ES2365924T3 - ANTIBODIES FOR THE ERYTHROPOYETIN RECEIVER AND USES THEREOF. - Google Patents

Abstract

An isolated antibody, or an antigen-binding portion thereof, that dissociates from the human erythropoietin receptor (EpoR) with a Koff rate constant greater than 1.3 x 10 -3 s -1 and that activates endogenous activity of said human EpoR in a mammal, said antibody or antigen-binding portion thereof comprising a heavy chain variable region (HCVR) and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of Formula I: QVQLQESGPGLVKPSE TLSLTCTVSGASISSY YWSWIRQPPGKGLEWI GYI-X1-X2-X3-GSTNYNPSLK SRVTISVDTSKNQFSL KLRSVTAADTAVYYCA RETLGIGDYS: GVLSS: GURS: (TO); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G) and alanine (A), glutamine (E), and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E) and threonine (T) with the proviso that X1 -X2 -X3 is other than YYS, and the chain variable region light comprises an amino acid sequence of SEQ ID NO: 17.

Description

Background of the Invention

Erythropoietin ("Epo") is a glycoprotein that is the main regulator of erythropoiesis. Specifically, Epo is responsible for promoting the growth, differentiation, and survival of erythroid progenitors, resulting in mature red blood cells. In response to changes in the level of oxygen in the blood and tissues, erythropoietin appears to stimulate both proliferation and differentiation of immature erythroblasts. It also acts as a growth factor, stimulating the mitotic activity of erythroid progenitor cells, such as colony-forming and erythroid burst-forming units. It also acts as a differentiation factor, triggering the transformation of an erythrocyte colony-forming unit into a proerythblast (See Erslev, A., New Eng. J. Med., 316: 101-103 (1987)).

Epo has a molecular weight of approximately 34,000 Daltons and can appear in three forms - alpha, beta, and asialo. During the middle and late stages of pregnancy, Epo is synthesized in the fetal liver. Subsequently, Epo is synthesized in the kidney, circulates in plasma, and is excreted in urine.

Human urinary Epo has been isolated and purified (see, Miyake et al., J. Biol. Chem., 252: 5558 (1977)). In addition, methods are known in the art for identifying, cloning, and expressing genes that encode Epo (see US Patent 4,703,008) as well as purify recombinant Epo from a cellular medium (see US Patent 4,667. 016).

Epo activity is mediated by the binding and activation of a cell surface receptor called erythropoietin receptor (EpoR). The Epo receptor belongs to the cytokine receptor superfamily and is believed to contain at least two distinct polypeptides, a 55-72 kDa species and an 85-100 kDa species (see US Patent 6,319,499, Mayeux et al., J. Biol. Chem, 266: 23380 (1991), McCaffery et al., J. Biol. 6,319,499, Mayeux et al., J. Biol. Chem, 266: 23380 (1991), McCaffery et al., J. Biol. Chem., 264: 10507 (1991)). Other studies have revealed other Epo receptor polypeptide complexes having molecular weights such as 110, 130, and 145 kDa (see US Patent 6,319,499).

Both murine and human Epo receptors have been cloned and expressed (see D'Andrea et al., Cell, 57: 277 (1989); Jones et al., Blood, 76: 31 (1990); Winkelmarm et al., Blood, 76: 24 (1990); WO 90/08822 / US Patent 5,278,065). The full-length human Epo receptor is a 483 amino acid transmembrane protein with a signal peptide of approximately 25 amino acids (see US Patent 6,319,499). The human receptor demonstrates approximately 82% amino acid sequence homology to the murine receptor. Same reference.

In the absence of ligand, the Epo receptor exists in a preformed dimer. The binding of Epo to its receptor causes a conformational change so that the cytoplasmic domains are placed in close proximity. Although not fully understood, this "dimerization" is believed to play a role in receptor activation. Activation of the Epo receptor results in various biological effects. Some of these activities include stimulation of proliferation, stimulation of differentiation, and inhibition of apoptosis (see US Patent 6,319,499, Liboi et al., PNAS USA, 90: 11351 (1993), Koury, Science: 248: 378 (1990)).

It is the relationship between Epo receptor dimerization and activation that can be used to identify compounds (ie, such as antibodies) other than Epo that are capable of: (1) dimerizing the Epo receptor; and (2) activate the receiver. These compounds would be useful in the treatment of mammals suffering from anemia and in the identification of mammals having a dysfunctional Epo receptor.

WO 96/40231 describes a method of generating antibodies that are receptor agonists. For example, an Epo agonist antibody is described that can be used in the treatment of anemia.

WO 99/55369 describes antibodies having reduced immunogenicity in humans and methods for their preparation comprising CDR grafts of donor antibodies from a non-human species. For example, the preparation of an anti-Epo receptor antibody by CDR-grafting of a murine donor antibody into a non-human primate acceptor antibody is described.

WO 97/048729 describes an antibody that activates Epo receptors and is useful for treating disorders related to Epo receptor activation. For example, the MAb34 antibody is described as capable of binding to the Epo receptor and exhibits a Koff of 1.7 x 10-2 s-1.

WO 04/035603 refers to antibodies and antibody fragments that bind and activate a receptor

Epo, such as antibody Ab 198 having a Kd of 13.9 nM.

Summary of the invention

The invention provides an isolated antibody, or an antigen-binding portion thereof, that dissociates from the human erythropoietin receptor (EpoR) with a Koff rate constant greater than about 1.3 x 10-3 s-1 and that activates an endogenous activity of said EpoR in a mammal, said antibody or antigen binding portion thereof comprising a heavy chain variable region (HCVR) and a light chain variable region, wherein the heavy chain variable region comprises a sequence of amino acids of Formula I:

Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15)

wherein X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G), alanine (A), glutamine (E), and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine (T) provided that X1-X2-X3 is other than YYS, and the variable region of the chain Light comprises an amino acid sequence of SEQ ID NO: 17. The antibodies of the invention are characterized by binding to EpoR with low affinity and dissociation of human erythropoietin receptor (EpoR) with a fast dissociation rate. Antibodies or antigen-binding portion thereof may be full length (eg, an IgG2) or may comprise only one antigen-binding portion (eg, an F (ab ') 2). In a preferred embodiment, the antibodies of the invention bind to EpoR with a Kd of approximately 7 nM

or older. In a more preferred embodiment, the antibody or antigen-binding portion thereof binds to EpoR with a Kd of approximately 8.5 nM or greater. In an even more preferred embodiment, the antibody or antigen-binding portion thereof binds EpoR with a Kd of approximately 20 nM and, more preferably, with a Kd of approximately 32 nM.

In a more preferred embodiment, the antibody or antigen-binding portion thereof dissociates from EpoR with a Koff rate constant selected from the group consisting of about 1.4 x 10-3 s-1 or greater, about 1, 9 x 10-3 s-1 and approximately 4.8 x 10-3 s-1 as determined by surface plasmon resonance.

The antibody or antigen-binding part thereof may be a monoclonal antibody. In a preferred embodiment, the antibody or antigen-binding portion thereof is an IgG2 isotype. In a further embodiment, the antibody or antigen-binding portion thereof is a human antibody.

In another embodiment, the antibody or antigen-binding portion thereof has a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

Another aspect of the invention relates to the antibody or antigen-binding portion thereof of the present disclosure to activate endogenous activity of a human erythropoietin receptor to treat anemia, hypoxemia, chronic tissue hypoxia, heart failure, ischemic heart disease, failure renal, neural cell damage, or neural tissue damage, or to promote wound healing in a mammal, by administering to the mammal a therapeutically effective amount of the antibody or antigen-binding portion thereof.

In another aspect, the invention relates to the antibody or antigen-binding portion thereof of the present disclosure for treating a mammal suffering from aplasia, by administration to the mammal in need of treatment of a therapeutically effective amount of the antibody or binding portion to antigen thereof.

In another aspect, the invention relates to the antibody or antigen-binding part thereof of the present disclosure for treating a mammal suffering from anemia, by administration to the mammal in need of treatment of a therapeutically effective amount of the antibody or binding part to antigen thereof.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding portion thereof of the invention and a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a purified or isolated polynucleotide sequence encoding a

polypeptide comprising an amino acid sequence of Formula I:

Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15)

wherein X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G), alanine (A), glutamine (E), and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine (T) provided that X1-X2-X3 is other than Y-Y-S. In other preferred embodiments, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: : 12, SEQ ID NO: 13 and SEQ ID NO: 14.

In another aspect, the invention relates to recombinant expression vectors comprising the polynucleotides set forth herein. The invention also relates to a host cell comprising the recombinant expression vectors. Preferably, the host cell is a eukaryotic cell, mammalian cell, yeast cell, or bacterial cell. More preferably, the host cell is a CHO cell, COS cell, or HEK-293 cell.

In another aspect, the invention relates to polypeptide sequences encoded by said polynucleotide sequences set forth herein.

Brief description of the drawings

FIG. 1 represents a schematic drawing of a scFv construct, including scFv and linkers.

FIG. 2 is a graph showing the equilibrium binding of soluble EpoR with various Ab12 scFv constructs expressed on the surface of yeast cells. In FIG. 2, - ■ -represents an Ab12 scFv construct comprising a Gly / Ser (WT) linker, GGGGSGGGGSGGGGS (SEQ ID NO: 1) at both the scFv linker and binding positions, - ● -represents a construct of Ab12 scFv comprising linker 41, GENKVEYAPALMALS (SEQ ID NO: 2) in the binding position and the Gly / Ser (WT) linker (SEQ ID NO: 1) in the position of the scFv linker, - ▲ -represents an Ab12 scFv construct comprising linker 41, (SEQ ID NO: 2) in the binding position and linker 40, GPAKELTPLKEAKVS (SEQ ID NO: 3) in the position of the scFv linker, and - ▼ -represents an Ab12 scFv construct comprising linker 41 (SEQ ID NO: 2) in the binding position and linker 34, GHEAAAVMQVQYPAS (SEQ ID NO: 4) in the position of the scFv linker.

FIG. 3 represents a 41/40 scFv dissociation rate analysis of Ab12.

FIG. 4 shows a schematic representation of the construction method for generating CDR mutagenic libraries in yeast.

FIG. 5 shows a schematic representation of Ab12 scFv heavy chain CDR mutagenic libraries. Library names are indicated to the left of each 3 amino acid sequence subjected to random selection. Ab12 CDR sequences are shown under each CDR.

FIG. 6 shows a schematic representation of Ab12 scFv light chain CDR mutagenic libraries. Library names are indicated to the left of each 3 amino acid sequence subjected to random selection. Ab12 CDR sequences are shown under each CDR.

FIG. 7 is a graph showing the amino acid sequences of the germline heavy chain variable regions from which Ab12.6 and Ab12.6 related antibodies were obtained (SEQ ID NO: 5), Ab12 (SEQ ID : 6), Ab12.6 (SEQ ID NO: 7), Ab12.56 (SEQ ID NO: 8), Ab12.118 (SEQ ID NO: 9), Ab12.119 (SEQ ID NO: 10), Ab12 .120 (SEQ ID NO: 11), Ab12.121 (SEQ ID NO: 12), Ab12.122 (SEQ ID NO: 13), Ab12.123 (SEQ ID NO: 14) and a consensus sequence (SEQ ID NO: : fifteen).

