Patent Application
20080038265
Erythropoietin (“Epo”) is a glycoprotein that is the primary regulator of erythropoiesis. Specifically, Epo is responsible for promoting the growth, differentiation and survival of erythroid progenitors, which give rise to 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 functions as a growth factor, stimulating the mitotic activity of erythroid progenitor cells, such as erythrocyte burst forming and colony-forming units. It also acts as a differentiation factor, triggering transformation of an erythrocyte colony-forming-unit into a proerythroblast (See Erslev, A., New Eng. J. Med., 316:101-103 (1987)).
Epo has a molecular weight of about 34,000 daltons and can occur in three forms—alpha, beta and asialo. During mid- to late gestation, Epo is synthesized in the fetal liver. Subsequently, Epo is synthesized in the kidney, circulates in the plasma and is excreted in the urine.
Human urinary Epo has been isolated and purified (See, Miyake et al., J. Biol. Chem., 252:5558 (1977)). Moreover, methods for identifying, cloning and expressing genes encoding Epo (See U.S. Pat. No. 4,703,008) as well as purifying recombinant Epo from a cell medium (See U.S. Pat. No. 4,667,016) are known in the art.
The activity of Epo is mediated through the binding and activation of a cell surface receptor referred to as the 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 a 85-100 kDa species (See U.S. Pat. No. 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 polypeptide complexes of Epo receptor having molecular weights such as 110, 130 and 145 kDa (See U.S. Pat. No. 6,319,499).
Both the 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); Winkelmann et al., Blood, 76:24 (1990); WO 90/08822/U.S. Pat. No. 5,278,065). The full length human Epo receptor is a 483 amino acid transmembrane protein with an approximately 25 amino acid signal peptide (See U.S. Pat. No. 6,319,499). The human receptor demonstrates about 82% amino acid sequence homology with the murine receptor. Id.
In the absence of ligands the Epo receptor exists in a preformed dimer. The binding of Epo to its receptor causes a conformational change such that the cytoplasmic domains are placed in close proximity. While not completely understood, it is believed that this “dimerization” plays a role in the activation of the receptor. The activation of the Epo receptor results in a number of biological effects. Some of these activities include stimulation of proliferation, stimulation of differentiation and inhibition of apoptosis (See U.S. Pat. No. 6,319,499, Liboi et al., PNAS USA, 90:11351 (1993), Koury, Science, 248:378 (1990)). Clearly, there is a need for a better understanding of the structural construct of the Epo receptor to assist in the identification of compound capable of (1) dimerizing the Epo receptor; and (2) activating the receptor. These compounds would be useful in treating mammals suffering from anemia and in identifying mammals having a dysfunctional Epo receptor. The present invention addresses these needs.
The invention provides antibodies, or an antigen-binding portion thereof that specifically bind to and activate human erythropoietin receptor (EpoR). The antibodies of the invention are characterized by binding to EpoR with low affinity and dissociating from human erythropoietin receptor (EpoR) with a fast off-rate. The antibodies or antigen-binding portion thereof can be full-length (e.g. an IgG2) or can comprise only an antigen-binding portion (e.g. an F(ab′)2). In a preferred embodiment, the antibodies of the invention bind to EpoR with a Kd of about 7 nM or greater. In a more preferred embodiment, the invention provides an isolated antibody or antigen-binding portion thereof that activates an endogenous activity of human erythropoietin receptor in a mammal and binds to a conformational epitope of the erythropoietin receptor. In an even more preferred embodiment, the invention provides an isolated antibody or antigen-binding portion thereof that activates an endogenous activity of human erythropoietin receptor in a mammal and competes with a second antibody or an antigen-binding portion thereof for binding to a conformational epitope of said human erythropoietin receptor or a fragment of said human erythropoietin receptor wherein the second antibody or antigen-binding portion thereof dissociates from human erythropoietin receptor (EpoR) with a Koff rate constant of greater than about 1.3×10−3 s−1. Preferably, the second antibody activates an endogenous activity of a human erythropoietin receptor in a mammal and comprises a heavy chain variable region (HCVR) having an amino acid sequence of Formula I:
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) with the proviso that X1-X2-X3 is other than Y—Y—S. In a preferred embodiment, the antibody or antigen-binding portion thereof comprises a HCVR having an amino acid sequence of Formula I wherein X1 is G and X2 and X3 are as defined therein. In other preferred embodiments, the antibody or antigen-binding portion thereof comprises a HCVR having an amino acid sequence of Formula I wherein X2 is G and X1 and X3 are as defined therein or X3 is E and X1 and X2 are as defined therein or X1 is G, X2 is G and X3 is as defined therein, or X2 is G, X3 is E and X1 is as defined therein. In particularly preferred embodiments, the antibody or antigen-binding portion thereof comprises a HCVR having an amino acid sequence of Formula I wherein X1 is G, X2 is G and X3 is E or X1 is A, X2 is G and X3 is T. Other preferred embodiments include an antibody or antigen-binding portion thereof comprising an amino acid sequence selected from the group consisting of
Preferably, the second antibody is Ab12.6. Preferably, the conformational epitope comprises amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 of said EpoR.
The aforementioned antibody or antigen-binding portion thereof may be a monoclonal antibody. Preferably, the antibody or antigen-binding portion thereof is an IgG2 isotype.
The invention also provides a method of activating an endogenous activity of a human erythropoietin receptor in a mammal, the method comprising the step of administering to the mammal a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding portions thereof.
The invention also provides a method of modulating an endogenous activity of a human erythropoietin receptor in a mammal, the method comprising the step of administering to the mammal a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding portions thereof.
The invention also provides a method of treating a mammal suffering aplasia, the method comprising the step of administering to a mammal in need of treatment a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding portions thereof.
The invention also provides a method of treating a mammal suffering anemia, the method comprising the step of administering to a mammal in need of such treatment a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding portions thereof.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any of the aforementioned antibodies or antigen-binding portions thereof and a pharmaceutically acceptable excipient.
The present invention further provides compositions comprising a crystallized EpoR, and particularly a crystalline composition of the human EpoR extracelluar domain (ECD) complexed with an antibody that specifically binds to EpoR, and methods for obtaining purified crystallized EpoR, as well as methods for using such compositions and crystals.
A further aspect of the present invention provides crystalline compositions of EpoR comprising a crystalline form of a polypeptide with an amino acid sequence spanning the amino acids 1 to 223 listed in SEQ ID NO:41, wherein the crystalline composition has a space group P212121 and unit cell dimensions a=117.95 b=156.17 and c=164.20 Å.
In another aspect the invention provides the structure coordinates of hEpoR in complex with an antibody that specifically binds to EpoR.
A further aspect of the invention provides methods for designing ligands, compounds, such as agonists and antagonists of the EpoR and variants of an antibody that specifically binds EpoR, or an antigen-binding portion thereof.
Yet another aspect of the invention provides a computer, which comprises a storage medium comprising a data storage material, for producing three-dimensional representations of molecular complexes comprising binding sites defined by structure coordinated of EpoR and an anti-EpoR antibody and methods for using these three-dimensional representations to design: 1) chemical entities and compounds that associate with EpoR or anti-EpoR antibody, 2) compounds, such as potential agonists or antagonists of EpoR; specifically, or 3) variants of anti-EpoR antibodies (such as variants of Ab12, Ab12.5, Ab12.56, Ab12.17, Ab12.25, Ab12.61, Ab12.70 and Ab12.76). Another aspect of the present invention provides method for crystallizing an EpoR-antibody complex. Preferably the methods for crystallization a EpoR polypeptide antibody complex comprising an amino acid sequence spanning the amino acids 1 to 223 listed in SEQ ID NO: 40 comprising: (a) preparing solutions of the polypeptide, antibody and precipitant; (b) growing a crystal comprising molecules of the polypeptide and said mixture solution; and (c) separating said crystal from said solution. The crystallization growth can be carried out by various techniques know by those skilled in the art, such as for example, batch crystallization, liquid bridge crystallization, or dialysis crystallization.
In yet another aspect, the present invention provides vectors useful in methods for preparing a substantially purified extracellular domain of EpoR comprising the polypeptide with an amino acid sequence spanning amino acids 1 to 223 listed in SEQ ID NO:41.
This invention pertains to isolated human antibodies, or antigen-binding portions thereof, that bind to human erythropoietin with low affinity, a fast off-rate and activation or agonistic activity to the EpoR. Various aspects of the invention relate to antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Methods of using the antibodies of the invention to stimulate erythropoietin activity either in vitro or in vivo, also are encompassed by the invention. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
Generally, nomenclatures used in connection with, and 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 art. The methods and techniques of the present invention are generally performed according to 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. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
All abstracts, references, patents and published patent applications referred to herein are hereby incorporated by reference.
In order that the present invention may be more easily understood, certain terms first are defined.
