethylenetriamine pentaacetic acid ("CHX-DTPA") derivatives. Other chelating agents include P-DOTA and EDTA derivatives. Particularly preferred radionuclides for indirect labeling include 111In and 90Y. Most imaging studies utilize 5mCi 111In labeled antibody, with this high dose being both safe and having increased imaging efficiency compared to lower doses, with optimal imaging occurring on days three to six after antibody administration. See, for example, Murray, (1985), j.nuc.med.26: 3328and Carragulilo et al, (1985), J.Nuc.Med.26: 67. a particularly preferred radionuclide for direct labeling is 131I. It will be appreciated by those skilled in the art that non-radioactive conjugates may also be assembled depending on the agent selected to be conjugated.

In certain embodiments, the diagnostic effector moiety is a FRET (fluorescence resonance energy transfer) probe. FRET has been used for a variety of diagnostic applications including the diagnosis of cancer. FRET probes may include cleavable linkers (enzyme sensitive or pH linkers) attached to the acceptor moieties of the donor and FRET probes, where cleavage results in enhanced fluorescence (including near infrared) (see, e.g., A. Cobos-Correa et al. Membrane-bound FRET probe visionalizates MMP12 Activity in molecular information, Nature Chemical Biology (2009),5(9), 628-63; S. Gehrig et al, spatialy Resolved Monitoring of neutral enzyme Activity with quantitative fluorescence detectors (2012) Angew. chem. int. Ed.,51, 6258-.

In one embodiment, the effector moiety is selected from:

c. functional effector moieties

In certain embodiments, the effector moieties of the present invention may be functionalized to contain additional groups in addition to the effector moiety itself. For example, the effector moiety may comprise a cleavable linker that releases the effector moiety from the binding polypeptide under specific conditions. In typical embodiments, the effector moiety may comprise a linker cleavable by a cellular enzyme and/or pH sensitive. Additionally or alternatively, the effector moiety may comprise a disulfide bond that is cleaved by intracellular glutathione when taken into the cell. Typical disulfide and pH sensitive linkers are provided below:

in another embodiment, the effector moiety may comprise a hydrophilic and biocompatible moiety such as a poly (glycine), poly (oxazoline) or PEG moiety. A typical structure ("Y") is provided below:

r ═ H, unsubstituted or alkyl-containing functional groups

P and Q ═ identical or different functional groups, useful for linking drugs, reporters and proteins

In certain embodiments, the effector moiety comprises an aminooxy group that facilitates coupling to the binding polypeptide via a stable oxime linker. Typical effectors that include aminooxy groups are set forth in part in table 2 herein.

TABLE 2 typical aminooxy effector moieties (where X can be any linker, Y is any spacer, and where X and/or Y are optional)

The agent herein may be any of the agents of Table 1 herein. Drug 1 and drug 2 may be the same or different drugs.

In other embodiments, the effector moiety comprises a hydrazine and/or an N-alkylated hydrazine group to facilitate coupling to the binding polypeptide via a stable hydrazone linkage. Typical effector moieties comprising aminooxy groups are set forth in table 14 herein.

TABLE 14 exemplary hydrazine and/or hydrazine effector moieties

Conjugation of effector moieties to binding polypeptides

In certain embodiments, the effector moiety is coupled (directly or through a linker moiety) to an oxidized glycan (e.g., an oxidized N-linked glycan) of the altered binding polypeptide (e.g., an engineered glycan at N298 of the antibody Fc domain). The term "oxidized glycan" means that the alcohol substituent on the glycan is oxidized to become a carbonyl substituent. The carbonyl substituent may be reacted with a suitable nitrogen nucleophile to form a carbon-nitrogen double bond. For example, reaction of a carbonyl group with an aminooxy or hydrazine group will form an oxime or hydrazone, respectively. In one embodiment, the carbonyl substituent is an aldehyde. Suitable oxidized glycans include oxidized galactose and oxidized sialic acid.

In one embodiment, the modified polypeptide of formula (II) may be of formula (II):

Ab(Gal-C(O)H)_x(Gal-Sia-C(O)H)_y

a compound of the formula (II),

wherein

A) Ab is an antibody or other binding polypeptide as defined herein;

B) gal is a component derived from galactose;

C) sia is a component derived from sialic acid;

D) x is 0 to 5; and is

E) y is a number of the groups 0 to 5,

wherein at least one of x and y is not 0.