FIG. 8 is a graph showing the amino acid sequences of the germline light chain variable regions (SEQ ID NO: 16) from which Ab12.6 and antibodies related to Ab12.6, Ab12 were obtained (SEQ ID No.: 17), Ab12.6 (SEQ ID NO: 17) and Ab12.56 (SEQ ID NO: 17), Ab12.118 (SEQ ID NO: 17), Ab12.119 (SEQ ID NO: 17), Ab12 .120 (SEQ ID NO: 17), Ab12.121 (SEQ ID NO: 17), Ab 12.122 (SEQ ID NO: 17), Ab12.123 (SEQ ID NO: 17).

FIG. 9 (a) - (i) shows the nucleic acid sequences of the heavy chain variable regions of Ab12, Ab12.6 and Ab12.6 related antibodies. The one letter codes representing the amino acids encoded by the nucleic acid sequences are shown at the top.

FIG. 10 shows the nucleic acid sequences of the light chain variable region of Ab12, Ab12.6 and Ab12.6 related antibodies. The one letter codes representing the amino acids encoded by the nucleic acid sequences are shown at the top.

FIG. 11 shows a graph of the EC50 and Emax values of Ab12.6 and antibodies related to Ab12.6. EC50 values represent the concentration of antibody or Epo at which 50% of maximum cell proliferation is achieved (50% of the slope of a sigmoid curve). The Emax values represent the maximum number of cells that this EC50 produces (as measured by absorbance).

FIG. 12 is a graph showing the formation of CFU-E (erythroid colony forming units) from human bone marrow in response to treatment with Epogen, Ab12, Aranesp ™, Ab12.6 and isotype control.

FIG. 13 is a graph showing the formation of CFU-E (erythroid colony forming units) from cells derived from transgenic mouse bone marrow mEpoR - / -, hEpoR + in response to treatment with Epogen, Ab12, Aranesp ™, Ab12.6 and isotype control.

FIG. 14 is a graph showing the change in hematocrit in mEpoR - / -, hEpoR + transgenic mice for 28 days after administration of a single dose of Ab12.6 on day 0 versus two doses of Aranesp ™ administered on day 0 and day 14. In FIG. 14, - ♦ -represents absence of treatment, - ■ represents isotype control, - ▲ -represents Aranesp ™ (3 μg / kg, 2x), -x-represents Ab12 (0.8 mg / kg) and -I-represents Ab12.6 (0.8 mg / kg).

FIG. 15 is a computer generated scan of a Western blot showing that Ab12.6 interacts with the recombinant EpoR extracellular domain only under native and non-denaturing conditions, indicating that Ab 12.6 recognizes a conformation-dependent epitope.

FIG. 16 is a graph showing that the Ab12.6-derived monomeric Fab fragment activates EpoR and stimulates proliferation of the human erythroleukemic cell line F36e.

Detailed description of the invention

The invention relates to isolated human antibodies or antigen-binding portions thereof, which bind human erythropoietin with low affinity, rapid dissociation rate, and activation or agonist activity for EpoR. Various aspects of the invention relate to antibodies and antibody fragments and pharmaceutical compositions thereof, as well as to nucleic acids, recombinant expression vectors and host cells to prepare such antibodies and fragments. Also included by the invention is the use of the antibody or antigen-binding portion thereof of the present disclosure to activate endogenous activity of a human erythropoietin receptor to treat anemia, hypoxemia, chronic tissue hypoxia, heart failure, ischemic heart disease, kidney failure, neuronal cell damage or tissue damage, to promote wound healing in a mammal, to treat aplasia or to treat anemia. Unless otherwise defined herein, the scientific and technical terms used in connection with the present invention will have the meanings commonly understood by those skilled in the art. Also, unless otherwise required by context, singular terms will include plurals and plural terms will include singular. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "include" as well as other forms such as "includes" and "included" is not limiting. In addition, terms such as "element" or "component" include both elements and components that comprise a unit and elements and components that comprise more than one subunit unless specifically indicated otherwise.

Generally, the nomenclatures used in connection with, and the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the technique. The methods and techniques of the present invention are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. another thing. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as is usually the case in the technique.

or as described in this document. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those that are well known and commonly used in the art. Conventional techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.

In order that the present invention may be more easily understood, certain terms are first defined.

The term "antibody" (abbreviated herein as Ab), as used herein, is understood to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains, and two interconnected light (L) chains by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a variable light chain region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariability regions, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed flanking regions (FR). Each VH and VL is composed of three CDRs and four FRs respectively, arranged from the amino terminus to the carboxy terminus in the following order. FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (sometimes called "I").

Furthermore, the term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments as long as they show activity. Biological desired.

The term "antigen binding part" of an Ab (or simply "antibody part"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen ( for example, human EpoR). It has been shown that the antigen binding function of an Ab can be performed by fragments of a full length Ab. Examples of binding fragments included within the term "antigen binding part" of an Ab include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, (ii) an F fragment (ab ') 2, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an Ab, (v) a dAb fragment (Ward et al. (1989) Nature 341: 544-546), consisting of a VH domain; and (vi) an isolated CDR. Furthermore, although the two Fv fragment domains, VL and VH, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be constructed as a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423426; and Huston et al. (1988) Proc. Natl. Acad. Sci. that such single-chain Abs are included within the term "antigen-binding portion" of an Ab. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies in which the domains VH and VL are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing domains to pair with complement domains aria from another chain and creating two antigen binding sites (see, eg, Holliger P., et al. (1993) Proc. Natl. Here. Sci. USA 90: 6444-6448; Poljak, R.J. et al. (1994) Structure 2: 1121-1123).

Furthermore, an antibody or antigen-binding part thereof may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or part of the antibody with one or more additional proteins or peptides. Examples of such immunoadhesion molecules include the use of the central streptavidin region to compose a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and the use of a residue of cysteine, a marker peptide, and a C-terminal polyhistidine marker to compose biotinylated and bivalent scFv molecules (Kipriyanov, SM, et al., (1994) Molecular Immunology 31: 1047-1058). Antibody portions, such as Fab and F (ab ') 2 fragments, can be prepared from whole antibodies using conventional techniques, such as digestion with papain or pepsin, respectively, of full length Ab. Furthermore, Ab, parts of Ab, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

The term "human antibody", as used herein, is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or specific mutagenesis in vitro or by somatic mutation in vivo), eg, in CDRs, and, in particular, in CDR2. However, the term "human antibody" as used herein is not intended to include antibodies in which germline derived CDR sequences from another mammalian species, such as a mouse, have been grafted into sequences flanking human.

The term "recombinant antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell. (further described in Section II, below), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15: 62-70; Azzazy H., and Highsmith WE (2002) Clin. Biochem . 35: 425-445; Gavilondo JV, and Larrick JW (2002) Bio Techniques 29: 128-145, Hoogenboom H., and Chames P. (2000) Immunology today 21: 371-378), antibodies isolated from an animal ( eg, a mouse) that is transgenic for human immunoglobulin genes (see, eg, Taylor, Ld, et al. (1992) Nucl. Acids Res.

20: 6287-6295; Kellermann S-A., And Green L.L. (2002) Current Opinion in Biotechnology 13: 593-597; Little M. et al (2000) Immunology Today 21: 364-370) or antibodies prepared, expressed, created or isolated by any other means involving splicing of immunoglobulin gene sequences into other DNA sequences. Such recombinant antibodies have variable and / or constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and VL regions. of the recombinant antibodies are sequences that, although derived from and related to germline VH and VL sequences, may not exist naturally within the germline repertoire of the antibody in vivo.

An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially without other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds EpoR is substantially free of antibodies that specifically bind to antigens other than EpoR). An isolated antibody that specifically binds EpoR, may, however, have cross-reactivity with other antigens, such as EpoR molecules from other species. Furthermore, an isolated antibody can be substantially without other cellular material and / or chemical compounds.

An "activating or agonist antibody" or "activating antibody" or antibody having "activating or agonistic capacity" is intended to refer to an antibody whose binding to EpoR results in stimulation or activation of the biological activity of EpoR. This biological activity can be assessed by measuring one or more indicators of EpoR biological activity including, but not limited to, antibody-induced proliferation of an Epo-responsive cell line, and antibody-induced changes in reticulocyte count, percent hematocrit and / or the binding of the antibody to Epo receptors. These indicators of biological activity of EpoR can be evaluated by one or more of several conventional in vitro or in vivo assays well known to those skilled in the art.

The term "chimeric antibody" refers to antibodies comprising light and heavy chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine light and heavy chain variable regions linked to constant regions. human.

The term "CDR-grafted antibody" refers to antibodies that comprise heavy and light chain variable region sequences of a species but in which the sequences of one or more of the VH and / or VL CDR regions are replaced with sequences CDRs from another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (eg, CDR3) have been replaced with human CDR sequences.

The term "humanized antibody" refers to antibodies that comprise heavy and light chain variable region sequences from a non-human species (eg, a mouse) but in which at least part of the VH and / or VL sequence is it has altered to be more "human-like," that is, more similar to the variable sequences of the human germline. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences have been introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. The means of making chimeric, CDR-grafted, and humanized antibodies are known to those of skill in the art (see, for example, US Patent Nos.

4,816,567 and 5,225,539). One method of making human antibodies employs the use of transgenic animals, such as a transgenic mouse. These transgenic animals contain a substantial part of the human antibody-producing genome inserted into their own genome, and the production of endogenous antibodies from the animal itself becomes deficient in the production of antibodies. Methods for obtaining such transgenic animals are known in the art. Such transgenic animals can be obtained using XenoMouse ™ technology or using a "minilocus" approach. Methods for obtaining Xenomice ™ are described in US Patent Nos. 6,162,963, 6,150,584, 6,114,598, and 6,075,181. Methods for obtaining transgenic animals using the "minilocus" approach are described in US Patents 5,545,807, 5,545,806 and 5,626,825. See also International Publication No. WO93 / 12227.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that enables the analysis of biospecific interactions in real time by detecting alterations in protein concentrations within a biosensing matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, New Jersey). For additional descriptions, see Example 8 and Jonsson, U., et al., (1993) Ann. Biol. Clin. 51. 19-26; Jonsson, U. et al. (1991) Biotechniques 11: 620-627; Johnsson,

B. et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

The term "Koff", as used herein, is intended to refer to the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex.

The term "Kon", as used herein, is intended to refer to the association constant of an antibody to an antigen.

The term "Kd", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. The value of Kd can be obtained by the following equation: Kd (M) = Koff (1 / s) / Kon (1 / M · s).