The term “antibody” (abbreviated herein as Ab), as used herein is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised 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 is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs respectively, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (sometimes referred to as “J”).
Furthermore, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
The term “antigen-binding portion” of an Ab (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g. 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 encompassed within the term “antigen-binding portion” of an Ab include (i) an Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, (ii) an F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at 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), which consists of a VH domain; and (vi) an isolated CDR. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. Such single chain Abs are intended to be encompassed within the term “antigen-binding portion” of an Ab. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123.
Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal poly-histidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., 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 papain or pepsin digestion, respectively, of whole Abs. Moreover, Abs, Ab portions 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 having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs and in particular CDR2. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
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 (described further in Section II, below), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., 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 that involves splicing of immunoglobulin gene sequences to 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 an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to germline VH and VL sequences, may not naturally exist within the antibody germline repertoire in vivo.
An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds EpoR is substantially free of antibodies that specifically bind antigens other than EpoR). An isolated antibody that specifically binds EpoR may, however, have cross-reactivity to other antigens, such as EpoR molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.
An “activating or agonistic antibody” or “antibody that activates” or antibody having “activating or agonistic capacity” is intended to refer to an antibody whose binding to EpoR results in stimulation or activation of EpoR biological activity. 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, antibody induced changes in reticulocyte count and/or percent hematocrit and/or antibody binding to Epo receptors. These indicators of EpoR biological activity can be assessed by one or more of several standard in vitro or in vivo assays well known to those of ordinary skill in the art.
The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
The term “humanized antibody” refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences. Means for making chimeric, CDR-grafted and humanized antibodies are known to those of ordinary skill in the art (see, e.g., U.S. Pat. Nos. 4,816,567 and 5,225,539). One method for making human antibodies employs the use of transgenic animals, such as a transgenic mouse. These transgenic animals contain a substantial portion of the human antibody producing genome inserted into their own genome and the animal's own endogenous antibody production is rendered deficient in the production of antibodies. Methods for making such transgenic animals are known in the art. Such transgenic animals can be made using XenoMouse® technology or by using a “minilocus” approach. Methods for making Xenomice™ are described in U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598 and 6,075,181. Methods for making transgenic animals using the “minilocus” approach are described in U.S. Pat. Nos. 5,545,807, 5,545,806 and 5,625,825. Also see International Publication No. WO93/12227.
The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further 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 Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.
The term “Koff”, as used herein, is intended to refer to the off rate constant for dissocation 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. 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” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A polypeptide may 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; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
The term “recovering” as used herein, refers to the process of rendering a chemical species such as a polypeptide substantially free of naturally associated components by isolation, e.g., using protein purification techniques well known in the art.
The term “endogenous activity of EpoR” as used herein, refers to any and all inherent biological properties of the erythropoietin receptor that occur as a consequence of binding of a natural ligand. Biological properties of EpoR include but are not limited to survival, differentiation and proliferation of hematopoeitic cells, an increase in red blood cell production and increase in hematocrit in vivo.
The terms “specific binding” or “specifically binding”, as used herein, in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing 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 specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. A “conformational epitope” as used herein, refers to an epitope whose amino acids are arranged in a non-linear or non-sequential manner. Typically, a conformation epitope has a 3-dimensional structure which is generated or produced upon proper folding of the protein or protein fragment in which the amino acids that form the conformational epitope reside.
The term “polynucleotide” as referred to herein means a polymeric form of two or more nucleotides, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA but preferably is double-stranded DNA.
The term “isolated polynucleotide” as used herein shall mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or some combination thereof) that, by virtue of its origin, the “isolated polynucleotide”: is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur 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 into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, 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, “expressions vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., 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 (for example, plasmid, recombinant expression vector) has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
The term “ligand” refers to any chemical moiety capable of binding a polypeptide. Preferably a ligand is an antigen. Antigens may possess one or more epitopes. Ligands to a first polypeptide sequence and second polypeptide sequence may be the same or different.
A “linking sequence” is a polypeptide sequence that connects two or more polypeptide sequences. The term “connects” refers to the joining of polypeptide sequences. Polypeptide sequences are joined preferably by peptide bonding.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein complex from the relevant portion of the backbone of the erythropoietin receptor polypeptide portion or the anti-erythropoietin receptor antibody portion of the erythropoietin receptor/anti-erythropoietin receptor antibody complex, as defined by the structure coordinates described herein.
The term “binding site”, as used herein, refers to a region of a protein, that, as a result of its shape, favorably associates with another protein, a chemical entity, a compound or an antibody, and an antigen binding fragment thereof. For example, the binding site on erythropoietin receptor for AB12.6 mAb is the epitope of AB12.6 mAb. This binding site could also be the binding site of a ligand, a compound or variant of AB12.6 mAb, or antigen binding fragments thereof.
The term “associating with” refers to a condition of proximity between two or more chemical entities, compounds and proteins, or portions thereof. The association may be non-covalent—wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it may be covalent.
I. Antibodies that Bind Human EpoR
The invention provides isolated antibodies, or antigen-binding portions thereof, that bind to human EpoR with low affinity, a fast off-rate and activating or agonistic capacity to EpoR. Preferably, the antibodies of the invention are recombinant, activating human anti-EpoR antibodies. More preferably, the antibodies or antigen-binding portions thereof also bind to human EpoR with a fast on-rate. Even more preferably, the antibodies have potency similar or comparable to Epo. The most preferred recombinant, activating 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 activating antibodies of EpoR, are summarized in Example 8 below.
The anti-EpoR antibody, and related antibodies, also exhibit a strong capacity to activate EpoR biological activity, as assessed by several 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. Ab12.6 activates EpoR in UT-7/Epo cells with an EC50 of 0.58 nM. Moreover, the activating capacity of the antibodies of the invention are maintained when the antibody is expressed as an Fab, F(ab′)2 or scFv fragment. Furthermore, such antibodies induce an increase in % hematocrit in mammals expressing human EpoR.
Regarding the binding specificity of Ab12.6 and variants thereof, this antibody binds to human EpoR in various forms, including soluble EpoR and transmembrane EpoR. Neither Ab12.6 nor its variants specifically binds to other cytokine receptors.
In one aspect, the invention pertains to Ab12.6 antibodies and antibody portions, Ab12.6-related antibodies and antibody portions, and other antibodies and antibody portions with equivalent properties to Ab12.6, such as low affinity binding to EpoR with fast dissociation kinetics and activating or agonist activity to EpoR. In one embodiment, the invention provides an isolated antibody, or an antigen-binding portion thereof, that dissociates from human EpoR with a Koff rate constant of greater than about 1.3×10−3 s−1 which may be determined by surface plasmon resonance. It is understood in the art that some variability (e.g. up to +20%) may occur in the calculation of EC50, Koff, and Kon values based on instrument variation and experimental design. Typically, such measurements are performed using duplicate or triplicate samples to minimize variability. In addition, such an antibody or antigen-binding portion thereof, binds in a manner sufficient to activate human EpoR as demonstrated by a standard in vitro proliferation assay.
More preferably, the isolated antibody, or antigen-binding portion thereof, dissociates from human EpoR with an off rate (Koff) of about 1.3×10−3 s−1 or greater, preferably, a Koff of about 1.4×10−3 s−1 or greater, more preferably, with a Koff of about 1.5×10−3 s−1 or greater, more preferably with a Koff of about 1.6×10−3 s−1 or greater, more preferably with a Koff of about 1.7×10−3 s−1 or greater, more preferably with a Koff of about 1.8×10−3 s−1 or greater, and even more preferably, with a Koff of about 1.9×10−3 s−1 or greater. In a particularly preferred embodiment, the isolated human antibody or antigen-binding portion thereof, dissociates from human EpoR with a Koff of about 4.8×10−3 s−1 or greater Even more preferably, the isolated human antibody, or antigen-binding portion thereof, dissociates from human EpoR with an off rate of at least 1.9×10−3 s−1 or at least 4.8×10−3 s−1.