Any art-recognized chemistry can be used to couple an effector moiety (e.g., an effector moiety comprising a linker moiety) to a glycan (see, e.g., Hermanson, g.t., Bioconjugate techniques. In certain embodiments, saccharide residues (e.g., sialic acid or galactose residues) of the glycans are first oxidized (e.g., treated with sodium periodate or galactose oxidase) to generate reactive aldehyde groups. The aldehyde group reacts with an effector moiety aminooxy or hydrazino to form an oxime or hydrazone linker, respectively. Typical methods for applying this general reaction scheme are illustrated in examples 10 to 15.

In certain embodiments, the polypeptide-binding native or engineered glycans are first pre-treated in vitro with glycosyltransferase to provide terminal sugar residues with appropriate reactivity. For example, sialylation may first be achieved using a combination of galactosyltransferase (Gal T) and sialyltransferase (Sial T). In certain embodiments, biantennary glycans lacking galactose (G0F or G0) or comprising only one galactose (G1F or G1) can be converted to hypervalent galactosylated or sialylated structures suitable for coupling (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, or G2S 2).

A typical conjugation scheme for producing sialylated glycoconjugates is shown in figure 25C. A combination of galactosyltransferase (Gal T) and sialyltransferase (Sial T) is used to enzymatically and site-specifically introduce sialic acid residues into the glycans of an antibody (e.g., engineered glycans at N298 of an Fc domain). The introduced sialic acid residues are then oxidized with a low concentration of sodium periodate to produce a reactive sialdehyde having appropriate reactivity with a drug linker (e.g., aminooxy drug linker) to produce an Antibody Drug Conjugate (ADC) (e.g., an oxime-linked ADC). By controlling glycan counts and sialic acid residue counts through in vitro remodeling, one skilled in the art can precisely control the drug-antibody ratio (DAR) of ADCs. For example, if 1 sialic acid is added to a single biantennary glycan (A1F) in each heavy chain, an antibody or binding polypeptide with a DAR of 2 can be uniformly obtained.

Modified binding polypeptides

In certain embodiments, the invention provides modified polypeptides that are the product of coupling (directly or through a linker moiety) an effector moiety to an oxidized glycan (e.g., an oxidized N-linked glycan) of an altered binding polypeptide (e.g., an engineered glycan at N298 of an antibody Fc domain).

In some embodiments of the present invention, the substrate is,

in one embodiment, the binding polypeptide may be of formula (III):

Ab--(Gal-C(H)＝N-Q-CON-X)_x(Gal-Sia-C(H)＝N-Q-CON-X)_y

a compound of the formula (III),

wherein:

A) ab is an antibody as defined herein;

B) q is NH or O;

C) CON is a linker moiety as defined herein; and is

D) X is a therapeutic or diagnostic agent as defined herein;

E) gal is a component derived from galactose;

F) sia is a component derived from sialic acid;

G) x is 0 to 5; and is

H) y is a number of the groups 0 to 5,

wherein at least one of x and y is not 0.

In one embodiment, the binding polypeptide may be of formula (III) may be of formula (IIIa):

Ab(Gal-C(H)＝N-Q-CH₂-C(O)-Z-X)_x(Gal-Sia-C(H)＝N-Q-CH₂-C(O)-Z-X)_y

in the formula (IIIa),

wherein:

A) ab is an antibody;

B) q is NH or O;

C) z is Cys- (MC)_a-(VC)_b-(PABC)_c-(C₁₆H₃₂O₈ C₂H₄)_f-, wherein

Cys is a component derived from cysteine amide;

mc is a component derived from maleimide;

vc is a component derived from valine coupled to citrulline;

PABC is a component derived from 4-aminobenzyl carbamate;

v.X is a therapeutic or diagnostic agent as defined herein;

a is 0 or 1;

b is 0 or 1;

c is 0 or 1; and is

ix.f is 0 or 1;

D) x is a therapeutic agent as defined herein;

E) gal is a component derived from galactose;

F) sia is a component derived from sialic acid;

G) x is 0 to 5; and is

H) y is a number of the groups 0 to 5,

wherein at least one of x and y is not 0.