The term "polypeptide", as used herein, refers to any polymeric chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term polypeptide and also refer to a polymeric chain of amino acids. The term "polypeptide" includes proteins, protein fragments, and analogs of native or artificial polypeptides of a protein sequence. A polypeptide can be monomeric or polymeric.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; it lacks substantially other proteins of the same species; it is expressed by a cell of a different species; or does not appear in nature. Therefore, a polypeptide that is chemically synthesized or synthesized in a different cellular system than the cell from which it is naturally derived will be "isolated" from its naturally associated components. A protein can also be made to be substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

The term "recovery", as used herein, refers to the process of making a chemical species such as a polypeptide substantially free of naturally associated components for isolation, for example, using protein purification techniques well known in the art. matter.

The term "endogenous EpoR activity", as used herein, refers to each and every one of the inherent biological properties of the erythropoietin receptor that occurs as a consequence of binding of a natural ligand. The biological properties of EpoR include, but are not limited to, survival, differentiation, and proliferation of hematopoietic cells, an increase in the production of red blood cells, and an increase in hematocrit in vivo.

The terms "specific binding" or "specifically binding", as used herein, in reference to the interaction of an antibody, protein or peptide with a second chemical species, means that the interaction is dependent on the presence of a particular structure (eg, an antigenic determinant or epitope) in the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins in general. If an antibody is specific for the "A" epitope, the presence of a molecule containing the A (or free unlabelled A) epitope in a reaction containing the labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "epitope" includes any polypeptide determinant capable of specifically binding to an immunoglobulin or T-lymphocyte receptor. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl. and, in certain embodiments, they may have specific three-dimensional structural characteristics and / or specific load characteristics. An epitope is a region of an antigen that is bound to an antibody. In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

The term "polynucleotide", as used herein, means a polymeric form of two or more nucleotides, ribonucleotides, or deoxyribonucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms of DNA, but is preferably double-stranded DNA.

The term "isolated polynucleotide", as used herein, shall mean a polynucleotide (eg, of genomic, cDNA, or synthetic origin, or some combination thereof) such that, by virtue of its origin, the " isolated polynucleotide ”: not associated with all or part of a polynucleotide with which the“ isolated polynucleotide ”occurs in nature; it is operably linked to a polynucleotide to which it is not naturally bound; or it does not appear in nature as part of a larger sequence.

The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and therefore replicate along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are frequently in the form of plasmids. In the present specification, "plasmids" and "vector" can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention seeks to include other forms of expression vectors such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a vector has been introduced (eg, plasmid or recombinant expression vector) . It should be understood that such terms are intended to refer not only to the particular cell, but also to the offspring of successive generations due to mutation or environmental influences. Such offspring may, in fact, not be identical to the parent cell, but still fall within the scope of the term "host cell" as used herein.

The term "ligand" refers to any chemical moiety capable of binding to a polypeptide. Preferably a ligand is an antigen. Antigens can possess one or more epitopes. The ligands for a first polypeptide sequence and a second polypeptide sequence may be the same or different.

A "linker sequence" is a polypeptide sequence that connects two or more polypeptide sequences. The term "connect" refers to the binding of polypeptide sequences. The polypeptide sequences are preferably linked by peptide bond.

I. Antibodies that Bind to Human EpoR

The invention provides an isolated antibody, or an antigen-binding portion thereof, that dissociates from the human erythropoietin receptor (EpoR) with a Koff rate constant of more than about 1.3 × 10-3 s-1 and that activates an endogenous activity of said human EpoR in a mammal, said antibody or antigen binding part thereof comprising a heavy chain variable region (HCVR) and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of Formula I:

Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15)

wherein X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G), alanine (A), glutamine (E), and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine (T) provided that X1-X2-X3 is other than YYS, and the variable region of the chain Light comprises an amino acid sequence of SEQ ID NO: 17. The most preferred recombinant activation antibody of the invention is referred to herein as Ab12.6. The binding properties of Ab12.6 and Ab12.6 related antibodies, all of which are "EpoR activating antibodies", are summarized in Example 8 below.

The anti-EpoR antibody, and related antibodies, also show a strong ability to activate the biological activity of EpoR, as evaluated by various in vitro and in vivo assays (see Examples 9-13). For example, these antibodies activate EpoR in UT-7 / Epo cells with EC50 values in the range of about 0.34 nM to 1,345 nM. Ab 12.6 activates EpoR in UT-7 / Epo cells with an EC50 of 0.85 nM. Furthermore, the activation capacity of the antibodies of the invention is maintained when the antibody is expressed as a Fab, F (ab ') 2 or scFv fragment. Furthermore, such antibodies induce an increase in the percentage of hematocrit in mammals expressing human EpoR.

With respect to the binding specificity of Ab12.6 and variants thereof, this antibody binds to human EpoR in various ways, including soluble EpoR and transmembrane EpoR. Neither Ab12.6 nor its variants specifically bind to other cytokine receptors.

It is understood in the art that there may be some variability (for example, up to ± 20%) in the calculation of the EC50, Koff and Kon values based on the variation of the instruments and the experimental design. Typically, such measurements are made using duplicate or triplicate samples to minimize variability. Furthermore, said antibody, or antigen-binding portion thereof, binds in a manner sufficient to activate human EpoR as demonstrated by a conventional in vitro proliferation assay.

More preferably, the isolated antibody, or antigen-binding portion thereof, dissociates from human EpoR with a dissociation rate (Koff) of approximately 1.4 x 10-3 s-1 or greater, more preferably, with a Koff approximately 1.5 x 10-3 s-1 or greater, more preferably, with a Koff of approximately 1.6 x 10-3 s-1 or greater, more preferably, with a Koff of approximately 1.7 x 10- 3 s-1 or greater, more preferably, with a Koff of approximately 1.8 x 10-3 s-1 or greater, and even more preferably, with a Koff of approximately 1.9 x 10-3 s-1 or greater . In a particularly preferred embodiment, the isolated human antibody or antigen-binding portion thereof is cleaved from the human EpoR with a Koff of approximately 4.8 x 10-3 s-1 or greater. Even more preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from EpoR at a dissociation rate of at least 1.9 x 10-3 s-1 or at least 4.8 x 10-3 s -one.

In another embodiment, said antibody or antigen-binding portion thereof is associated with human EpoR with a Kd rate constant equal to or greater than about 7 nM and, more preferably, with a Kd rate constant between about 7-32 nM, inclusive. More preferably, an antibody or antigen-binding portion thereof associates with human EpoR with a rate constant Kd at least equal to 7 nM and up to 32 nM, inclusive. The Kd can be calculated from the Koff and Kon velocity constants, these constants being determined by surface plasmon resonance or other methodologies well known to those of ordinary skill in the art. In a more preferred embodiment, an antibody or antigen-binding portion thereof dissociates from EpoR with a Koff of approximately 1.9 x 10-3 s-1 and a Kd of approximately 20 nM. In a preferred embodiment, an antibody or antigen-binding portion thereof is cleaved from human EpoR with a Koff of approximately 4.8 x 10-3 s-1 and a Kd of approximately 32 nM. In most preferred embodiments, an antibody or antigen-binding portion thereof dissociates from human EpoR with a Koff of at least 1.9 x 10-3 s-1 and a Kd of at least 20 nM. In a preferred embodiment, an antibody or antigen-binding portion thereof is cleaved from human EpoR with a Koff of at least 4.8 x 10-3 s-1 and a Kd of at least 32 nM.

More preferably, the isolated antibody or antigen-binding portion thereof activates human EpoR in a standard in vitro proliferation assay using a human erythroleukemic cell line, such as for example F36E or UT-7 / Epo. In a preferred embodiment, the antibody is an isolated human recombinant antibody or an antigen-binding portion thereof.

Surface plasmon resonance analysis to determine Kd and Koff is well known to those of ordinary skill in the art and can be performed as described herein (see Example 8). Example 9 describes a conventional in vitro assay to determine cell proliferation. Examples of recombinant human antibodies that meet, or are predicted to meet, the aforementioned activation and kinetic criteria include antibodies that have the following pairs [VH / VL], the sequences of which are shown in FIGS. 7 and 8: SEQ ID NO: 15 / SEQ ID NO: 17, SEQ ID NO: 7 / SEQ ID NO: 17, SEQ ID NO: 8 / SEQ ID NO: 17, SEQ ID NO: 9 / SEQ ID NO: 17, SEQ ID NO: 10 / SEQ ID NO: 17, SEQ ID NO: 11 / SEQ ID NO: 17, SEQ ID NO: 12 / SEQ ID NO: 17, SEQ ID NO: 13 / SEQ ID NO: 17 and SEQ ID NO: 14 / SEQ ID NO: 17.

In a preferred embodiment, Ab12.6 and Ab12.6-related antibodies comprise the heavy chain CDR2 sequences shown in Figure 7. In a more preferred embodiment, Ab12.6 and Ab12.6-related antibodies comprise VE sequences. : shown in Figure 9. In an even more preferred embodiment, Ab12.6 and Ab12.6-related antibodies further comprise the VL sequences shown in Figure 10.

II. Antibody Expression

An antibody, or antibody part, of the invention can be prepared by recombinant expression of immunoglobulin heavy and light chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors bearing DNA fragments encoding the immunoglobulin heavy and light chains of the antibody such that the light and heavy chains are expressed in the cell. host and, preferably, they are secreted in the medium in which the host cells are grown, the antibodies can be recovered from said medium. Conventional recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning : A Laboratory Manual, Second Edition, Cold Spring Harbor, New Your, (1989), Ansubel, FM et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing associates (1989) and in US Patent No. 4,816,397 to Boss et al.

To express an anti-EpoR antibody of the invention, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of variable germline heavy and light chain sequences using the polymerase chain reaction (PCR) and as described herein. To express Ab12.6 or an antibody related to Ab12.6, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of the germline heavy and light chain variable sequences using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, eg, "Vbase" human germline sequence database; see also Kabat, BA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, LM, et al., (1992) The Reportoire of Human Germiline VH Sequences Reveals about Fifty Groups of V body portion of the invention can be functionally linked (by Segments with Different Hypervariable Loops "J. Mol. Biol. 227: 776-798; and Cox, JPL et al. (1994)" A Directory of Human Germ-line V78 Segments Reveals a Strong Bias in their Usage ”Eur. J. Immunol. 24: 827-836).

To obtain a DNA fragment encoding the Ab12.6 heavy chain variable region or an Ab12.6-related antibody, the human germline sequence VH4-59 is amplified by standard PCR. Furthermore, the A30 germline sequence of the Vκ1 family is amplified by conventional PCR. Suitable PCR primers can be designed for use in amplification of the VH4-59 germline sequence and the Vκ1 family germline sequence A30 based on the nucleotide sequences described in the references mentioned above, using standard methods.

Alternatively, DNA from the Ab12-expressing cell line can be obtained and modified by means well known in the art (such as site-directed mutagenesis) to generate Ab12.6 and Ab12.6-related antibodies. A cell line expressing Ab12 antibody was deposited at the American Type Culture Collection (ATCC), 1081 University Boulevard, Manassas, Virginia 20110, under the terms of the Budapest Treaty, on September 30, 2003, and was assigned the number of access PTA-5554. This deposit is provided for the convenience of those skilled in the art and is not an admission that such a deposit is required for the practice of the invention or that equivalent embodiments are not within the skill of the art in view of the present description. The public availability of this deposit is not a grant of a license to make, use, or sell the material deposited under this or any other patent. The nucleic acid sequence of the deposited material prevails if it conflicts with any sequence described in this document.