In another embodiment, such an antibody or antibody binding portion thereof associates with human EpoR with a Kd rate constant equal to or greater than about 7 nM and more preferably, with a Kd rate constant of between about 7-32 nM, inclusive. More preferably, an antibody or antibody binding portion thereof associates with human EpoR with a Kd rate constract at least equal to 7 nM and up to 32 nM, inclusive. Kd may be calculated from Koff and Kon rate constants, which constants may be determined by plasmon surface resonance or other methodologies well know to those of ordinary skill in the art. In a more preferred embodiment, an antibody or antigen-binding portion thereof dissociates from human EpoR with a Koff of about 1.9×10−3 s−1 and a Kd of about 20 nM. In a preferred embodiment, an antibody or antigen-binding portion thereof dissociates from human EpoR with a Koff of about 4.8×10−3 s−1 and a of about 32 nM. In most preferred embodiments, an antibody or antigen-binding portion thereof dissociates from human EpoR with a Koff of at least 1.9×10−3 s−1 and a Kd of at least 20 nM. In a preferred embodiment, an antibody or antigen-binding portion thereof dissociates from human EpoR with a Koff of at least 4.8×10−3 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 for determining Kd and Koff is well known to those of ordinary skill in the art and can be performed as described herein (see Example 8). A standard in vitro assay for determining cell proliferation is described in Example 9. Examples of recombinant human antibodies that meet, or are predicted to meet, the aforementioned kinetic and activation criteria include antibodies having the following [VH/VL] pairs, the sequences of which are shown in
In another aspect, the invention relates to Ab12.6 and Ab12.6 related (i.e. variants) antibodies which comprise a heavy chain variable region comprising an amino acid sequence of Formula I:
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)
with the proviso that X1—X2—X3 is other than Y—Y—S. In a preferred embodiment, Ab12.6 and Ab12.6 related antibodies comprise the heavy chain CDR2 sequences shown in
In another aspect, the invention relates to isolated antibodies, or antigen-binding portions thereof, that have activating or agonistic capacity to EpoR and bind to a conformational epitope of EpoR. The conformational epitope may be encompassed within an isolated full-length EpoR or any fragment of EpoR, provided such fragment is capable of forming a functional conformational epitope. A functional conformational epitope refers to a conformational epitope of sufficient size and proper folding to allow binding of an antibody or antigen-binding fragment as described herein. An example of such a functional conformational epitope includes but is not limited to a fragment comprising the EpoR extracellular domain. More preferably, the antibody or antigen-binding fragment thereof binds to a functional conformational epitope comprising amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 of EpoR (SEQ ID NO:41). Preferably, the isolated antibody or antigen-binding portion thereof activates an endogenous activity of human erythropoietin receptor in a mammal and competes with a second antibody or an antigen-binding portion thereof for binding to a conformational epitope of the human erythropoietin receptor or a fragment of the human erythropoietin receptor wherein the second antibody or antigen-binding portion thereof dissociates from human erythropoietin receptor (EpoR) with a Koff rate constant of greater than about 1.3×10−3 s−1. Preferably, the second antibody is Ab12.6. Methods for performing such competition determinations are well known to those of ordinary skill in the art.
In another aspect, the invention relates to a method of screening or identifying an antibody or antigen-binding portion thereof that interacts with a conformational epitope of EpoR comprising the steps of providing a functional conformational epitope as described herein, reacting the functional conformational epitope with the antibody or antigen-binding portion thereof for a time and under conditions sufficient to allow the conformational epitope and antibody or antigen-binding portion thereof to interact and determining whether the antibody or antigen-binding portion thereof interacts with the functional conformational epitope. Methods of screening for antibody binding in this manner are well known to those of ordinary skill in the art.
In another aspect, the invention relates to an isolated or purified protein fragment of EpoR comprising amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112, and H114 of EpoR wherein these amino acids form a functional conformational epitope in the protein fragment. Such protein fragments may be used for screening or identifying new antibodies to the epitope by methodologies well known to those of ordinary skill in the art.
II. Expression of Antibodies
An antibody, or antibody portion, of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultures, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expressions 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), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates (1989) and in U.S. Pat. No. 4,816,397 by 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 germline light and heavy chain variable sequences using the polymerase chain reaction (PCR) and as described herein. To express Ab12.6 or an Ab12.6-related antibody, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy 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 e.g., the “Vbase” human germline sequence database; see also Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al. (1992) “The Repertoire of Human Germline 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, J. P. L. et al. (1994) “A Directory of Human Germ-line V78 Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol. 24:827-836; the contents each of which are expressly incorporated herein by reference). To obtain a DNA fragment encoding the heavy chain variable region of Ab12.6, or an Ab12.6-related antibody, the VH4-59 human germline sequence is amplified by standard PCR. In addition, the A30 germline sequence of the Vκ1 family is amplified by standard PCR. PCR primers suitable for use in amplifying the VH4-59 germline sequence and A30 germline sequence of the Vκ1 family can be designed based on the nucleotide sequences disclosed in the references cited supra, using standard methods.
Alternatively, DNA may be obtained from the cell line expressing Ab12 and modified by means well known in the art (such as site-directed mutagenesis) to generate Ab12.6 and Ab12.6-like antibodies. A cell line expressing Ab12 antibody was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, under the terms of the Budapest Treaty, on Sep. 30, 2003 and was accorded accession number PTA-5554. This deposit is provided for the convenience of those skilled in the art and is neither an admission that such deposit is required to practice the invention nor that equivalent embodiments are not within the skill of the art in view of the present disclosure. The public availability of this deposit is not a grant of a license to make, use or sell the deposited material under this or any other patents. The nucleic acid sequence of the deposited material is incorporated in the present disclosure by reference and is controlling if in conflict with any sequence described herein.
Once the germline or Ab12 VH and VL fragments are obtained, these sequences can be mutated to encode the Ab12.6 or Ab12.6-related amino acid sequences disclosed herein. The amino acid sequences encoded by the germline or Ab12 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 Ab12.6 or Ab12.6-related sequence that differ. The appropriate nucleotides of the germline or Ab12 DNA sequences are mutated such that the mutated sequence encodes the Ab12.6 or Ab12.6-related amino acid sequence, using the genetic code to determine which nucleotide changes should be made. Mutagenesis of the germline or Ab12 sequences is carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis.
Once DNA fragments encoding Ab12.6 or Ab12.6-related VH and VL segments are obtained (by amplification and mutagenesis of VH and VL genes, as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as antibody constant region or a flexible linker. The term “operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
In an alternative method, an scFv gene may be constructed with wild type CDR regions (e.g. of Ab12) and then mutated in the manner described in Example 3 below.
The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The present invention further encompasses all known human heavy chain constant regions, including but not limited to all known allotypes of the human heavy chain constant region. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG2 constant region. For a Fab fragment heavy chain, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.
The 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 linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The present invention encompasses all known human light chain constant regions, including but not limited to all known allotypes of the human light chain constant region. DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
It is to 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 (e.g. Chothia, Kabat, Oxford Molecular's AbM modeling software, all of which are known to those of ordinary skill in the art). Such designations, however, are not critical to the invention.
To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence GENKVEYAPALMALS (SEQ ID NO:2) such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by, for example, a second flexible linker GPAKELTPLKEAKVS (SEQ ID NO:3). For other linkers sequences also see e.g., 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 portions of the invention, DNA's encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen 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. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to the insertion of the Ab12.6 or Ab12.6-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the Ab12.6 or Ab12.6-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH “segment” within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can 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 linked in-frame to the amino terminus of the antibody chain gene. The single peptide can be an immunoglobin signal peptide or a heterologous signal peptide (i.e., 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 (e.g., polyadenylation signals) that control the transcription or translation of the 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 such factors as the choice of the host cell to be transformed, the level of the expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression 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), adenovirus, (e.g. the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S. Pat. No. 4,968,615 by 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 replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on 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 the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it's theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Prokaryotic expression of antibody genes has been reported to be ineffective for production of high yields of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include the Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, HEK-293 cells, 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 for expression of the antibody in the host cells or, more preferably, secretion of the antibody into 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 portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to EpoR. The molecules expressed from such truncated DNA molecules also are encompassed 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 heavy and light chain genes are each operatively linked to CMV enhancer/AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are culture to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium.
In view of forgoing, another aspect of the invention pertains to nucleic acid, vector and host cell compositions that can be used for recombinant expression of the antibodies and antibody portions of the invention. The nucleotide sequence encoding the heavy chain variable region of Ab12.6 and variants thereof is shown in
In one embodiment, the invention provides an isolated nucleic acid encoding a heavy chain variable region comprising an amino acid sequence of Formula I:
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)
with the proviso that X1—X2—X3 is other than Y—Y—S.
This nucleic acid can encode only the CDR2 region or, more preferably encodes an entire antibody heavy chain variable region (HCVR). For example, the nucleic acid can encode a HCVR having a CDR2 domain comprising the amino acid sequence of SEQ ID NO:18 and a CDR1 domain comprising amino acid sequence from position 26 to position of 35 of SEQ ID NO:15 and a CDR3 domain comprising the amino acid sequence from position 102 to position 109 of SEQ ID NO:15.
In yet another embodiment, the invention provides isolated nucleic acids encoding an Ab12.6-related CDR2 domain, e.g., comprising amino acid sequences selected from the group consisting of:
In still another embodiment, the invention provides an isolated nucleic acid encoding antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:17. The nucleic acid can encode only the HCVR or can also encode an antibody light chain constant region, operatively linked to the LCVR. In one embodiment, this nucleic acid is in a recombinant expression vector. Those of ordinary skill 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 DNAs which encode the polypeptide sequences described herein. The degeneracy of the genetic code is well established in the art. (See, e.g. 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 any and all degenerate codons at any and all positions in the nucleotide, provided that such codons encode the amino acids sequences as set forth herein.