It should be understood that formula (III) is not intended to imply that the antibody, Gal substituent and Gal-Sia substituent are linked in a chain-like manner. Rather, when these substituents are present, the antibody is directly linked to each substituent. For example, a binding polypeptide of formula (III) wherein x is 1 and y is 2 can have the arrangement shown below:

the CON substituents in formula (III) and components therein are described with reference to formula (I) for the effector moiety.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is 0.

The antibody Ab of formula (III) may be any suitable antibody as described herein.

In one embodiment, a method is provided for preparing a binding polypeptide of formula (III), the method comprising reacting an effector moiety of formula (I):

NH₂-Q-CON-X

formula (I)

Wherein

A) Q is NH or O;

B) CON is a linker moiety; and is

C) X is a therapeutic or diagnostic agent as defined herein,

reacting with a modified antibody of formula (II) as follows:

Ab(OXG)_r

formula (II)

Wherein

A) OXG is an oxidized glycan; and is

B) r is selected from 0 to 4;

in one embodiment, a method is provided for preparing a binding polypeptide of formula (III), the method comprising reacting an effector moiety of formula (I):

NH₂-Q-CON-X

a compound of the formula (I),

wherein:

A) q is NH or O;

B) CON is a linker moiety; and is

C) X is a therapeutic or diagnostic agent as defined herein,

reacting with a modified antibody having the following formula (IIa):

Ab(Gal-C(O)H)_x(Gal-Sia-C(O)H)_y

a compound of the formula (IIa),

wherein

A) Ab is an antibody as described herein;

B) gal is a component derived from galactose;

C) sia is a component derived from sialic acid;

D) x is 0 to 5; and is

E) y is a number of the groups 0 to 5,

wherein at least one of x and y is not 0.

IX. methods of treatment with modified antibodies

In one aspect, the invention provides a method of treating or diagnosing a patient comprising administering an effective amount of a binding polypeptide disclosed herein. Preferred embodiments of the present disclosure provide kits and methods for diagnosing and/or treating disorders, such as neoplastic disorders, in a mammalian subject in need of such treatment. Preferably, the subject is a human.

The binding polypeptides of the present disclosure are very effective in many different applications. For example, in one embodiment, the binding polypeptides described can be used to reduce or eliminate cells bearing an epitope recognized by the binding domain of the binding polypeptide. In another embodiment, the binding polypeptide is effective to eliminate circulating soluble antigen or to reduce the concentration of circulating soluble antigen. In one embodiment, the binding polypeptide reduces tumor size, inhibits tumor growth, and/or prolongs the survival of an animal bearing the tumor. Accordingly, the disclosure also relates to methods of treating tumors in humans or other animals by administering to such humans or animals an effective, non-toxic amount of the modified antibody. One skilled in the art will be able to determine by routine experimentation what an effective, non-toxic amount of the modified binding polypeptide to use for the purpose of treating malignancies. For example, the amount of modified antibody or fragment thereof that is therapeutically active may vary according to factors such as the stage of the disease (e.g., stage I versus stage IV), age, sex, medical complications (e.g., immune suppression status or disease), and the weight of the subject and the ability of the modified antibody to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, multiple divided doses may be administered daily, or the dose may be reduced proportionally as indicated by the exigencies of the therapeutic situation.

In general, the compositions provided in the present disclosure can be used to prophylactically or therapeutically treat any neoplasm that comprises an antigenic marker that allows the modified antibody to target cancer cells.

Method of administering a modified antibody or fragment thereof

Methods of making and administering the binding polypeptides of the disclosure to a subject are well known in the art and can be readily determined by one of skill in the art. The route of administration of the binding polypeptides of the present disclosure may be oral, parenteral, by inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Parenteral administration in intravenous, intra-arterial, subcutaneous and intramuscular forms is generally preferred. While administration of all of these forms is expressly contemplated as within the scope of the present disclosure, the form for administration will be a solution for injection, particularly for intravenous or intra-arterial injection or instillation. Generally, suitable pharmaceutical compositions for injection may include buffers (e.g., acetate, phosphate, or citrate buffers), surfactants (e.g., polysorbates), optional stabilizers (e.g., human albumin), and the like. However, in other methods compatible with the teachings herein, the modified antibody can be delivered directly to the site of the undesirable cell population thereby increasing exposure of the diseased tissue to the therapeutic agent.