Once the Ab12 germline or VH and VL fragments have been obtained, these sequences can be mutated to encode Ab12.6 or Ab12.6-related amino acid sequences described herein. The amino acid sequences encoded by the Ab12 or germline VH and VL DNA sequences are first compared to the Ab12.6 or Ab12.6-related VH and VL amino acid sequences to identify amino acid residues in the sequence of Ab12.6 or related to Ab12.6 that differ. The appropriate nucleotides of the germline or Ab12 DNA sequences are mutated such that the mutated sequences encode the amino acid sequence of Ab12.6 or related to Ab12.6, using the genetic code to determine what nucleotide changes should be made. Mutagenesis of germline sequences

or Ab12 is carried out by conventional methods, such as PCR-mediated mutagenesis (in which mutated nucleotides are incorporated into the PCR primers so that the PCR product contains the mutations) or targeted mutagenesis.

Once the DNA fragments encoding VH and VL segments of Ab12.6 or related to Ab12.6 have been obtained (by amplification and mutagenesis of VH and VL genes, as described above), those DNA fragments they can be further manipulated by conventional recombinant DNA techniques, for example to convert variable region genes to full-length antibody chain genes, Fab fragment genes, or a scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked", as used in this context, is intended to mean that the two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in phase.

In an alternative method, a scFv gene can be constructed with wild-type CDR regions (eg, from Ab12) and then mutated in the manner described in Example 3 below.

Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2, and CH3). Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, No. of Publication NIH 91-3242). The present invention further includes all known human heavy chain constant regions, including but not limited to all known allotypes of the human heavy chain constant region. DNA fragments that include these regions can be obtained by standard PCR amplification. The heavy chain constant region may be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD, but more preferably it is a constant region of IgG2. For a Fab fragment heavy chain, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively binding the DNA encoding VL to another DNA molecule encoding the light chain constant region. , CL. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, No. NIH Publication No. 913242). The present invention includes all known human light chain constant regions, including but not limited to all known allotypes of the human light chain constant region. DNA fragments that include these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but more preferably it is a kappa constant region.

It should be understood that the specific designations of FR and CDR regions within a particular heavy or light chain variable region may vary depending on the convention or numbering system used to identify such regions (eg Chothia, Kabat, model formation software from AbM from Oxford Molecular, all of which are known to those of skill in the art.) Such designations, however, are not critical to the invention.

To create a scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, eg, encoding the amino acid sequence GENKVEYAPALMALS (SEQ ID NO: 2) so that the sequences VH and VL can be expressed as a contiguous single chain protein, with the VL and VH regions linked by, for example, a second flexible linker GPAKELTPLKEAKVS (SEQ ID NO: 3). For other linker sequences see also, for example, Bird et al. (1988) Science 242: 423-426, Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883 and McCafferty et al., Nature (1990) 348: 552

554.

To express the antibodies or antibody parts of the invention, DNA encoding the full or partial length heavy and light chains, obtained as described above, are inserted into expression vectors so that the genes are operably linked to sequences of transcription and translation control. In this context, the term "operably linked" is intended to mean that an antibody gene is linked to a vector such that vector transcription and translation control sequences fulfill their intended function of regulating gene transcription and translation. of antibody. The expression vector and the expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. Antibody genes are inserted into the expression vector by conventional methods (eg, ligation of complementary restriction sites on the antibody and vector gene fragment or blunt-ended ligation if no restriction sites are present). Before insertion into the Ab12.6 heavy or light chain sequence

or related to Ab12.6, the expression vector may already carry constant region sequences of the antibody. For example, one approach to convert Ab12.6-related or Ab12.6-related VH and VL sequences into full-length antibody genes is to insert them into expression vectors that already encode the heavy chain constant regions and the constant regions of light chain, respectively, so that the VH segment is operably linked to the CH "segment" within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is phase linked with the amino terminal end of the antibody chain gene. The single peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenoviruses (eg, the adenovirus major late promoter (AdMLP)) and polyoma. For a further description of viral regulatory elements and sequences thereof, see, for example, US Patent No. 5,168,062 to Stinski, US Patent No. 4,510,245 to Bell et al. and United States Patent No. 4,968,615 to Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see for example, US Patent Nos. 4,399,216; 4,634,665 and 5,179,017, all from Axel et al.). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene for use in dhfr-host cells with methotrexate selection / amplification and the neomycin (neo) gene for G418 selection.

For expression of light and heavy chains, the expression vector or vectors encoding the heavy and light chains are introduced by transfection into a host cell by standard techniques. The various forms of the term "transaction" are intended to include a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, eg, electroporation, calcium phosphate precipitation, DEAE-dextran transfection. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, the expression of antibodies in eukaryotic cells, and more preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular the cells of mammal, they are more likely than prokaryotic cells to assemble and secrete an immunologically active and appropriately folded antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for the production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6: 12-13).

Preferred mammalian host cells to express the recombinant antibodies of the invention include Chinese Hamster Ovary cells (CHO cells) (including CHO dhfr- cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a selectable DHFR marker, for example as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621), NSO myeloma cells, COS cells, HEK cells -293 and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or, more preferably, secretion of the antibody in the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to produce intact antibody parts, such as Fab fragments or scFv molecules. It will be understood that variations of the above procedure are included within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding the light chain or heavy chain (but not both) of an antibody of the present invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding the light or heavy chains.

or both, which is not necessary to join EpoR. Molecules expressed from such truncated DNA molecules are also included by the antibodies of the invention.

In a preferred system for recombinant expression of an antibody, or antigen-binding portion thereof, of the invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr CHO cells. -by calcium phosphate mediated transfection. Within the recombinant expression vector, the antibody's heavy and light chain genes are each operably linked to CMV enhancer / AdMLP promoter regulatory elements to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, which allows selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transforming host cells are cultured to allow expression of the antibody heavy and light chains and the intact antibody is recovered from the culture medium. Conventional molecular biology techniques are used to prepare the recombinant expression vector, transfect host cells, select transformants, culture host cells, and recover antibody from the culture medium.

In view of the foregoing, another aspect of the invention relates to nucleic acid, vector and host cell compositions that can be used for the recombinant expression of the antibodies and antibody parts of the invention. The nucleotide sequence encoding the Ab12.6 heavy chain variable region and variants thereof is shown in Figure 9. The nucleotide sequence encoding the Ab12.6 light chain variable region is shown in Figure 10. The CDR1 domain of the HCVR of Ab12.6 includes nucleotides 26-35 of SEQ ID NO: 7, the CDR2 domain includes nucleotides 50-65 of SEQ ID NO: 7 and the CDR3 domain includes nucleotides 98-105 from SEQ ID NO: 7. It will be appreciated by the person skilled in the art that nucleotide sequences encoding Ab12.6-related antibodies, or part thereof (eg, a CDR domain, such as a CDR2 domain), they can be derived from nucleotide sequences encoding the Ab12.6 LCVR and HCVR using the genetic code and conventional molecular biology techniques.

In one embodiment, the invention provides an isolated nucleic acid encoding a heavy chain variable region comprising an amino acid sequence of Formula I:

Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y

Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15)

in which:

X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G), alanine (A), glutamine

(AND)

 and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine

(T)

with the proviso that X1-X2-X3 is different from Y-Y-S.

In yet another embodiment, the invention provides isolated nucleic acids encoding a polypeptide selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

In yet another embodiment, the invention provides an isolated nucleic acid encoding an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 17. The nucleic acid may encode only the HCVR or it may also encode a region antibody light chain constant, operably linked to LCVR. In one embodiment, this nucleic acid is in a recombinant expression vector. Those skilled in the art will appreciate that the nucleic acids encoding the antibodies of the invention are not limited to those specifically described herein, but also include, due to the degeneracy of the genetic code, any DNA encoding the polypeptide sequences in this document. Degeneracy of the genetic code is well established in the art (See, eg, Bruce Alberts et al. (Eds)., Molecular Biology of the Cell, Second Edition, 1989, Garland Publishing Inc., New York and London). Accordingly, the nucleotide sequences of the invention include those comprising each and every degenerate codon at each and every position on the nucleotide, provided that such codons encode the amino acid sequences set forth herein.

In yet another embodiment, the invention provides an isolated nucleic acid encoding a light chain variable region of the antibody comprising the amino acid sequence of SEQ ID NO: 17 (i.e., the LCVR of Ab12.6) although skilled in the art. the subject will appreciate that due to the degeneracy of the genetic code, other nucleotide sequences can encode the amino acid sequence of SEQ ID NO: 17. Nucleic acid can encode LCVR operably linked to HCVR. For example, the nucleic acid can comprise a constant region of IgG1 or IgG2 or IgG4. In a preferred embodiment, the nucleic acid comprises a constant region of IgG2. In yet another embodiment, this nucleic acid is in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding both an antibody heavy chain and an antibody light chain. For example, in one embodiment, the invention provides a recombinant expression vector that encodes:

a) an antibody heavy chain having a variable region comprising the amino acid sequence of SEQ ID NO: 7 (ie the HCVR of Ab12.6); and

b) an antibody light chain having a variable region comprising the amino acid sequence of SEQ ID NO: 7 (ie the LCVR of Ab12.6).

The invention also provides host cells into which one or more of the recombinant expression vectors of the invention have been introduced. Preferably, the host cell is a mammalian host cell, more preferably the host cell is a CHO cell, an NSO cell, or a HEK-293 cell or COS cell. Furthermore, the invention provides a method of synthesizing a recombinant human antibody of the invention by culturing a host cell of the invention in a suitable culture medium until a recombinant human antibody of the invention is synthesized. The method may further comprise isolating the recombinant human antibody from the culture medium.

III. Selection of Recombinant Antibodies

Recombinant antibodies of the invention, in addition to the Ab12.6 antibodies or Ab12.6 related antibodies described herein, can be isolated by scanning a recombinant combinatorial antibody library, preferably a yeast display library of scFv, prepared using cDNA from Chimeric, humanized, or human VL and VH (eg, Ab12). Methodologies for preparing and exploring such libraries are known in the art. In addition to commercially available vectors for generating yeast display libraries (eg, vector pYD1, Invitrogen, Carlsbad, California) there are examples of methods and reagents particularly susceptible for use in the generation and screening of antibody display libraries. in, for example, Boder ET and Wittrup KD, Yeast surface display for directed evolution of protein expression, affinity and stability, Methods Enzymol., 328: 430-440 (2000) and Boder ET and Wittrup KD, Yeast surface display for screening combinatorial polypeptide libraries, Nat Biotechnol. 15 (6): 553-7 (June 1997).