In still 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 (i.e., the Ab12.6 LCVR although the skilled artisan 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. The nucleic acid can encode the LCVR operatively linked to the HCVR. For example, the nucleic acid can comprise an IgG1, or IgG2 or IgG4 constant region. In a preferred embodiment, the nucleic acid comprises an IgG2 constant region. 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 encoding:
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 a COS cell. Still further 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 can 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 or Ab12.6-related antibodies disclosed herein can be isolated by screening of a recombinant combinatorial antibody library, preferably a scFv yeast display library, prepared using chimeric, humanized or human (e.g. Ab12) VL and VH cDNAs. Methodologies for preparing and screening such libraries are known in the art. In addition to commercially available vectors for generating yeast display libraries (e.g., pYD1 vector, Invitrogen, Carlsbad, Calif.) examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in, for example, Boder E. T. and Wittrup K. D., Yeast surface display for directed evolution of protein expression, affinity, and stability, Methods Enzymol., 328:430-44 (2000) and Boder E. T. and Wittrup K. D., 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 off-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 scFvs on the surface of yeast (preferably Saccaromyces cerevisiae). Ab12 scFvs are analyzed to determine those having the highest expression levels. Such constructs then are screened, preferably using soluble recombinant human EpoR. Those scFv constructs having the highest degree of binding of soluble EpoR are selected for subsequent mutagenesis of the heavy and light chain variable regions to generate CDR mutagenic libraries.
To further increase the off-rate constant for EpoR binding, the VH and VL segments of the preferred VH/VL pair(s) can be randomly mutated, preferably within the CDR2 region of VH, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. This in vitro affinity maturation can be accomplished by replacing a portion of each CDR with a degenerate single-stranded oligonucleotide encoding three amino acids within the CDR being targeted. The replacement of a portion of each CDR with a new randomized sequence (up to 8000 possibilities) can be accomplished by homologous recombination in yeast (see, e.g. Example 3). These randomly mutated VH segments can be analyzed for binding to EpoR in the context of an scFv; scFvs exhibiting an improved fluorescence and a fast off-rate can then be isolated and the CDR mutation identified by sequencing.
Following screening of a recombinant scFv display library, clones having the desired characteristics are selected for conversion, preferably to immunoglobulin gamma type 2/kappa light chain (IgG2/K) antibodies. Nucleic acid encoding the selected antibody can be recovered from the display package (e.g., 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 antibody forms of the invention (e.g., linked to nucleic acid encoding additional immunoglobulin domains, such as additional constant regions). To express a recombinant human antibody isolated by screening of a combinatorial library, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cells, as described in further detail in Section II above.
IV. Uses of Anti-EpoR Antibodies
The antibodies or antigen-binding portion thereof, of the present invention have a number of uses. In general, the antibodies or antigen-binding portion thereof may 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 for treating disorders characterized by low red blood cell levels and/or decreased hemoglobin levels (e.g. anemia). In addition, such antibodies or antigen-binding portions thereof may be used for treating disorders characterized by decreased or subnormal levels of oxygen in the blood or tissue, such as, for example, hypoxemia or chronic tissue hypoxia and/or diseases characterized by inadequate blood circulation or reduced blood flow. Antibodies or antigen-binding portions thereof also may be useful in promoting wound healing or for protecting against neural cell and/or tissue damage, resulting from brain/spinal cord injury, stroke and the like. Non-limiting examples of conditions that may be treatable by the antibodies of the invention include anemia, such as chemotherapy-induced anemia, cancer associated anemia, anemia of chronic disease, HIV-associated anemia, bone marrow transplant-associated anemia and the like, heart failure, ischemic heart disease and renal failure. As such, the invention includes methods of treating any of the aforementioned diseases or conditions comprising the step of administering to a mammal a therapeutically effective amount of said antibody. Preferably, the mammal is a human.
The antibodies or an antigen-binding portions thereof, of the present invention also can be used to identify and diagnose mammals that have a dysfunctional EPO receptor. Mammals that have a dysfunctional EPO receptor are characterized by disorders such as anemia. Preferably, the mammal being 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. Red blood cell aplasia 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 method involves the step of administering to a mammal suffering from said 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 also can be used to detect EPO receptor (e.g., in a biological sample, such as tissue specimens, intact cells, or extracts thereof), using a conventional immunoassay, such as an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA) or tissue immunohistochemistry. The invention provides a method for detecting EPO receptor in a biological sample comprising contacting a biological sample with an antibody or antigen-binding portion of the invention and detecting either the antibody (or antibody portion), to thereby detect EPO receptor in the biological sample. The antibody or antigen-binding portion directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody or antibody fragment. A variety of immunoassay formats may be practiced (such as competitive assays, direct or indirect sandwich immunoassays and the like) and are well known to those of ordinary skill 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, B-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 to human EpoR, the anti-EpoR antibodies, or portions thereof, of the invention can be used to activate or stimulate EpoR activity. The antibodies and antigen-binding portions thereof preferably are capable of activating EpoR activity both in vitro and in vivo. Accordingly, such antibodies and antibody portions can be used to activate EpoR activity, e.g., in a cell culture containing EpoR, in human subjects or in other mammalian subjects having EpoR with which an antibody of the invention cross-reacts.
In another embodiment, the invention provides a method of activating an endogenous activity of a human erythropoietin receptor in a mammal, the method comprising the step of 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. Moreover, 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. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).
In another aspect, the invention provides a method for treating a mammal suffering from aplasia, the method comprising the step of administering to the mammal in need of treatment a therapeutically effective amount of an antibody or antigen-binding portion thereof, of the invention. In addition, the invention provides a method for treating a mammal suffering from anemia, the method comprising the step of administering to the mammal in need of treatment 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-portions 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 portion of the invention along with a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion also may be included. Optionally, disintegrating agents can be included, such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate and the like. In addition to the 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 this invention may be in a variety of forms. They include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g. injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and 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 (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or 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 the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. 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 enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered 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 brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
The antibodies and antibody portions 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 intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. (See, e.g. 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 may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, buccal tablets, troches, capsules, elixiers, suspensions, syrups, wafers, and the like. To administer an antibody or antibody fragment of the invention by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
Supplementary active compounds also can 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, thus avoiding possible toxicities or complications associated with monotherapies or alternatively, act synergistically or additively to enhance the therapeutic effect.
As used herein, the term “therapeutically effective amount” or “pharmaceutically effective amount” means an amount of antibody or antibody portion effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. The exact dose will be ascertainable by one skilled in the art. As known in the art, adjustments based on age, body weight, sex, diet, time of administration, drug interaction and severity of condition may be necessary and will be ascertainable 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 amount effective, at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be tested; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent 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 art of compounding 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 is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
VI. Novel Linker Sequences
The invention also provides novel linker sequences for connecting a first polypeptide sequence and a second polypeptide sequence to form a single polypeptide. In a preferred embodiment the novel linking sequence connects a first polypeptide sequence and a second polypeptide sequence to form a single polypeptide chain, wherein said first polypeptide sequence is capable of binding a ligand, and said second polypeptide sequence is capable of binding a ligand, and wherein said linking sequence comprises one or more amino acid sequences selected from the group consisting of Gly-Phe-Lys-Asp-Ala-Leu-Lys-Gln-Pro-Met-Pro-Tyr-Ala-Thr-Ser (SEQ ID NO: 27); Gly-His-Glu-Ala-Ala-Ala-Val-Met-Gln-Val-Gln-Tyr-Pro-Ala-Ser (SEQ ID NO:2); Gly-Pro-Ala-Lys-Glu-Leu-Thr-Pro-Leu-Lys-Glu-Ala-Lys-Val-Ser (SEQ ID NO:3); and Gly-Glu-Asn-Lys-Val-Glu-Tyr-Ala-Pro-Ala-Leu-Met-Ala-Leu-Ser (SEQ ID NO:4).
VII. Crystal Structures and Methods for Using the Structure Coordinated that Define the Three-Dimensional Structure of an Erythropoietin Receptor in Complex with an Anti-Erythropoietin Receptor Antibody
The crystallizable compositions provided by this invention are amenable to X-ray crystallography. Therefore, this invention also encompasses crystals of the crystallizable compositions. This invention also provides the three dimensional structure as obtained by X-ray crystallography of an erythropoietin receptor/anti-erythropoietin receptor antibody complex at high resolution, such as at 3.2 Å resolution. See Example 21. In a preferred embodiment, the erythropoietin receptor polypeptide is the extracellular domain of human erythropoietin receptor (for example, amino acids 1 to 223 of SEQ ID NO: 41) and the anti-erythropoietin receptor antibody, or an antigen binding fragment thereof, is the Fab fragment of a human Ab12.6.
The three dimensional structures of other crystallizable compositions of this invention may also be determined by X-ray crystallography using X-ray crystallographic techniques routine in the art.