In one embodiment, the binding polypeptide administered is a binding polypeptide of the following formula (III):

Ab(Gal-C(H)＝N-Q-CON-X)_x(Gal-Sia-C(H)＝N-Q-CON-X)_y

formula (III)

Wherein:

A) ab is an antibody as defined herein;

B) q is NH or O;

C) CON is a linker moiety as defined herein; and is

D) X is a therapeutic or diagnostic agent as defined herein;

E) gal is a component derived from galactose;

F) sia is a component derived from sialic acid;

G) x is 0 to 5; and is

H) y is a number of the groups 0 to 5,

wherein at least one of x and y is not 0.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol/water solutions, emulsions or suspensions, including saline and buffered media. In the compositions and methods of the present disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the in situ preparation of sterile injectable solutions or dispersions. In these cases, the composition must be sterile and should flow to the extent that easy syringability exists. It should be stable under processing and storage conditions and will preferably be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersants, and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

In any event, sterile injectable solutions can be prepared by incorporating the active compound (e.g., the modified binding polypeptide by itself or in combination with other active agents) in the required amount in a suitable solvent with one or a combination of ingredients enumerated herein, followed by filtered sterilization, if desired. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes and vials, and sealed under sterile conditions according to methods known in the art. In addition, the preparations can be packaged and sold in kit form, such as those described in co-pending U.S. patent nos. 09/259,337 and U.S. patent No. 09/259,338, each of which is incorporated herein by reference. These articles of manufacture will preferably have a label and package insert indicating that the relevant composition is useful for treating a subject suffering from or susceptible to an autoimmune or neoplastic disorder.

The effective dosage of the compositions of the present disclosure for the treatment of the above conditions will vary depending on a number of different factors, including the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, the administration of other drugs, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals including transgenic mammals can also be treated. Therapeutic dosages can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

For passive immunization with a binding polypeptide, the dosage can range, for example, from about 0.0001 to 100mg/kg, and more typically 0.01 to 5mg/kg (e.g., 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, lmg/kg, 2mg/kg, etc.) of the host weight. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight or in the range of 1-10mg/kg, preferably at least 1 mg/kg. Intermediate doses within the above ranges are also intended to be included within the scope of the present disclosure. The subject may administer such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Typical treatment means administration in multiple doses over an extended time course, for example, at least six months. Other typical treatment regimens mean administration once every two weeks or once a month or once every 3 to 6 months. A typical dosage schedule includes 1-10mg/kg or 15mg/kg daily continuously, 30mg/kg every other day or 60mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are used simultaneously, in which case the dose administered for each antibody falls within the indicated ranges.

The binding polypeptides of the present disclosure can be administered in a variety of circumstances. The interval between single doses may be weekly, monthly or yearly. The intervals may be irregular, as indicated by measuring blood levels of the modified binding polypeptide or antigen in the patient. In some methods, the dose is adjusted to achieve a plasma concentration of the modified binding polypeptide of 1-1000 μ g/ml and in some methods 25-300 μ g/ml. Alternatively, the binding polypeptide may be administered as a slow release formulation, in which case less frequent administration is required. For antibodies, the dose and frequency will vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.

The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising an antibody of the invention or cocktail thereof can be administered to a patient not in a disease state to enhance resistance in the patient. Such an amount is defined as a "prophylactically effective dose". In this use, the precise amount will also depend on the health and general immune status of the patient, but will generally be in the range of 0.1 to 25mg per dose, especially 0.5 to 2.5mg per dose. Relatively low doses are administered at relatively infrequent intervals over a long time course. Some patients continue to receive treatment for the remainder of their life. In therapeutic applications, relatively high doses (e.g., about 1 to 400mg/kg antibody per dose, more typically 5 to 25mg doses for radioimmunoconjugates and higher doses for cytotoxic drug-modified antibodies) are sometimes required at relatively short intervals until disease progression is reduced or terminated, and preferably until the patient exhibits partial or complete remission of disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient.