In a preferred embodiment, to isolate human antibodies with low affinity and a fast dissociation rate for EpoR, a human agonist antibody (such as, for example, Ab12) is first used to generate human heavy and light chain sequences expressed as scFv in the surface of a yeast (preferably Saccaromyces cerevisiae). Ab12 scFv are analyzed to determine those with the highest levels of expression. Such constructs are then scanned, preferably using soluble recombinant human EpoR. Those scFv constructs that have the highest degree of soluble EpoR binding are screened for subsequent mutagenesis of the heavy and light chain variable regions to generate CDR mutagenic libraries.

To further increase the dissociation constant for EpoR binding, the preferred VH and VL segments of the pair or VH / VL pairs can be randomly mutated, preferably within the CDR2 region of VH, in a process analogous to the somatic mutation process in I live responsible for the affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be achieved by replacing a portion of each CDR with a degenerate single-stranded oligonucleotide that encodes three amino acids within the targeted CDR. Replacement of part of each CDR with a new randomly selected sequence (up to 8000 possibilities) can be accomplished by homologous recombination in yeast (see, eg, Example 3). These randomly mutated VH segments can be analyzed for EpoR binding in the context of a scFv; scFv that show improved fluorescence and fast dissociation rate can then be isolated and the CDR mutation can be identified by sequencing.

After scanning a recombinant scFv display library, clones having the desired characteristics for conversion are selected, preferably into kappa light chain / kappa immunoglobulin type 2 (IgG2 / K) antibodies. The nucleic acid encoding the selected antibody can be recovered from the display package (eg, from the yeast expression vector) and subcloned into other expression vectors by standard recombinant DNA techniques. If desired, the nucleic acid can be further manipulated to create other forms of the antibody of the invention (eg, linked to nucleic acids that encode additional immunoglobulin domains, such as additional constant regions). To express an isolated recombinant human antibody by scanning a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into mammalian host cells, as described in greater detail in Section II above.

IV. Uses of anti-EpoR antibodies

The antibodies or antigen-binding portions thereof of the present invention have various uses. In general, the antibodies or antigen-binding portions thereof can be used to treat any condition treatable by erythropoietin or a biologically active variant or analog thereof. For example, antibodies

or antigen-binding portions thereof, of the invention, are useful in treating disorders characterized by low levels of red blood cells and / or reduced levels of hemoglobin (eg, anemia). Furthermore, such antibodies or antigen-binding portions thereof can be used to treat disorders characterized by reduced or below normal levels of oxygen in the blood or tissues, such as, for example, hypoxemia or chronic tissue hypoxia and / or or diseases characterized by inadequate blood circulation or reduced blood flow. Antibodies or antigen-binding parts thereof may also be useful to promote wound healing or to protect against damage to neural tissues and / or cells, due to brain / spinal cord injury, stroke. Non-limiting examples of conditions that can be treated with antibodies of the invention include anemia, such as chemotherapy-induced anemia, anemia associated with cancer, anemia of chronic disease, anemia associated with HIV, anemia associated with bone marrow transplantation and the like, insufficiency. heart disease, ischemic heart disease and kidney failure. As such, the antibodies of the invention can be used to treat any of the aforementioned diseases or conditions by administering to a mammal a therapeutically effective amount of said antibody. Preferably, the mammal is a human.

The antibodies or antigen-binding portions thereof of the present invention can also be used to identify and diagnose mammals having a dysfunctional EPO receptor. Mammals that have a dysfunctional EPO receptor are characterized by disorders such as anemia. Preferably, the mammal that is identified and diagnosed is a human. Additionally, the antibodies of the present invention can be used in the treatment of anemia in mammals suffering from red blood cell aplasia. Aplasia of red blood cells may result from the formation of neutralizing anti-erythropoietin antibodies in patients during treatment with recombinant erythropoietin (Casadevall, N. et al., N. Eng. J. Med. 346: 469 (2002)). The antibodies of the invention can be used to treat aplasia by administering to a mammal suffering from such aplasia and in need of treatment a therapeutically effective amount of the antibodies of the present invention.

In one embodiment of the invention, the EPO receptor antibodies and antigen-binding portions thereof can also be used to detect the EPO receptor (eg, in a biological sample, such as tissue test samples, intact cells, or extracts thereof), using a standard immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or tissue immunohistochemistry. The invention provides a method for detecting the EPO receptor in a biological sample comprising contacting a biological sample with an antibody or antigen binding part of the invention and detecting the antibody (or part of the antibody), for detecting thereof mode the EPO receptor in the biological sample. The antibody or antigen-binding part is directly or indirectly labeled with a detectable substance to facilitate detection of the antibody or the bound or unbound antibody fragment. A variety of immunoassay formats can be practiced (such as competitive assay, direct sandwich immunoassays

or indirect) and are well known to those skilled in the art.

Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine, dansyl chloride, or phycoerythrin; and an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125I, 131I, 35S, or 3H. Given their ability to bind human EpoR, the anti-EpoR antibodies, or parts thereof, of the invention can be used to activate or stimulate EpoR activity. Antibodies or antigen-binding portions thereof are preferably capable of activating EpoR activity both in vitro and in vivo. Accordingly, such antibodies and antibody parts can be used to activate EpoR activity, for example, in an EpoR-containing cell culture, in human subjects, or in other EpoR-bearing mammalian subjects with which an antibody of the invention reacts cross shape.

In another embodiment, the invention provides the antibody or antigen-binding portion thereof of the present disclosure to activate endogenous activity of a human erythropoietin receptor to treat anemia, hypoxemia, chronic tissue hypoxia, heart failure, ischemic heart disease, failure kidney, tissue damage or neural cell damage or to promote wound healing in a mammal, by administering to said mammal a therapeutically effective amount of an antibody or antigen-binding portion thereof, of the invention. Preferably, the mammal is a human subject.

An antibody of the invention can be administered to a human subject for therapeutic purposes. Furthermore, an antibody of the invention can be administered to a non-human mammal with which the antibody is capable of binding for veterinary purposes or as an animal model of human disease. With respect to the latter, such animal models may be useful in evaluating the therapeutic efficacy of antibodies of the invention (eg, to test dosages and timing of administration).

In another aspect, the invention provides the antibody or antigen-binding portion thereof of the present disclosure to treat an aplasia-like mammal, by administration to a mammal in need of treatment of a therapeutically effective amount of an antibody or part of antigen binding thereof of the invention. Furthermore, the invention provides the antibody or antigen-binding portion thereof of the present disclosure for treating a mammal suffering from anemia by administration to the mammal in need of treatment of a therapeutically effective amount of an antibody or antigen-binding portion thereof. , of the invention.

V. Pharmaceutical compositions and pharmaceutical administration

The antibodies and antibody parts of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises a therapeutically or pharmaceutically effective amount of an antibody or antibody part of the invention together with a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes each and every solvent, dispersion media, coatings, antibacterial and antifungal agent, absorption retardant and isotonic agent, which are physiologically compatible . Examples of pharmaceutically acceptable carriers or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, and ethanol as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars and polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as humectants or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers may also be included, which increase the shelf life or efficacy of the antibody or part of the antibody. Optionally, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, may be included. In addition to excipients, the pharmaceutical composition can include one or more of the following, carrier proteins such as serum albumin, buffers, binding agents, sweeteners, and other flavoring agents; coloring agents and polyethylene glycol.

The compositions of the present invention can be in a variety of forms. They include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (eg, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and the therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by infusion or intravenous injection. In another preferred embodiment, the antibody or antibody fragment is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration of drug. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody fragment) in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing 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 lyophilization which produces an active ingredient powder plus any additional desired ingredients of a previously filtered sterile solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prolonged absorption of injectable compositions can be achieved by including an absorption retarding agent in the composition, for example, monostearate salts and gelatin.

The antibodies and antibody parts of the invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is induction or intravenous infusion. As will be appreciated by the person skilled in the art, the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound can be prepared with a vehicle that protects the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biocompatible, biodegradable polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods of preparing such formulations are patented or generally known to those of skill in the art. (See, eg, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed. Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an antibody or antibody portion of the invention can be administered orally, for example, with an inert diluent or an assimilable edible vehicle. The compound (and other ingredients if desired) can also be included in a hard or soft shell gelatin capsule, compressed into a tablet, oral tablets, troches, capsules, elixirs, suspensions, syrups, or wafers. To administer an antibody or antibody fragment of the invention by administration other than parenteral, it may be necessary to coat the compound with, or co-administer with, a material that prevents its inactivation.

Complementary active compounds can also be incorporated into the compositions. In certain embodiments, the antibody or antibody portion is co-formulated with and / or co-administered with one or more additional therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with monotherapies or, alternatively, acting synergistically or additively to enhance the therapeutic effect.

As used herein, the term "therapeutically effective amount" or "pharmaceutically effective amount" means an effective amount of antibody or part of antibody, at dosages and for periods of time necessary to achieve the desired therapeutic result. The exact dose may be determined by an expert in the field. As is known in the art, adjustments based on age, body weight, sex, diet, time of administration, drug interaction, and severity of the condition may be necessary and may be determined with routine experimentation by those skilled in the art. A therapeutically effective amount is also one in which the therapeutically beneficial effects outweigh any toxic or detrimental effects of the antibody or antibody fragment. A "prophylactically effective amount" refers to an effective amount, dosages, and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Dosage regimens can be adjusted to provide the optimal desired response (eg, a therapeutic or prophylactic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased proportionally as indicated by the demands of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in unit dosage form to facilitate administration and allow uniformity of dosage. The unit dosage form as used herein refers to physically discrete units suitable as unit dosages for the mammalian subjects to be tested; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for dosage unit forms of the invention are dictated by and depend directly on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved and (b) the limitations inherent in the technique of forming said Such an active compound for the treatment of sensitivity in individuals.

An exemplary non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1-20 mg / kg, more preferably 0.5-10 mg / kg. It should be noted that dosage values may vary with the type and severity of the condition to be alleviated. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time in accordance with the needs of the individual and the professional judgment of the person administering or supervising the administration of the compositions, and that the ranges of Dosages set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

EXAMPLES

Example 1: Conversion of Ab12 into a single chain antibody fragment

The initial objective of the present study was to reduce the rate of dissociation of Ab12 IgG2 / K using the yeast display technology. To meet this objective, Ab12 IgG2 / K was converted into a scFv using linker sequences. Several different linker sequences were explored during the construction of Ab12 scFv (FIG. 1). Each linker combination was evaluated by analysis of Ab12 scFv expression on the yeast surface (Saccaromyces cerevisiae). The linker combination that resulted in the highest surface expression of Ab12 scFv was selected as the construct to use for subsequent mutagenesis of CDR regions and fluorescence activated cell sorting (FACS). FIG. 1 depicts a schematic description of a scFv construct, showing the location of the scFv linkers and binding and the selection of available sequences. The linker sequences were combined in various orders to obtain the highest expression of scFv on the yeast surface.