X-ray crystallography is a collection of techniques, which allow the determination of the structure of a molecular entity. The techniques include crystallization of the entity, collection and processing of X-ray diffraction intensities, determination of phases (by, e.g., multiple isomorphous replacement, molecular replacement or difference Fourier techniques) and model building and refinement.
The three-dimensional structure of the extracellular domain of an erythropoietin receptor/Fab fragment of human Ab12.6 mAb complex is defined by a set of structure coordinates as set forth in
As shown in Example 21, the epitope on erythropoietin receptor for Ab12.6 mAb comprises erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114.
A binding site defined by structure coordinates of erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 according to
One embodiment of the present invention provides a molecular complex comprising a binding site defined by structure coordinates of erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 according to
Another embodiment of the present invention provides a molecular complex comprising a binding site, defined by structure coordinates of erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 according to
The present invention further provides a molecular complex comprising a binding site, defined by structure coordinates of erythropoietin receptor amino acids, wherein: (a) amino acid R99 of the erythropoietin receptor is associated with amino acid Y33 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a face/face stacking; (b) amino acid R99 of the erythropoietin receptor is associated with amino acid Y50 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a edge stacking interaction; (c) amino acid W64 of the erythropoietin receptor is associated with amino acid Y33 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is an edge stacking interaction; (d) amino acid E97 of the erythropoietin receptor is associated with amino acid L100 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a weak hydrogen bond; (e) amino acid V112 of the erythropoietin receptor is associated with amino acid L100 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a van der walls interaction; (f) amino acid P107 of the erythropoietin receptor is associated with amino acid D58 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a van der walls interaction; and (g) amino acid H110 of the erythropoietin receptor is associated with amino acid G101 of the heavy chain of the anti-erythropoietin receptor antibody, wherein said association is a van der walls interaction, according to
The present invention yet further provides a molecular complex comprising a binding site, defined by structure coordinates of erythropoietin receptor amino acids, wherein: (a) amino acid H110 of the erythropoietin receptor is associated with amino acid H91 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a face/face stacking interaction; (b) amino acid P107 of the erythropoietin receptor is associated with amino acid Y94 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a van der waals interaction; (c) amino acid R111 of the erythropoietin receptor is associated with amino acid E31 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a hydrogen bond; (d) amino acid R111 of the erythropoietin receptor is association with amino acid E32 of the light chain of the anti-erythropoietin receptor antibody, wherein said associated is a hydrogen bond; (e) amino acid E25 of the erythropoietin receptor is associated with amino acid R30 of the light chain of the anti-erythropoietin receptor antibody, wherein said associated is a hydrogen bond; (f) amino acid L26 of the erythropoietin receptor is associated with amino acid R30 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a hydrogen bond; (g) amino acid V112 of the erythropoietin receptor is associated with amino acid A50 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a van der waals interaction; and (h) amino acid H114 of the erythropoietin receptor is associated with amino acid C53 of the light chain of the anti-erythropoietin receptor antibody, wherein said association is a hydrogen interaction.
Another embodiment of the present invention provides a molecular complex defined by structure coordinates of one or more anti-erythropoietin receptor antibody amino acids Y33, Y50, D58, L100 and G101 of the heavy chain and amino acids R30, E31, E32, A50, H91 and Y94 of the light chain according to
Yet another embodiment of the present invention provides a molecular complex defined by at least a portion or all of the structure coordinates of all the erythropoietin receptor and anti-erythropoietin receptor antibody amino acids set forth in
Those of skill in the art will understand that a set of structure coordinates for a polypeptide complex is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape.
The variations in coordinates discussed above may be generated due to mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in
Alternatively, modification in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three dimensional shape is considered to be the same as that of the unmodified crystal.
Various computational analyses are therefore necessary to determine whether a molecular complex or a portion thereof is sufficiently similar to all or parts of the extracellular domain of a erythropoietin receptor/Fab fragment of human Ab12.6 mAb structure described above as to be considered the same. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in its accompanying User's Guide.
The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results.
Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention, equivalent atoms such as protein backbone atoms (N, C.alpha., C and O) will be defined for all conserved residues between the two structures being compared. Also, only rigid fitting operations will be considered.
When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
For the purpose of this invention, any molecular complex that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) between 0.00 Å and 1.50 Å, preferably between 0.00 Å and 1.00 Å, more preferably between 0.00 Å and 0.50 Å, when superimposed on the relevant backbone atoms described by the structure coordinates listed in
Once the structure coordinates of a protein crystal have been determined, they are useful in solving the structures of other crystals.
In accordance with the present invention, the structure coordinates of a complex comprising the extracellular domain of erythropoietin receptor and Fab fragment of, for example, human Ab12.6 mAb, and portions thereof, is stored in a machine-readable storage medium. A machine could be a computer. Such data may be used for a variety of purposes, such as drug discovery, discovery of Ab12.6 mAb variants with improved properties, such as improved specific binding to erythropoietin receptor, and X-ray crystallographic analysis of other protein crystals.
In order to use the structure coordinates generated for the erythropoietin receptor/anti-erythropoietin receptor antibody complex or one of its binding sites or homologues thereof, it is necessary to convert them into a three-dimensional shape. This is achieved through the use of commercially available software that is capable of generating a three-dimensional graphical representation of molecular complexes, or portions thereof, from a set of structure coordinates.
Accordingly, one embodiment of this invention provides a machine-readable data storage medium comprising a data storage material encoded with machine-readable data comprising a portion of or the entire set of the structure coordinates set forth in
A computer of this invention comprises a machine-readable data storage medium encoded with machine-readable data, wherein said data comprises one of the following four structure coordinates: (1) the structure coordinates of erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 according to
This invention also provides a computer for determining at least a portion of the structure coordinates corresponding to X-ray diffraction data obtained from a molecular complex of erythropoietin receptor/anti-erythropoietin receptor antibody, wherein said computer comprises: a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates of erythropoietin receptor and/or anti-erythropoietin receptor antibody according to
Preferably, the computer further comprises a display for displaying said structure coordinates.
This invention also provides a computer for determining at least a portion of the structure coordinates corresponding to an X-ray diffraction pattern of a molecular complex, wherein said computer comprises: a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises at least a portion of the structure coordinates according to
For the first time, the present invention permits the use of structure-based and rational drug design techniques to design, select, and synthesize chemical entities, compounds (such as agonists or antagonists of erythropoietin receptor), and AB12.6 mAb variants with improved properties, such as higher or lower binding affinity for erythropoietin receptor as compared to Ab12.6 mAb. Additionally, the present invention permits the use of structure-based or rational drug design techniques to make improvements of currently available erythropoietin receptor antagonists, that are capable of binding to the extracellular domain of erythropoietin receptor/Fab fragment of human Ab12.6 mAb complex, or any portion thereof.
One particularly useful drug design technique enabled by this invention is iterative drug design. Iterative drug design is a method for optimizing associations between a protein and a compound (that compound includes an antibody) by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.
In iterative drug design, crystals of a series of protein/compound or antibody complexes are obtained and then the three-dimensional structure of each new complex is solved. Such an approach provides insight into the association between the proteins and compounds or antibodies of each new complex. This is accomplished by selecting compounds or antibodies with inhibitory activity, obtaining crystals of the new protein/compound or antibody complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound or antibody complex and previously solved protein/compound or antibody complexes. By observing how changes in the compound or antibody affect the protein/compound or antibody associations, these associations may be optimized.
In some cases, iterative drug design is carried out by forming successive protein-compound or antibody complexes and then crystallizing each new complex. Alternatively, a pre-formed protein crystal is soaked in the presence of an inhibitor, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound or antibody complex.
The structure coordinates set forth in
The structure coordinates set forth in
Therefore, another embodiment of this invention provides a method of utilizing molecular replacement to obtain structural information about a crystallized molecular complex whose structure is unknown comprising the steps of: a) generating an X-ray diffraction pattern from said crystallized molecular complex; and b) applying at least a portion of the structure coordinates set forth in
Preferably, the crystallized molecular complex comprises an erythropoietin receptor polypeptide and an anti-erythropoietin receptor antibody polypeptide.
By using molecular replacement, all or part of the structure coordinates of the extracellular domain of the erythropoietin receptor/Fab fragment of the human Ab12.6 mab complex provided by this invention (and set forth in
Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.
Thus, molecular replacement involves generating a preliminary model of a molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the extracellular domain of the erythropoietin receptor/Fab fragment of the human Ab12.6 mAb complex according to
The structure of any portion of any crystallized molecular complex that is sufficiently homologous to any portion of the extracellular domain of an erythropoietin receptor/Fab fragment of human Ab12.6 mAb complex can be solved by this method.
In a preferred embodiment, the method of molecular replacement is utilized to obtain structural information about a molecular complex, wherein the complex comprises an erythropoietin receptor-like polypeptide. Preferably the erythropoietin receptor-like polypeptide is erythropoietin receptor, a mutant thereof or a homologue thereof.