The binding polypeptides of the present disclosure can optionally be administered in combination with other agents that are effective to treat a disorder or condition in need of (e.g., prophylactic or therapeutic) treatment. An effective monotherapeutic dose (i.e., a therapeutically effective amount) of a 90Y-labeled modified antibody of the present disclosure ranges between about 5 to about 75mCi, more preferably between about 10 to about 40 mCi. An effective monotherapeutic non-bone marrow ablative dose range of the 131I-modified antibody is between about 5 and about 70mCi, more preferably between about 5 and about 40 mCi. An effective monotherapeutic ablative dose of the 131I-labeled antibody (i.e., possibly requiring autologous bone marrow transplantation) ranges from about 30 to about 600mCi, more preferably between about 50 and less than about 50 mCi. In conjugation to a chimeric antibody, the effective monotherapeutic non-myeloablative dose of an iodine-131 labeled chimeric antibody is between about 5 and about 40mCi, more preferably less than about 30mCi, due to the longer circulating half-life relative to mouse antibodies. Imaging standards for, for example, 111In labels are typically less than 5 mCi.

Although the binding polypeptide may be administered as described above, it must be emphasized that in other embodiments the binding polypeptide may be administered as a first line therapy to otherwise healthy patients. In such embodiments, the binding polypeptide can be administered to a patient having normal or average red bone marrow reserve and/or to a patient who has never undergone and is not undergoing other treatment. As used herein, the administration of a modified antibody or fragment thereof coupled to or in combination with an adjunctive therapy means the sequential, simultaneous, co-extensive, concomitant or contemporaneous administration or use of the therapies and disclosed antibodies. One skilled in the art will appreciate that the administration or application of the various components of the combination treatment regimen may be timed to enhance the overall effectiveness of the treatment. For example, the chemotherapeutic agent may be administered in a standard, known course of therapy followed by administration of the radioimmunoconjugates of the present disclosure over the next several weeks. In turn, cytotoxins associated with the binding polypeptides can be administered intravenously, followed by external beam irradiation for tumor localization. In another embodiment, the modified binding polypeptide can be administered concurrently with one or more selected chemotherapeutic agents in a single office treatment (office view). One skilled in the art (e.g., an experienced oncologist) can readily identify effective combination treatment regimens based on the selected adjunctive therapy and the teachings of the present specification without undue experimentation.

In this regard it is understood that the combination of binding polypeptide and chemotherapeutic agent may be administered in any order, within any time frame that provides therapeutic benefit to the patient. That is, the chemotherapeutic agent and binding polypeptide can be administered in any order or simultaneously. In selected embodiments, the binding polypeptides of the present disclosure will be administered to a patient who has previously undergone chemotherapy. In other embodiments, the binding polypeptide and the chemotherapeutic treatment are administered substantially simultaneously or concurrently. For example, the binding polypeptide can be administered to a patient while undergoing a course of chemotherapy. In preferred embodiments, the modified antibody will be administered within one year of any chemotherapeutic agent or treatment. In other preferred embodiments, the binding polypeptide will be administered within 10, 8,6, 4 or 2 months of any chemotherapeutic agent or treatment. In other preferred embodiments, the binding polypeptide will be administered within 4, 3, 2, or 1 week of any chemotherapeutic agent or treatment. In other preferred embodiments, the binding polypeptide will be administered within 5, 4, 3, 2 or 1 day of any chemotherapeutic agent or treatment. It should be further understood that the two agents or treatments may be administered to the patient within about a few hours or minutes (i.e., substantially simultaneously).

It is further understood that the binding polypeptides of the present disclosure may be coupled to or used in combination with any chemotherapeutic agent or agents that eliminate, reduce, inhibit or control the growth of neoplastic cells in vivo (e.g., to provide a combined treatment regimen). Typical chemotherapeutic agents compatible with the present disclosure include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate, and prednisone. The combination MOPP of four drugs (mechlorethamine (nitrogen mustard), vincristine (Oncovin)), procarbazine and prednisone) is very effective in treating many types of lymphoma and includes the preferred embodiments of the present disclosure. In anti-MOPP patients, a combination of ABVD (e.g., doxorubicin, bleomycin, vinblastine and dacarbazine), ChIVPP (chlorambucil, vinblastine, procarbazine and prednisone), CABS (lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) may be used. Arnold S.freedman and Lee M.Nadler, Malignant Lymphomas, in 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 for standard dosing and scheduling. These therapeutic agents may be used unchanged or may be altered according to the needs of a particular patient, in combination with one or more binding polypeptides of the disclosure as described herein.