Various single-stranded oligonucleotides encoding Ab12 scFv were co-transformed with a linearized "gap" vector derived from pYD1 (Invitrogen, Carlsbad, CA) in yeast by techniques well known to those skilled in the art. Protein expression on the functional cell surface was compared by incubating the transformed yeast with increasing concentrations of soluble EpoR (EposR) at 37 ° C (FIG. 2). Bound antigen was detected using an EpoR monoclonal antibody, MAB307 obtained commercially from R and D Systems (Minneapolis, MN) followed by anti-mouse phycoerythrin (PE, Southern Biotech, Birmingham, AL). The Ab12 scFv construct that showed the highest expression employed linker 41 (SEQ ID NO: 2) as the binding linker and linker 40 (SEQ ID NO: 3) as the scFv linker (hereinafter). Ab12 41/40). This construct was used in all subsequent FACS experiments as described below.

Example 2: Analysis of the rate of dissociation of ScFv from Ab12 in yeast Measurements of the rate of dissociation of scFv from Ab12 41/40 were made by incubation of 0.5 μM EposR with 0.1 D.O. yeast. (approximately 1 x 106 yeast cells) for 1.5 hours at 37 ° C; these cells were then chilled on ice and washed at 4 ° C. A 10,000-fold excess of Ab12 IgG1 (Abbott Laboratories, Abbott Park, IL), heated to 37 ° C, was added to the cells and individual samples were removed at various time points, cooled, and then read on an Epics XL1 flow cytometer. (Beckman Coulter, Fullerton, CA). The experiment was designed so that as EposR dissociated from ScFv from Ab12, it immediately bound to Ab12 IgG1 (present at saturation concentration) and would no longer be detected on the yeast surface. The remaining bound EposR was detected by the addition of MAB307 followed by the addition of anti-mouse PE. Figure 3 depicts a ScFv dissociation rate analysis of Ab12 41/40. As Figure 3 shows, most of EposR had dissociated 20 minutes after the competition and this parameter was taken into account in the dissociation rate FACS analyzed later

Example 3: Creation of AbF ScFv CDR Mutagenic Libraries

The 6 Ab12 41/40 CDRs (three in the heavy chain and three in the light chain) were randomized, and libraries composed of 8 members each were generated. The linearized "gapped" pYD1 vector (Invitrogen) was modified to include a TEV protease site and also to contain a ScFv sequence of Ab12 41/40 (ie pYD1Tev-Ab12-41 / 40). Next, pYD1-Tev-Ab12-41 / 40 vectors with gaps, lacking specific regions of each CDR, were prepared by PCR and the gap was replaced by a degenerate single-stranded oligonucleotide encoding three amino acids within the CDR to which directed. The replacement of a part of each CDR with a new randomly selected sequence (up to 8,000 possibilities) is

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achieved by homologous recombination in yeast. A schematic of this library construction method is shown in Figure 4, indicating that the gapped vector and the single-stranded oligonucleotide are introduced into yeast by co-transformation. The gapped vector and the oligonucleotide undergo homologous recombination, thereby generating a library of randomly selected CDRs. A total of 50 libraries were generated using this method. Libraries are schematically shown in Figures 5 and 6.

Example 4: Ab12 41/40 ScFv Library FACS

All 50 Ab12 ScFv libraries and wild-type Ab12 ScFv yeast were subjected to dissociation rate FACS analysis on a MoFlo high speed cell separator (Dako Cytomation California Inc. Carpinteria, CA). Transformed yeast cells (0.6 O.D.) were incubated with 0.5 µM EposR at 37 ° C until equilibrium was reached (2 hours). The cells were then cooled, washed and a 10,000 fold molar excess (5 µg / ml) of IgG1 Ab12 preheated to 37 ° C was added. After a 20 minute incubation at 37 ° C, the cells were cooled again, washed and labeled depending on whether you were preparing for "one-color" FACS or "two-color" FACS. For the first, cells were labeled with a mixture of MAB307 and anti-mouse PE. For the second, cells were first labeled with a mixture of MAB307 and rabbit anti-6-his antibody (Research Diagnostics, Flanders, NJ), followed by a mixture of anti-mouse PE and goat anti-rabbit FITC (Southern Biotech, Birmingham, AL). Individual control samples were also prepared to establish MoFlo compensation and to ensure that there was no non-specific background staining.

For Cycle 1 dissociation FACS, each library sample was compared to Ab12 ScFv yeast (WT control) for tests of a population of cells that had increased FL2 fluorescence (and therefore, a potentially longer dissociation). In each case, the brightest 1% of the cells on the FL2 axis were selected, collected and re-cultured in medium (Cycle 1 production). For Cycle 2 cleavage FACS, an identical cell incubation procedure was performed on each Cycle 1 library production for some libraries; for others, FACS Cycle 2 involved additional reagents to detect surface expression. For each Cycle 2 cleavage FACS analysis, about a 0.1% higher selection of cells was made on the FL2 axis and this selection was imposed on all Cycle 1 library productions, when applicable. Libraries presenting a population of cells having a higher FL2 than those of the WT selection for FACS were selected, those without cells within the reference range were not further analyzed. For the selected libraries, the brightest 0.1% of cells on the FL2 axis were selected and collected. An aliquot was plated in yeast selective medium (SD or "individual exclusion") for the isolation of yeast colonies and the rest were grown as liquid cultures for future cell analysis.

Example 5: Analysis of Isolated Clones after Dissociation Separation

Selected crude Cycle 2 productions were grown in liquid medium and subjected to dissociation analysis (data not shown). Productions exhibiting improved dissociation curves were selected for further analysis. Individual clones of these productions were recovered after plating in selective medium and plasmid DNA isolation. PCR was used to amplify the scFv region of each clone and the products were sequenced to identify amino acid substitutions. Table 1 highlights the sequencing results of each Cycle 2 production. All unique clones were named and the frequency of their prevalence was noted.

Table 1 CDRH2 sequence of Ab12 Y I Y Y S G S T N Y N P S L K S

Library name and number Future IgG2 / K name Sequence

CDR replaced in

cloned sequencing

mutagé library unique

H2-1-1 WT

Y I Y

H2-1-1 R2 number 1,8

Ab12.26 Y V G

H2-1-1 R2 number 2

Ab12.27 Y A S

H2-1-1 R2 number 3

Ab12.28 R V G

H2-1-1 R2 number 4

Ab12.29 V R A

H2-1-1 R2 number 5

Ab12.30 K C G

H2-1-1 R2 number 6

Ab12.31 G V G

H2-1-1 R2 number 7

Ab12.32 H R R

H2-1-1 R2 number 9

Ab12.33 A G L

H2-1-1 R2 number 10

Ab12.34 Y G A

H2-1-2 WT

I Y Y

H2-1-2 R2 number 1

Ab12.35 T G P

H2-1-2 R2 number 2

Ab12.36 G G V

H2-1-2 R2 number 6

Ab12.37 V A I

H2-1-2 R2 number 7

Ab12.38 A Y G

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H2-1-2 R2 number 8

Ab12.39 V G M

H2-1-2 R2 number 9

Ab12.40 V G A

H2-1-2 WT

I Y Y

H2-1-2 R2 number 11

Ab12.41 Q G H

H2-1-2 R2 number 12

Ab12.42 V W G

H2-1-2 R2 number 13

Ab12.43 G T S

H2-1-2 R2 number 14, 15

Ab12.44 YOU SEE

H2-1-2 R2 number 16

Ab12.45 V H M

H2-1-2 R2 number 17

Ab12.46 V G L

H2-1-2 R2 number 18

Ab12.47, C A G

H2-1-2 R2 number 19

Ab12.48 Y G G

H2-1-2 R2 number 20 (number 5 from 1c / 2c)

Ab12.49 T T E

H2-1-3 WT

Y Y S

H2-1-3 R2 number 1

Ab12.1 A S G

H2-1-3 R2 number 2

Ab12.2 G A G

H2-1-3 R2 number 3

Ab12.3 G N G

H2-1-3 R2 number 4

Ab12.4 A G G

H2-1-3 R2 number 5

Ab12.5 G G H

H2-1-3 R2 number 6

Ab12.6 G G E

H2-1-3 R2 number 7, 8, 9

Ab12.7 G G G

H2-1-3 R2 number 10

Ab12.8 M G G

H2-1-3 WT

Y Y S

H2-1-3 R2 number 11

Ab12.55 A G E

H2-1-3 R2 number 12, 13, 24, 25, 27-31)

Ab12.56 A G T

H2-1-3 R2 number 14, 15

Ab12.107 G V G

H2-1-3 R2 number 16

Ab12.108 A D E

H2-1-3 R2 number 17

Ab12.109 E V G

H2-1-3 R2 number 18

Ab12.110 A D G

H2-1-3 R2 number 19

Ab12,111 A G G

H2-1-3 R2 number 20

Ab12.112 G V S

H2-1-3 R2 number 21

Ab12,113 G V T

H2-1-3 R2 number 22

Ab12,114 E G G

H2-1-3 R2 number 23

Ab12,115 G E E

H2-1-3 R2 number 26

Ab12,116 T E R

H2-4-1 WT

Y S G

H2-4-1 R2 number 1

Ab12.64 P F S

H2-4-1 R2 number 2

Ab12.65 S P V

H2-4-1 R2 number 3

Ab12.66 P P F

H2-4-1 R2 number 5

Ab12.67 P G V

H2-4-1 R2 number 6

Ab12.68 S P I

H2-4-1 R2 number 7

Ab12.69 P F T

H2-4-1 R2 number 8, 9

Ab12.70 S P S

H2-4-1 R2 number 10

Ab12.71 P S I

H2-4-1 WT number 4

Ab12 Y S G

To determine which affinity maturation clones would be converted to an IgG2 / K format, the productions of each library were analyzed and considered for the following parameters: isolation frequency, consensus sequence change in CDR, and fluorescence shift of raw productions and individual yeast clones. Clones appearing at a higher frequency, containing a consensus change representative of the CDR sequence, and having the highest overall FL2 signal in equilibrium binding and dissociation analysis were selected for conversion.

Example 6: Cloning and Expression of Antibodies Derived from Yeast Presentation

Selected scFv were converted to IgG2 / K antibodies by PCR amplification of the variable domains, followed by ligation of these domains to an intact IgG2 constant region or K region present in the pBOS vector (Mizushima and Nagata, Nucleic Acids Research, Vol 18, p. 5322, 1990). The pBOS plasmids encoding both the heavy and light chain reactions were introduced by transient transfection into COS cells and the resulting supernatants from cell cultures were purified on a protein A sepharose column. The purified antibodies were dialyzed in phosphate buffered saline. (PBS) and were quantified by 280 optical density spectrophotometric reading (OD 280). Each antibody was subjected to BIAcore affinity measurements and used as a test article in F36E and UT-7 / Epo cell proliferation assay.

Example 7: BIAcore Analysis of Yeast Presentation Derived Antibodies

5 BIAcore analyzes were performed on a BIAcore 2000 using the BIAcontrol software version 3.1.0 and on a BIAcore 000 using the BIAcontrol software version 4.0.1 (BIAcore, Uppsala, Sweden) using EposR as the test antigen. Table 2 highlights the affinity parameters in each mutated Ab12 clone compared to Ab12.