The structure coordinates of the extracellular domain of an erythropoietin receptor/Fab fragment of a human Ab12.6 mAb complex as provided by this invention are particularly useful in solving the structure of other crystal forms of erythropoietin receptor-like polypeptide, preferably other crystal forms of erythropoietin receptor; erythropoietin receptor-like polypeptide/anti-erythropoietin receptor antibody-like polypeptide, preferably the extracellular domain of erythropoietin receptor/Fab fragment of human Ab12.6 mAb; or complexes comprising any of the above.
Such structure coordinates are also particularly useful to solve the structure of crystals of erythropoietin receptor-like polypeptide/anti-erythropoietin receptor antibody-like polypeptide complexes, particularly the extracellular domain of a erythropoietin receptor/Fab fragment of a human Ab12.6 mAb, co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities and interaction of candidate erythropoietin receptor agonists or antagonists with erythropoietin receptor or the extracellular domain of erythropoietin receptor/Fab fragment of human Ab12.6 mAb complex. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvent allows determination of the location where each type of solvent molecule resides. Small molecules that bind tightly to these sites can then be designed and synthesized and tested for their erythropoietin receptor antagonist activity.
In another preferred embodiment, methods for generating the structure coordinates of protein homologues of erythropoietin receptor using the X-ray coordinates of erythropoietin receptor described in
All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3.5 Å resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, 01992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)). This information may thus be used to optimize known erythropoietin receptor antagonists, such as anti-erythropoietin receptor antibodies, and more importantly, to design new or improved erythropoietin receptor antagonists.
A chemical entity, a compound (including an agonist or antagonist of erythropoietin receptor) or a variant of the Ab12.6 mAb, or an antigen binding fragment thereof, or human Ab12.6 mAb, or an antigen binding fragment thereof, or variants of another anti-erythropoietin receptor antibody, or an antigen binding fragment thereof, can be designed by computational means by performing fitting operations. A compound includes macromolecules such as proteins or polypeptides.
The present invention also encompasses methods of evaluating the potential of a chemical entity to associate with a molecular complex of this invention, or a homologue of said molecular complex.
This invention provides a method for evaluating the potential of a ligand to associate with a molecular complex of this invention, or a homologue of said molecular complex, comprising the steps of: (i) employing computational means to perform a fitting operation between the chemical entity and a binding site (the binding site could be a binding site for Ab12.6 mAb, or an antigen binding fragment thereof, or human Ab12.6 mAb, or an antigen binding fragment thereof) of the molecular complex or a binding site of the homologue of the molecular complex; and (ii) analyzing the results of said fitting operation to quantify the association between the chemical entity and either binding site.
The present invention also encompasses methods for identifying a potential ligand of erythropoietin receptor comprising the steps of: a) using the structure coordinates of erythropoietin receptor amino acids E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 according to
This invention also encompasses methods for evaluating the potential of a variant of Ab12.6 mAb, or an antigen binding fragment thereof, or another anti-erythropoietin receptor antibody, or an antigen binding fragment thereof, to associate with a molecular complex of this invention or a homologue of said molecular complex; comprising the steps of: (i) employing computational means to perform a fitting operation between the variant and a binding site (the binding site could be a binding site for Ab12.6 mAb, or an antigen binding fragment thereof, of a molecular complex of this invention or a binding site (the binding site could be a binding site for Ab12.6 mAb, or an antigen binding fragment thereof, of a homologue of the molecular complex; and (ii) analyzing the results of said fitting operation to quantify the association between the binding site of the molecular complex or the binding site of the homologue of the molecular complex.
For the first time, the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities, compounds, including agonists or antagonists of erythropoietin receptor, and variants of Ab12.6 (or another anti-erythropoietin receptor antibody), and antigen binding fragments thereof, capable of binding to erythropoietin receptor.
The design of chemical entities, compounds including agonists or antagonists of erythropoietin receptor and variants of Ab12.6 mAb (or another anti-erythropoietin receptor antibody), and antigen binding fragments thereof, that bind to erythropoietin receptor according to this invention generally involves consideration of two factors. First, the chemical entity, compound or AB12.6 mAb variant must be capable of physically and structurally associating with erythropoietin receptor. Non-covalent molecular interactions important in the association of a protein, such as erythropoietin receptor, with its binding partner include hydrogen bonding, van der Waals and hydrophobic interactions.
Second, the chemical entity, compound or Ab12.6 mAb variant must be able to assume a conformation that allows it to associate with erythropoietin receptor directly. Although certain portions of the chemical entity, compound or Ab12.6 mAb variant or humanities Ab12.6 mAb variant will not directly participate in these associations, those portions of the chemical entity, Ab12.6 mAb variant or compound may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity, Ab12.6 mAb variant or compound in relation to all or a portion of the binding site, e.g., active site or accessory binding site of erythropoietin receptor, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with erythropoietin receptor.
An erythropoietin receptor-binding entity, compound or variant of Ab12.6 mAb, or antigen binding fragments of either, can be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the binding sites of erythropoietin receptor as defined by this invention.
One skilled in the art can use one of several methods to screen chemical entities or fragments for their ability to associate with erythropoietin receptor and more particularly with the binding sites of erythropoietin receptor. This process may begin by visual inspection of, for example, the binding sites for anti-erythropoietin receptor antibody, on the computer screen based on the erythropoietin receptor coordinates in
The initial objective of this study was to decrease the off-rate of Ab12 IgG2/K using yeast display technology. To meet this objective, Ab12 IgG2/K was converted into an scFv using linker sequences. Several different linker sequences were scrutinized during the construction of Ab12 scFv (
Various single stranded oligonucleotides encoding Ab12 scFvs were co-transformed with a linearized “gapped” vector derived from pYD1 (Invitrogen, Carlsbad, Calif.) into yeast by techniques well known to practitioners in the art. Functional cell surface protein expression was compared by incubating the transformed yeast with increasing concentrations of soluble EpoR (EposR) at 37° C. (
Off-rate measurements of Ab12 41/40 scFv were performed by incubation of 0.5 μM EposR with 0.1 O.D. yeast (approximately 1×106 yeast cells) for 1.5 hours at 37° C.; following this cells were chilled on ice and washed at 4° C. A 10,000 fold excess of Ab12 IgG1 (Abbott Laboratories, Abbott Park, Ill.), warmed to 37° C., was added to the cells and individual samples were withdrawn at various time points, chilled and later read on an Epics XL1 flow cytometer (Beckman Coulter, Fullerton, Calif.). The experiment was designed so that as EposR dissociated from Ab12 scFv, it would immediately bind to Ab12 IgG1 (present at a saturating concentration) and would no longer be detected on the surface of yeast. The remaining bound EposR was detected by addition of MAB307 followed by addition of anti-mouse PE.
All 6 CDRs of Ab12 41/40 (three in the heavy chain and three in the light chain) were subjected to randomization, and libraries composed of 8000 members each were generated. Linerarized “gapped” pYD1 vector (Invitrogen) was modified to include a TEV protease site and also to contain Ab12 41/40 scFv sequence (i.e. pYD1Tev-Ab12-41/40). Thereafter, gapped pYD1-Tev-Ab12-41/40 vectors, missing 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 being targeted. The replacement of a portion of each CDR with a new randomized sequence (up to 8000 possibilities) was accomplished by homologous recombination in yeast. A schematic of this library construction method is shown in
All 50 Ab12 scFv libraries and wild type Ab12 scFv yeast were subjected to off-rate FACS analysis on a MoFlo high-speed cell sorter. (Dako Cytomation California Inc. Carpinteria, Calif.) Transformed yeast cells (0.6 O.D.) were incubated with 0.5 μM EposR at 37° C. until equilibrium was reached (2 hours). Cells were then chilled, washed, and a 10,000 fold molar excess (5 μg/mL) of Ab12 IgG1 prewarmed to 37° C. was added. After a 20-minute incubation at 37° C., cells were again chilled, washed and labeled depending on whether they were being prepared for “one-color” FACS or “two-color” FACS. For the former, cells were labeled with a mixture of MAB307 and anti-mouse PE. For the latter, cells were labeled first with a mixture of MAB307 and rabbit anti-6-his antibody (Research Diagnostics, Flanders, N.J.), followed by a mixture of anti-mouse PE and goat anti-rabbit FITC (Southern Biotech, Birmingham, Ala.). Individual control samples were also prepared to set MoFlo compensation and to ensure no non-specific background staining existed.