Other regimens useful in the context of the present disclosure include the administration of a monoalkylating agent, e.g., cyclophosphamide or chlorambucil, or a combination such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and adriamycin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), proace-MOPP (prednisone, methotrexate, adriamycin, cyclophosphamide, etoposide and leucovorin plus standard MOPP), proce-cytabo (prednisone, adriamycin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate and leucovorin), and MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, fixed doses of prednisone, bleomycin and leucovorin). The standard dosage and timing for each of these regimens can be readily determined by one skilled in the art. CHOP can also be 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' -deoxyhelpesmycin, and fludarabine.

Rescue treatment may be used for patients with moderate and high NHL who fail to achieve remission or relapse. Rescue therapy uses drugs such as cytosine arabinoside, carboplatin, cisplatin, etoposide, and ifosfamide, administered alone or in combination. In the recurrent or aggressive (aggressive) forms of certain neoplastic diseases, the following protocol is often used: IMVP-16 (ifosfamide, methotrexate, and etoposide), MIME (methyl-gag, ifosfamide, methotrexate, and etoposide), DHAP (dexamethasone, high-dose cytarabine and cisplatin), ESHAP (etoposide, methylprednisolone, HD cytarabine, cisplatin), cepp (b) (cyclophosphamide, etoposide, procarbazine, prednisone, and bleomycin), and CAMP (lomustine, mitoxantrone, cytarabine, and prednisone), each using known dose ratios and timing schedules.

The amount of chemotherapeutic agent to be used in combination with the modified antigens of the present disclosure may vary from subject to subject or may be administered according to methods known in the art. See, for example, Bruce A. Chabner et al, Antineoplastic Agents, inodoman & GILMAN' S THE PHARMACOLOGICAL BASIS OF THERAPEUTIC 1233-1287(Joel G. Hardman et al, eds.,9th ed. 1996).

As previously discussed, the binding polypeptides of the present disclosure, immunoreactive fragments thereof, or combinations thereof can be administered in a pharmaceutically effective amount for the in vivo treatment of a disorder in a mammal. In this regard, it is understood that the disclosed binding polypeptides will be formulated to facilitate administration and to promote stability of the active agent.

Preferably, the pharmaceutical compositions of the present invention include pharmaceutically acceptable, non-toxic, sterile carriers such as physiological saline, non-toxic buffers, preservatives, and the like. For the purposes of the present invention, a pharmaceutically effective amount of a modified binding polypeptide, immunoreactive fragment thereof, or combination thereof (conjugated or unconjugated to a therapeutic agent) will mean an amount sufficient to achieve effective binding to the antigen and to achieve a benefit, such as improvement of symptoms of a disease or disorder or detection of a substance or cell. In the case of tumor cells, the modified binding polypeptide will preferably be capable of interacting with a selected immunoreactive antigen on a neoplasm or immunoreactive cell and causing increased death of such cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified binding polypeptide.

In keeping with the scope of the disclosure, the binding polypeptides of the disclosure may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect in accordance with the methods of treatment described previously. The binding polypeptides of the present disclosure can be administered to these humans or other animals in conventional dosage forms prepared by combining the antibodies of the present disclosure with conventional pharmaceutically acceptable carriers or diluents according to known techniques. One skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent are determined by the amount of active ingredient with which it will be combined, the route of administration, and other known variables. One skilled in the art will further appreciate that cocktail mixtures comprising one or more of the binding polypeptide species described in the present disclosure can prove particularly effective.

V. expression of binding Polypeptides

In one aspect, the invention provides polynucleotides encoding the binding polypeptides disclosed herein. Also provided are methods of making binding polypeptides comprising expression of these polynucleotides.

Polynucleotides encoding the binding polypeptides disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce the desired number of claimed antibodies and fragments thereof. Accordingly, in certain aspects, the invention provides expression vectors comprising the polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.

The term "vector" or "expression vector" as used herein means, for the purposes of the specification and claims, a vector used according to the present invention as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, these vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to replicate into and/or in eukaryotic or prokaryotic cells.

A variety of expression vector systems can be employed for purposes of the present invention. For example, one type of vector employs DNA elements derived from an animal virus, such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV), or SV40 virus. Others include the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow the transfected host cells to be selected. The marker may provide prototrophy, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper to an auxotrophic host. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Other elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers, and termination signals. In a particularly preferred embodiment, the cloned variable region genes are inserted into an expression vector together with a composition of heavy and light chain constant region genes (preferably human) as described above.