10 Table 2

Name

Kon (1 / M x s) Koff (1 / s) Kd (nM)

Ab12

1.4 x 105 1.3 x 10-3 eleven

Ab12.6

1.5 x 105 4.8 x 10-3 32

Ab12.56

9.4 x 104 1.9 x 10-3 twenty

Ab12.17

1.4 x 105 4.5 x 10-5 0.33

Ab12.25

6.5 x 104 7 x 10-5 one

Ab12.61

8.5 x 104 9.0 x 10-5 one

Ab12.70

1.6 x 105 9.9 x 10-4 6

Ab12.76

2.1 x 105 9.9 x 10-5 0.48

As Table 2 shows, Ab12.6 and Ab12.56 showed faster dissociation rates and higher Kd values relative to Ab12.

Example 8: Generation of Sub-variants of Ab12.6

To determine the contribution of amino acid substitutions present in the Ab12.6 sequence, sub-variants were synthesized using Ab12.6 IgG2 / K DNA and suitable PCR primers designed to create substitutions when appropriate. Sub-variants were also subjected to BIAcore analysis as has been

20 described above. Table 3 highlights the affinity parameters of each sub-variant clone.

Table 3

Name

Kon (1 / M x s) Koff (1 / s) Kd (nM)

Ab2.118

2.5 x 105 5.5 x 10-3 22

Ab12,119

2.1 x 105 4.4 x 10-3 twenty-one

Ab12,120

2.7 x 105 2 x 10-3 7

Ab12,121

2.1 x 105 6.3 x 10-3 31

Ab12,122

2.2 x 105 4.9 x 10-3 2. 3

Ab12,123

1.3 x 105 3.3 x 10-3 25

Example 9: Antibody Dependent Human Cell Proliferation Assay

Ab12, Ab12.6 and Ab12.6 related variants were tested in established in vitro cell proliferation assays. Stock cultures of erythroleukemic cell lines, UT-7 / Epo or F36E cells were maintained in DMEM or RPMI 1640 medium respectively with 10% fetal bovine serum and 1 unit per ml of erythropoietin

Recombinant human. Before assays, cells were grown overnight at a density of 4.0 to 5.0 x 105 cells per ml in growth medium without Epo. Cells were recovered, washed and resuspended at a density of 1.0 x 10 6 cells per ml in the assay medium (RPMI 1640 or DMEM + 10% FBS) and 50 µl of cells were added to the wells of a plate. 96-well microtiter. 50 µl of each Ab or Epo standard (Recombinant Human Epo (rHuEpo)) in assay medium was added to the wells at final concentrations that

35 ranged from 25 nM to 0.098 nM and the plates were incubated in a humidified incubator at 37 ° C with a 5% CO2 atmosphere. After 72 hours, 20 µl of Promega Cell Titer 96 Aqueous® Reagent (prepared according to the manufacturer's instructions, Madison, Wisconsin) was added to all wells. The plates were incubated at 37 ° C with a 5% CO2 atmosphere for 4 hours and the optical density at 490 nm was determined in a Spectra Max 190 plate reader.

The EC50 and Emax values (shown in Table 4 below) were determined from graphs generated with the spectrophotometric data. The highest affinity antibodies (Ab12.17, Ab12.25, Ab12.61 and Ab12.76) produced bell-shaped curves from which data for EC50 and / or Emax could not be obtained. In contrast, the curves generated from the lower affinity antibodies (shown in

Table 4) produced sigmoid curves (as the native Epo ligand). Furthermore, as Table 4 and Figure 11 show, Ab12.6 and variants related to Ab12.6 (with the exception of AB12.119) unexpectedly stimulated cell proliferation to a greater extent than Ab12.

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50 Table 4

Test Material

CE50 Emax

Epo

0.297 2.82

Ab12

1.29 1.98

Ab12.6

 0.58 2.81

AB12.56

1.17 2,512

Ab12,118

1.13 2.65

Ab12,119

1.34 2.53

Ab12,120

0.34 2

Ab12,121

0.465 2.3

Ab12,122

0.42 2.4

Ab12,123

0.91 2.7

Example 10: Construction of Transgenic MEpoR Mice - / -, hEopR +

Transgenic mice producing only human EpoR (hEopR +, single allele) and non-endogenous mouse EpoR (mEpoR - / -, double allele mutation) were generated as described in Liu, C. et al., Jounal of Biological Chemistry, 272: 32395 (1997) and Yu, X., et al., Blood, 98 (2): 475 (2001). Breeding colonies were established to generate mice for in vivo studies of erythropoiesis.

Example 11: Human Bone Marrow CFU-E Assay Fresh human bone marrow red blood cells obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD) were removed by methods well known in the art and resuspended at 2.5 x 106 cells / ml in 2% IMDM-FBS. Cells (0.1 ml) were added to 17 100 ml culture tubes (VWR, West Chester, PA) containing 2.4 ml of Methocult (StemCell Technologies, Vancouver, Canada), 0.6 ml of IMDM-FBS 2% 0.066 mL Stem Cell Growth Factor (Sigma, St. Louis, Missouri, 1 μg / mL), and Epogen ™ (Dik Drug Co., Chicago, IL), Aranesp ™ (Dik Drug Co.) , Ab12, Ab12.6 or Ab isotype control at the indicated concentrations. After mixing, 1.1 ml of the Methocult suspension was added to a sterile 35 mm non-tissue culture treated petri dish and incubated at 37 ° C, 5% CO 2 for 2 weeks. The colonies, identified by microscopy, were red. The results in Figure 12 indicate that Ab12.6 was more effective than Ab12 in supporting the formation of human CFU-E colonies.

Example 12: Transgenic Mouse Bone Marrow CFU-E Assay

Freshly collected bone marrow red blood cells from femurs of transgenic mEpoR - / -, hEpoR + mice were removed by methods well known in the art and resuspended at 2 x 10 6 cells / ml in 2% IMDM-FBS. Cells (0.1 ml) were added to 17 100 mm culture tubes (VWR, West Chester, PA) containing 3.0 ml of Methocult (StemCell Technologies, Vancouver, Canada), 0.165 ml of cell growth factor mother (Sigma, St. Louis, Missouri, 1 μg / ml) and Epogen ™ (Dik Drug Co., Chicago, IL), Aranesp ™ (Dik Drug Co.), Ab12, Ab12.6 or Ab for isotype control a the indicated concentrations. After mixing, 1.1 ml of the Methocult suspension was added to a sterile petri dish treated with 35mm non-tissue culture and incubated at 37 ° C, 5% CO 2 for 2 weeks. Colonies stained with benzidine (Reference Fibach, E., 1998 Hemoglobin, 22: 5-6, 445458) were identified by microscopy. The results in Figure 13 indicate that Ab12.6 was more effective than Ab12 in supporting colony formation of transgenic mouse CFU-E similar to the results observed in the human CFU-E assay (see Figure 12 above). ).

Example 13: Effect of Administration of Ab12.6 on Hematocrit Change in Transgenic Mouse mEpoR- /, hEpoR +

Experiments were performed to determine the effect of a single dose of Ab12.6 on erythropoiesis with respect to Aranesp ™ (Amgen, Thousand Oaks, CA), a longer acting variant of Epogen. Transgenic mice (mEpoR - / -, hEpoR + as described in Example 10) were injected subcutaneously once with Ab12, Ab12.6 or an isotype control Ab at 0.8 mg / kg 0.2 ml of vehicle (phosphate buffered saline [PBS] containing 0.2% bovine serum albumin [BSA]). Control animals were injected in the same way Aranesp ™ at 3 µg / kg, only a second dose of Aranesp ™ was also administered on day 14 (standard Aranesp ™ therapeutic dosing regimen is ~ 3 µg / kg administered biweekly). Blood samples were taken on days 0, 7, 14, 21, and 28 to determine hematocrits by methods well known in the art. As shown in Figure 14, compared to Ab12, Ab12.6 had improved potency in raising and maintaining hematocrit levels over a 28 day time period. Ab12.6 at 0.8 mg / kg also caused a faster rate of hematocrit increase measured on day 7 than a single dose of Aranesp ™ administered at 3 µg / kg. Furthermore, a single dose of Ab12.6 was at least as effective in raising the hematocrit on day 28 as Aranesp ™ administered twice on day 0 and day 14.

Example 14: Generation of linker library Degenerate oligonucleotide linkers, 45 nucleotides in length, were generated according to the following design: 5 'GGA NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS NHS AGT 3' (SEQ ID NO. : 28) and 5 'GGA VNS VNS VNS VNS VNS VNS VNS VNS VNS VNS VNS VNS VNS AGT 3' (SEQ ID NO: 29) where N is A or G or C or T; V is A or C or G; H is A or C or T; and S is C or G.

In the first linker sequence, the use of the NHS codon prevents the creation of GGC and GGG, the two possible codons for glycine in this codon selection preferably. Furthermore, the NHS codon prevents the creation of TGC (only possible codon for cysteine) and TGG (only possible codon for tryptophan), TGC, CGG and AGG (all possible codons for arginine) and AGC (one of the three serine codons). possible). In the lower linker sequence, the use of the VNS codon limits the creation of TCC and TCG, two of the three possible serine codons. Furthermore, the VNS codon prevents the creation of TTC (only possible codon for phenylalanine), TAC (only possible codon for tyrosine), TGC (only possible codon for cysteine), TGG (only possible codon for tryptophan), TAG (single codon for stop possible) and TGG (one of the three possible codons for leucine). These linker sequences were synthesized as part of a longer synthetic oligonucleotide that also contained elements complementary to a part of a control scFv DNA sequence LT28-A that had a linker sequence (G4S) 3. LT28SA was generated using standard molecular biology techniques by replacing the CDR3 sequence of LT28 Ala-Ala-Trp-Asp-Asp-Ser-Leu-Ser-Gly-Pro-Val (described in WO 01/58956, published on May 16, August 2001) with Ala-Ala-Gly-Asp-Asp-Phe-Leu-Val-Ser-Met-Leu. The linker sequence (G4S) 3 is described in US Patent Nos. 5,258,498 and 5,482,858.

Oligonucleotides from the extended linker library were incorporated into the LT-28-8A scFv by PCR.

VNS and NHS linker PCR products were generated, purified and mixed with restriction-digested yeast display plasmid (pYD-1) containing homologous regions of complementary DNA sequences present at both the 5 'and 3' ends of the VNS and NHS linker PCR products. PCR-generated products encoding the entire LT28-8A scFv (with a linker encoded by NHS or VNS) were inserted by homologous recombination into the galactose-inducible pYD-1 vector so that they were in-phase. Homologous recombinants were selected by subsequent culture in medium without tryptophan and without uracil. The titles of the resulting BNS and NHS linker libraries were evaluated by colony counting and the libraries were prepared for analysis by a fluorescence activated cell sorter (FACS).