For Round 1 off-rate FACS, each library sample was compared to Ab12 scFv yeast (WT control) for evidence of a population of cells having an increased FL2 fluorescence (and, therefore, a potentially longer off-rate). In each case, the brightest 1% of cells in the FL2 axis were gated, collected, and re-grown in media (Round 1 output). For Round 2 off-rate FACS, the identical cell incubation procedure was performed on each Round 1 library output for some libraries; for others, the Round 2 FACS involved additional reagents to detect surface expression. For each Round 2 off-rate FACS analysis, a gate was drawn around the top 0.1% of cells in the FL2 axis, and this gate was superimposed on all Round 1 library outputs, where applicable. Libraries displaying a population of cells having a higher FL2 than those in the WT gate were selected for FACS, those with no cells inside of the reference gate were not analyzed further. For those selected libraries, the brightest 0.1% of cells in the FL2 axis were gated and collected. An aliquot was plated on selective media for yeast (SD or “single dropout”) for yeast colony isolation and the remainder were grown as liquid cultures for future cell analysis.
Selected bulk Round 2 outputs were grown in liquid media and subjected to off-rate analysis (data not shown). Outputs displaying improved off-rate curves were chosen for further analysis. Individual clones from these outputs were recovered following plating on selective media and plasmid DNA isolation. PCR was used to amplify the scFv region of each clone and products were sequenced to identify the amino acid substitutions. Table 1 highlights sequencing results from each Round 2 output. All unique clones were named and the frequency of their prevalence noted.
To determine which clones from the affinity maturation would be converted into an IgG2/K format, outputs from each library were analyzed and considered for the following parameters: frequency of isolation, consensus sequence change in the CDR, and overall fluorescent shift of bulk outputs and individual yeast clones. Those clones appearing at a higher frequency, containing a representative consensus change in CDR sequence and having the highest overall FL2 signal in off-rate and equilibrium binding analyses were chosen for conversion.
Selected scFvs were converted into 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 vector pBOS (Mizushima and Nagata, Nucleic Acids Research, Vol 18, pg 5322, 1990). pBOS plasmids encoding both heavy and light chain regions were transfected transiently into COS cells and resulting supernatants from cell cultures were purified over a protein A sepharose column. Purified antibodies were dialyzed into phosphate buffered saline (PBS) and quantitated by optical density 280 (O.D.280) spectrophotometric reading. Each antibody was subjected to affinity measurements by BIAcore and used as a test article in UT-7/Epo and F36E cell proliferation assays.
BIAcore analyses were performed on a BIAcore 2000 utilizing the BIAcontrol software version 3.1.0 and on a BIAcore 3000 utilizing the BIAcontrol software version 4.0.1. (BIAcore, Uppsala, Sweden) using EposR as the test antigen. Table 2 highlights the affinity parameters of each mutated Ab12 clone compared to Ab12.
As Table 2 shows, Ab12.6 and Ab12.56 showed faster off-rates and higher Kd values relative to Ab12.
To determine the contribution of the 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 where appropriate. Sub-variants also were subjected to BIAcore analyses as described above. Table 3 highlights the affinity parameters of each subvariant clone.
Ab12, Ab12.6 and Ab12.6-related variants were tested in established in vitro cell proliferation assays. Stock cultures of the human erythroleukemic cell lines, UT-7/Epo, or F36E cells were maintained in DMEM or RPMI 1640 media respectively with 10% fetal bovine serum and 1 unit per mL of recombinant human erythropoietin. Prior to assays, cells were cultured overnight at a density of 4.0 to 5.0×105 cells per mL in growth medium without Epo. Cells were recovered, washed and resuspended at a density of 1.0×106 cells per mL in assay medium (RPMI 1640 or DMEM+10% FBS) and 50 uL of cells added to wells of a 96 well microtiter plate. 50 uL of each of Ab or Epo standard (recombinant human Epo (rHuEpo)) in assay medium were added to wells at final concentrations ranging 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 (as prepared per manufacturer's instructions, Madison, Wis.) was added to all wells. 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.
EC50 and Emax values (shown in Table 4 below) were determined from graphs generated from the spectrophotometric data. Higher affinity antibodies (Ab12.17, Ab12.25, Ab12.61 and Ab12.76) produced bell-shaped curves from which EC50 and/or Emax data could not be obtained. In contrast, curves generated from the lower affinity antibodies (shown in Table 4) produced sigmoidal curves (as does the native ligand Epo). Furthermore, as Table 4 and
Transgenic mice that produced only human EpoR (hEpoR+, single allele) and no endogenous mouse EpoR (mEpoR−/−, double allele mutation) were generated as described in Liu, C. et al, Journal 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.
Fresh human bone marrow obtained from Cambrex Bio Science Walkersville, Inc. (Walkersville, Md.) were cleared of red blood cells by methods well known in the art and resuspended at 2.5×106 cells/mL in IMDM-2% FBS. Cells (0.1 mL) were added to 17×100 mm culture tubes (VWR, West Chester, Pa.) containing 2.4 mL Methocult (StemCell Technologies, Vancouver, Canada), 0.6 mL of IMDM-2% FBS, 0.066 mL stem cell growth factor (Sigma, St. Louis, Mo., 1 μg/mL), and Epogen™ (Dik Drug Co., Chicago, Ill.), Aranesp™ (Dik Drug Co.), Ab12, Ab12.6 or isotype control Ab at the concentrations indicated. After mixing, 1.1 mL of the Methocult suspension was added to a 35 mm non tissue culture treated sterile petri dish and incubated at 37° C., 5% CO2 for 2 weeks. Colonies, identified microscopically, were red in color. The results in
Fresh harvested bone marrow collected from femurs of mEpoR−/−, hEpoR+ transgenic mice were cleared of red blood cells by methods well known in the art and resuspended at 2×106 cells/mL in IMDM-2% FBS. Cells (0.1 mL) were added to 17×100 mm culture tubes (VWR, West Chester, Pa.) containing 3.0 ml Methocult (StemCell Technologies, Vancouver, Canada), 0.165 mL stem cell growth factor (Sigma, St. Louis, Mo., 1 μg/mL), and Epogen™ (Dik Drug Co., Chicago, Ill.), Aranesp™ (Dik Drug Co.), Ab12, Ab12.6, or isotype control Ab at the concentrations indicated. After mixing, 1.11 mL of the Methocult suspension was added to a 35 mm non tissue culture treated sterile petri dish and incubated at 37° C., 5% CO2 for 2 weeks. Colonies stained with benzidine (Reference Fibach, E., 1998 Hemoglobin, 22:5-6, 445-458) were identified microscopically. The results in
Experiments were performed to determine the effect of a single dose of Ab12.6 on erythropoiesis relative to Aranesp™ (Amgen, Thousand Oaks, Calif.), a longer acting variant of Epogen. Transgenic mice (mEpoR−/−, hEpoR+ as described in Example 10) were injected subcutaneously once with Ab-12, Ab12.6 or an isotype control Ab at 0.8 mg/kg in 0.2 mL vehicle (phosphate buffered saline [PBS] containing 0.2% bovine serum albumin [BSA]). Control animals were injected the same way with Aranesp™ at 3 μg/kg only a second Aranesp™ dose also was administered on day 14 (the standard of care Aranesp™ dosing regimen is 3 μg/kg administered biweekly). Sample bleeds were taken on day 0, 7, 14, 21 and 28 for determining hematocrits by methods well known in the art. As shown in
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) wherein 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 biased codon selection. In addition, the NHS codon prevents creation of TGC (only possible codon for cysteine), and TGG (only possible codon for tryptophan), CGC, CGG, and AGG (all possible codons for arginine), and AGC (one of three possible serine codons). In the lower linker sequence, the use of the VNS codon limits the creation of TCC and TCG, two of three possible serine codons. In addition, the VNS codon prevents 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 (only possible stop codon), and TTG (one of three possible codons for leucine). These linker sequences were synthesized as part of a longer synthetic oligonucleotide which also contained complementary elements to a portion of a control scFv DNA sequence LT28-8A having a (G4S)3 linker sequence. LT28-8A was generated using standard molecular biological 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 Aug. 16, 2001 and incorporated herein by reference) with Ala-Ala-Gly-Asp-Asp-Phe-Leu-Val-Ser-Met-Leu. Linker sequence (G4S)3 is described in U.S. Pat. Nos. 5,258,498 and 5,482,858 which patents are incorporated herein by reference. The extended linker library oligonucleotides were incorporated into the LT-28-8A scFv by PCR.
NHS- and VNS-linker PCR products were generated, purified and mixed with restriction-digested yeast-display plasmid (pYD-1) containing homologous regions of complementary DNA sequence present in both the 5′ and 3′ termini of NHS- and VNS-linker PCR products. PCR generated products encoding the entire LT28-8A scFv (with an NHS or VNS-encoded linker) were inserted by homologous recombination into the galactose-inducible pYD-1 vector such that they were in-frame. Homologous recombinants were selected by subsequent growth in tryptophan- and uracil-minus media. Titers of the resulting NHS- and VNS-linker libraries were assessed by colony counts and the libraries were prepared for analysis by a fluorescense-activated cell sorter (FACS).