In other preferred embodiments, the binding polypeptides of the invention may be expressed using a polycistronic construct. In such expression systems, multiple gene products of interest, such as heavy and light chains of an antibody, can be generated from a single polycistronic construct. These systems advantageously use an Internal Ribosome Entry Site (IRES) to provide relatively high levels of the polypeptides of the invention in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. patent 6,193,980, which is incorporated herein by reference. One skilled in the art will appreciate that such an expression system can be used to efficiently produce the full range of polypeptides disclosed herein.

More generally, once a vector or DNA sequence encoding the antibody or fragment thereof has been prepared, the expression vector may be introduced into a suitable host cell. That is, the host cell may be transformed. Introduction of the plasmid into the host cell can be accomplished by a number of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection with intact viruses. See, Ridgway, A.A.G. "Mammalian Expression Vectors" Chapter 24.2, pp.470-472 Vectors, Rodriguezand Denhardt, Eds. (Butterworks, Boston, Mass.1988). More preferably, the plasmid is introduced into the host via electroporation. The transformed cells were grown under conditions suitable for light and heavy chain production, and tested for synthesis of heavy and/or light chain proteins. Typical experimental techniques include enzyme-linked immunosorbent assays (ELISA), Radioimmunoassays (RIA) or Fluorescence Activated Cell Sorting (FACS), immunohistochemistry, and the like.

The term "transformation" is used herein in its broadest sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and, in turn, results in alteration of the recipient cell.

In these same cell lines, "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA techniques and encoding at least one heterologous gene. In the description of the methods for isolating polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody unless clearly indicated otherwise. That is, recovery of the polypeptide from "cells" can refer to recovery from centrifuged whole cells or from cell cultures that include both culture media and suspension cells.

In one embodiment, the host cell line used for antibody expression is of mammalian origin; one skilled in the art will be able to determine the particular host cell line best suited for the desired gene product to be expressed therein. Typical host cell lines include, but are not limited to, DG44 and DUXB11 (chinese hamster ovary cell line, DHFR-), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (derivative of CVI with SV40T antigen), R1610 (chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), 293 (human kidney). In one embodiment, the cell line provides altered glycosylation of the antibody expressed therefrom, e.g., afucosylation (e.g., per. c6.rtm. (brucell) or FUT8 knockout CHO cell lines (potelligent. rtm. cells) (Biowa, Princeton, n.j.). in one embodiment, NS0 cells may be used.

In vitro production allows scale-up to provide large quantities of the desired polypeptide. Techniques for mammalian cell culture under tissue culture conditions are well known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or a continuous stirred reactor, or fixed or embedded cell culture, e.g., in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges. If necessary and/or desired, the polypeptide solution can be purified by chromatography methods using, for example, gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose and/or (immuno) affinity chromatography.

The gene encoding the binding polypeptide of the invention may also be expressed in a non-mammalian cell, such as a bacterial or yeast or plant cell. In this regard, it is understood that a variety of unicellular non-mammalian microorganisms, such as bacteria, may also be transformed; i.e. those capable of growing in culture or fermentation. Bacteria susceptible to transformation include members of the family Enterobacteriaceae (Enterobacteriaceae), such as Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); strains of the genera Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus fluuenzae). It is further understood that the polypeptide may become part of an inclusion body when expressed in bacteria. Polypeptides must be isolated, purified, and then assembled into functional molecules.

In addition to prokaryotic cells, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although several other strains are also frequently available. For expression in Saccharomyces (Saccharomyces), for example, the plasmid YRp7(Stinchcomb et al, Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper et al, Gene, 10: 157(1980)) is often used. The plasmid already contains the TRP1 gene which provides a selection marker for mutant yeast lacking the ability to grow in tryptophan, for example ATCC No.44076 or PEP4-1(Jones, Genetics, 85: 12 (1977)). the trpl lesion is present as a characteristic of the yeast host cell genome, which in turn provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Examples

The invention is further illustrated by the following examples, which are to be regarded as illustrative rather than restrictive. The contents of the sequence listing, drawings, and all references, patents, and published patent applications throughout this application are expressly incorporated herein by reference.