Example 15: Library Analysis

Two library plots of LT28-8A scFv from VNS and NHS linkers from Example 14 were compared to those of LT28-8A scFv when induced yeast cells from all groups were incubated with V5-FTTC monoclonal antibody (Invitrogen, Carlsbad , CA). The epitope marker V5 was encoded within the scFv and was at the 3 'end of the polypeptide, and as a result the presence of this epitope indicated that the scFv was fully translated and the signal generated by antibody binding was representative of the expression levels of the scFv on the yeast surface. The percentage of cells that stained positively for FICT: LT28-8A was 58%; the NHS library was 31%; and the VNS library was 47%.

FACS analyzes of cells from the three test groups were compared after the addition of biotinylated IL-18, prepared as described in WO 01/58956, and streptavidin R-phycoerythrin (RPE) (Jackson ImmunoResearch, West Grove, PA) and V5-FITC. RPE fluorescence represents antigen binding to scFv on yeast surface and, together with the presence of the V5 epitope, a dual color signal is generated by clones expressing full length scFv and binding antigen. A concentration of 30 nM biotinylated IL-18 was selected for this analysis because the LT-28-8A scFv had a KD of 30 nM on the yeast surface. The percentage of cells that stained positively for both FITC and RPE: LT28-8A was 55%; NHS library was 25%; and VNS library was 36%.

Cells from the LT-28-8A-NHS or -VNS scFv libraries that demonstrated identical fluorescence to the control were individually selected and harvested using a cell separator. These collected populations (called "productions") were amplified in a liquid culture and aliquots of the culture were seeded in solid medium to isolate individual colonies. DNA was extracted from individual colonies and the nucleotide sequence of the linker was determined by DNA sequencing.

Example 16: Comparison of scFv Containing Variable Linker Sequences

To conclude whether linkers containing variable amino acids had any effect on the behavior of scFv in vitro or in vivo, 11 random NHS-R1 production scFv clones from Example 15 were selected, containing linkers with only one glycine and serine, and they were tested in a series of tests.

Example 17: Kd measurement on yeast surface

Dissociation constants (Kd) of 11 clones of scFv producing NHS-R1 and scFv of LT-28-8A (with the

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

linker (G4S) 3) were measured in a seven-point titration analysis. This included the NHS-R1 producing scFv clones: 13, 19, 22, 23, 30, 33, 34, 38, 40, 41 and 44. Antigen binding was assayed as described in Example 14. All NHS-R1 producing scFv clones and control scFv showed a Kd of approximately 22-26 nM.

Example 18: expression and purification of soluble scFv in bacteria

The in vivo expression of 10 NHS-R1 production clones (10, 13, 19, 30, 33, 34, 38, 40, 41, 44) and scFv of LT-28-8A were analyzed after construction of constructs. of expression encoded by scFv. All 10 LT28-8A scFv sequences were ligated into the inducible expression vector pUC19 / pCANTAB (US Patent No. 5,872,215) and used to transform TG-1 cells. After growth in restricted expression, induction of scFv was initiated by addition of 1mM IPTG and soluble scFv was affinity purified from periplasmic preparations of induced TG-1 cells. Clones 13, 19 and 30 grew very little and were not induced. Purified scFv was tested for protein concentration by BCA assay:

Table 5

Shows

Concentration (μg / ml)  Shows Concentration (μg / ml)

NHS-R1-10

41 NHS-R1-40 4156

NHS-R1-33

826 NHS-R1-41 607

NHS-R1-34

1600 NHS-R1-44 55

NHS-R1-38

619 LT-28-8A 516

Example 19: Activity of soluble scFv in a bioassay

Soluble scFv produced by LT-28-8A and NHS-R1 production scFv clones 33, 34, 38, 40, 41 and 44 were tested in a neutralization bioassay as described in WO 01/58956. All scFv preparations showed IC50 values of about 1x10-7 to 2x10-7 M. Linker sequence 33 is SEQ ID NO: 27; linker sequence 34 is SEQ ID NO: 4; linker sequence 40 is SEQ ID NO: 3; and linker sequence 41 is SEQ ID NO: 2.

Example 20: Ab12.6 recognizes a conformation dependent epitope

Extracellular domain of recombinant expressed EpoR was produced through expression in CHO cells and purified to homogeneity. 3 µg of EpoR extracellular domain per lane were electrophoresed on 4-20% polyacrylamide gels under denaturing conditions (in SDS buffer) or native conditions (without SDS buffer). For Western blot analysis, the gels were transferred to PVDF membranes, blocked with 5% milk powder, and incubated with Ab 12.6 (10 µg / ml) for 1-2 hours at room temperature. Membranes were washed 4 times with PBS / Tween, incubated with HRP-conjugated goat anti-human antibody (1: 2,500) and developed with 4-chloro-1-naphthol as substrate. As Figure 15 shows, Ab12.6 interacts with the recombinant EpoR extracellular domain only under native and non-denaturing conditions, indicating that Ab12.6 recognizes a conformation-dependent epitope.

Example 21: Fab of monomeric Ab12.6 activates EpoR

Monomeric Ab12.6 Fab and bivalent F (ab ') 2 fragments were prepared and purified using standard papain and pepsin digestion conditions (Pierce ImmunoPure Fab and F (ab') 2 Preparation Kits; Pierce, Rockford IL ). Stock cultures of the human erythroleukemic cell line F36E were maintained in RPMI 1640 medium with 10% fetal bovine serum and one unit per ml of recombinant human erythropoietin. Before assays, cells were grown overnight at a density of 4.0 to 5.0 x 105 cells per ml in growth medium without EPO. Cells were recovered, washed and resuspended at a density of 1.0 x 106 cells per ml in assay medium (RPMI 1640 + 10% FBS) and 50 µl of cells were added to wells of a 96 microtiter plate. wells. 50 µl each of Ab12.6, Ab12.6 Fab, Ab12.6 F (ab ') 2 or EPO standards (recombinant human EPO (rHuEPO)) in assay medium were added to the wells and plates they were incubated in a humidified incubator at 37 ° C with a 5% CO2 atmosphere. After 72 hours, 20 µl of Promega Cell Titer 96 Aqueous® Reagent (prepared according to the manufacturer's instructions, Madison, Wisconsin) was added to all wells. The plates were incubated at 37 ° C with a 5% CO2 atmosphere for 4 hours and the optical density at 490 nm was determined using a microplate reader (Wallac Victor 1420 Multilabel Counter, Wallac Company, Boston, MA). The results, seen in Figure 16, showed that the monomeric Ab12.6 Fab stimulated the proliferation of the F36E cell line.

The present invention is illustrated by the foregoing description and examples. SEQUENCE LIST

<110> Abbot Laboratories Reilly, Edward B. Lacy, Susan E. Fung, Emma Belk, Johathan P. Roguska, Michael

<120> Erythropoietin Receptor Antibodies and Uses Thereof

<130> 7349W001

<140>

<141>

<150> 10 / 821,497

<151> 04-09-2004

<150> 10 / 822,306

<151> 04-12-2004

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<170> FastSEQ for Windows Version 4.0

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<213> Homo sapiens

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 116 15 <212> PRT

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<221> VARIANT 10 <222> 52, 53, 54

<223> Xaa = Any Amino Acid

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<222> 3, 4, 5 20

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Claims (21)

1. An isolated antibody, or an antigen-binding portion thereof, that dissociates from the human erythropoietin receptor (EpoR) with a Koff rate constant greater than 1.3 x 10-3s-1 and that activates endogenous activity of said human EpoR in a mammal, said antibody or antigen-binding portion thereof comprising a heavy chain variable region (HCVR) and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of Formula I: Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15) wherein: X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G) and alanine (A), glutamine

(AND)

 and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine

(T)

with the proviso that X1 -X2 -X3 is different from Y-Y-S, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 17.

2.

The antibody or antigen binding part thereof of claim 1, wherein said Koff rate constant is selected from the group consisting of 1.4 x 10-3 s-1 or greater, 1.9 x 10-3 s-1 and 4.8 x 10-3 s-1.

3.

The antibody or antigen-binding portion thereof of claim 1, wherein said antibody is a monoclonal antibody.

Four.

The antibody or antigen binding part thereof of claim 3, wherein said antibody is an IgG2 isotype.

5.

The antibody or antigen binding part thereof of claim 1 which is a human antibody.

6.

The antibody or antigen-binding portion thereof of claim 1 that binds human EpoR with a Kd of 7 nM or greater.

7.

The antibody or antigen-binding part thereof of claim 6 wherein said Kd is 7 to 32 nM inclusive.

8.

The antibody or antigen-binding portion thereof of claim 1, wherein the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

9.

The antibody or antigen-binding portion thereof according to any one of claims 1 to 8 to activate an endogenous activity of a human erythropoietin receptor to treat anemia, hypoxemia, chronic tissue hypoxia, heart failure, ischemic heart disease, failure kidney, damage to cells or neural tissue, or to promote wound healing in a mammal, by administering to said mammal a therapeutically effective amount of said antibody or antigen-binding portion thereof.

10.

The antibody or antigen-binding part thereof according to any one of claims 1 to 8 for treating a mammal suffering from aplasia, by administration to the mammal in need of treatment of a therapeutically effective amount of said antibody or part of binding to antigen thereof.

eleven.

The antibody or antigen-binding part thereof according to any one of claims 1 to 8 for treating a mammal suffering from anemia, by administration to the mammal in need of treatment of a therapeutically effective amount of said antibody or part of binding to antigen thereof.

12.

A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding portion thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

13.

An isolated or purified polynucleotide sequence encoding a heavy chain variable region and a

light chain variable region, in which the heavy chain variable region comprises an amino acid sequence of Formula I: Q-V-Q-L-Q-E-S-G-P-G-L-V-K-P-S-E T-L-S-L-T-C-T-V-S-G-A-S-I-S-S-Y Y-W-S-W-I-R-Q-P-P-G-K-G-L-E-W-I G-Y-I-X1-X2-X3-G-S-T-N-Y-N-P-S-L-K S-R-V-T-I-S-V-D-T-S-K-N-Q-F-S-L K-L-R-S-V-T-A-A-D-T-A-V-Y-Y-C-A R-E-R-L-G-I-G-D-Y-W-G-Q-G-T-L-V T-V-S-S (SEQ ID NO: 15) wherein: X1 is independently selected from the group consisting of tyrosine (Y), glycine (G), and alanine (A); X2 is independently selected from the group consisting of tyrosine (Y), glycine (G) and alanine (A), glutamine

(AND)

 and aspartic acid (D); and X3 is independently selected from the group consisting of serine (S), glycine (G), glutamine (E), and threonine

(T)

with the proviso that X1 -X2 -X3 is different from Y-Y-S, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 17.

14.

 The polynucleotide of claim 13, wherein said polynucleotide encodes a heavy chain variable region selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.

fifteen.

A recombinant expression vector comprising the polynucleotide of claim 13.

16.

A host cell comprising the recombinant expression vector of claim 15.

17.

The host cell of claim 16 which is a mammalian cell.

18.

The host cell of claim 17 selected from the group consisting of a CHO cell, a COS cell and a HEK-293 cell.

19.

The host cell of claim 16 which is a yeast cell.

twenty.

 The host cell of claim 16 which is a bacterial cell.

twenty-one.

A polypeptide sequence encoded by said polynucleotide sequence of claim 13.