Dot plots of NHS- and VNS-linker LT28-8A scFv libraries from Example 14 were compared with those of LT-28-8A scFv when induced yeast cells from all groups were incubated with V5-FITC monoclonal antibody (Invitrogen, Carlsbad, Calif.). The V5 epitope tag 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 expression levels of the scFv on the surface of yeast. Percent of cells staining positive for FITC: LT-28-8A was 58%; NHS-library was 31%; and VNS-library was 47%.
FACS analysis of cells from the three test groups were compared following the addition of biotinylated IL-18, prepared as described in WO 01/58956, and streptavidin R-phycoerythin (RPE) (Jackson ImmunoResearch, West Grove, Pa.) and V5-FITC. The fluorescence of RPE represents the binding of antigen to the scFvs on the surface of yeast, and, in conjunction with the presence of the V5 epitope, a dual-color signal is generated by clones that express full-length scFv and bind antigen. A concentration of 30 nM biotinylated IL-18 was chosen for this analysis because the LT-28-8A scFv had a KD of 30 nM on the surface of yeast. Percent of cells staining positive for both FITC and RPE: LT-28-8A was 55%; NHS-library was 25%; and VNS-library was 36%.
Cells from the NHS- or VNS-LT28-8A scFv libraries that demonstrated fluorescence identical to control were individually gated and collected using a cell sorter. These collected populations (termed the “outputs”) were amplified in liquid culture and aliqouts of culture were plated on to solid media to isolate individual colonies. DNA was extracted from individual colonies and the linker nucleotide sequence was determined by DNA sequencing.
To determine if linkers containing variable amino acids had any effect on the behavior of scFvs in vitro or in vivo, 11 random NHS-R1 output scFv clones from Example 15, containing linkers with only one glycine and serine, were selected and tested in a series of assays.
The dissociation constants (Kd) of 11 NHS-R1 output scFv clones and LT-28-8A scFv (with the (G4S)3 linker) were measured in a seven-point titration analysis. These included NHS-R1 output scFv clones: 13, 19, 22, 23, 30, 33, 34, 38, 40, 41, and 44. Binding of antigen was assayed as described in Example 14. All NHS-R1 output scFv clones and control scFv showed Kds of about 22-26 nM.
Expression, in vivo, of 10 NHS-R1 output clones (10, 13, 19, 30, 33, 34, 38, 40, 41, 44) and LT-28-8A scFv were analyzed following the construction of expression constructs encoding the scFvs. All 10 LT28-8A scFv sequences were ligated into the pUC19/pCANTAB (U.S. Pat. No. 5,872,215) inducible expression vector and transformed into TG-1 cells. Following growth under restricted expression, scFv induction was initiated by addition of 1 mM IPTG and soluble scFv was affinity purified from periplasmic preparations of induced TG-1 cells. Clones 13, 19 and 30 grew very poorly and were not induced. Purified scFv was assayed for protein concentration by BCA assay:
The soluble scFvs produced by LT-28-8A and NHS-R1 output 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 1×10−7 to 2×10−7 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; Linker sequence 41 is SEQ ID NO:2.
Recombinant-expressed EpoR extracellular domain was produced through CHO cell expression and purified to homogeneity. Three micrograms of EpoR extracelluar domain per lane were electrophoresed on 4-20% poly-acrylamide gels under either denaturing conditions (in SDS buffer) or native conditions (no SDS buffer). For Western blot analysis, gels were transferred to PVDF membranes, blocked with 5% dry milk and incubated with Ab12.6 (10 μg/ml) for 1-2 h at room temperature. Membranes were washed four times with PBS/Tween, incubated with HRP conjugated goat anti-human antibody (1:2500) and developed with 4-chloro-1-naphthol as substrate. As
In order to map the EpoR binding site and provide a molecular basis for the interaction of Ab12.6 with this site, a soluble form of mature EpoR extracellular domain (ECD) (SEQ ID NO: 40) including a his-tag, was expressed in E. coli and purified as described (Johnson, D. L. et al. Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli. Protein Expr. Purif. 7, 104-113 (1996)). To facilitate the generation of Fab fragments, Ab12.6 was re-engineered as an IgG1 human antibody and subjected to papain cleavage essentially as described in Harlow, E. & Lane, D. Antibodies, A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988). Samples for crystallization contained 1:1 complexes of EpoR ECD and Ab12.6 Fab fragments at a concentration of 14 mg/mL in 20 mM HEPES, 150 mM NaCl, 1 mM NaN3 at pH7.5. Crystallization was carried out using the hanging drop vapor diffusion method at 17° C. combining 2 μL protein with 2 μL of reservoir solution consisting of 15% PMME5000 and 600 mM Li2SO4. Protein crystals grew to approximately 0.8×0.1×0.1 mm in two weeks time. The cryopreservative was made using 80% reservoir solution and 20% glycerol. Crystals were flash frozen in liquid nitrogen for data collection after quick passage through the cryopreservative. Data were collected at the IMCA beamline ID-17 at Argonne National Laboratory and diffraction data were collected and processed to 3.2 Å resolution using HKL2000 (Otwinowski, Z. & Minor, W. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-326 (1997).) The crystals are space group P212121 and unit cell parameters a=117.95, b=156.17, c=164.20 with three Fab's bound to three EpoR's in the asymmetric unit based on Matthews parameter calculations.
The structure was solved using a combination of Phaser (McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol. Crystallogr. 61, 458-464 (2005)) and Molrep (Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022-1025 (1997)) for molecular replacement. The search model used in Phazser for the Fab fragment was 1JPT, and an ensemble of EPOR structures (1CN4, 1EBA, 1EBP and 1EER) were used to search for the EPOR portions. This procedure identified two Fab/EpoR complexes in the asymmetric unit. One of these Fab/EpoR complexes was then used as a search model in Molrep to identify the third Fab/EpoR complex in the asymmetric unit with the first two complexes from Phaser held fixed. The resulting structure showed well determined electron density for three copies of EpoR, two well-defined copies of the Ab12.6 Fab, while the third copy has well-defined density of the L and H chains in the CDR domains, the conserved domains of the L and H chains of the third copy are solvent exposed and not well ordered. Refinement was initiated with multiple rounds of visual inspection and manual fitting in Quanta (Accelrys Software, Inc., San Diego, Calif.) and refinement using CNX (Brunger, A. T. et al. Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905-998 (1998) and Badger, J., Berard, D., Kumar, R. A., Szalma, S., Yip, P., Griesinger, C., Junker, J., in CNX Software Manual, Molecular Simulations, Inc. (1999), San Diego, Calif. Badger J, Berard D, Kumar R A, Szalma S, Yip P, Griesinger C, et al. CNX software manual. San Diego, Calif.: Molecular Simulations, 1999) followed by a final refinement using refmac (Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S., & Dodson, E. J. Efficient anisotropic refinement of Macromolecular structures using FFT. Acta Crystallogr. D Biol. Crystallogr. 55, 247-255 (1999)) to refine the structure to 3.2 Å resolution with an Rwork=25% and Rfree=32%.
This crystal structure of the Fab-EpoR confirmed that Ab12.6 binds EpoR through a non-linear, conformationally defined epitope that includes residues E25, L26, W64, E97, R99, P107, H110, R111, V112 and H114 of EpoR. (See
Monomeric Fab and bivalent F(ab′)2 fragments of Ab12.6 were prepared and purified using standard papain and pepsin digestion conditions (Pierce ImmunoPure Fab and F(ab′)2 Preparation Kits; Pierce, Rockford Ill.). Stock cultures of the human erythroleukemic cell line, F36E cells were maintained in RPMI 1640 media with 10% fetal bovine serum and 1 unit per mL of recombinant human erythropoietin. Prior to assays, cells were cultured overnight at a density of 4.0 to 5.0×105 cells per mL in growth medium without EPO. Cells were recovered, washed and resuspended at a density of 1.0×106 cells per mL in assay medium (RPMI 1640+10% FBS) and 50 uL of cells added to wells of a 96 well microtiter plate. Fifty uL of 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 wells 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 (as prepared per manufacturer's instructions, Madison, Wis.) was added to all wells. 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, Mass.). The results, seen in
The present invention is illustrated by way of the foregoing description and examples. The foregoing description is intended as a non-limiting illustration, since many variations will become apparent to those skilled in the art in view thereof. Changes can be made to the composition, operation and arrangement of the method of the present invention described herein without departing from the concept and scope of the invention.
This application is a continuation-in-part of U.S. patent application Ser. No. 11/102,424, filed Apr. 8, 2005, which claims priority to U.S. provisional application Ser. Nos. 60/561,084 and 60/561,313, filed Apr. 9, 2004 and Apr. 12, 2004, respectively, the specifications of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60561084 | Apr 2004 | US | |
| 60561313 | Apr 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11102424 | Apr 2005 | US |
| Child | 11614772 | Dec 2006 | US |
