WO1998038211A2

WO1998038211A2 - Permuted hemoglobin-like proteins

Info

Publication number: WO1998038211A2
Application number: PCT/US1998/003836
Authority: WO
Inventors: Elaine A. Best; Peter O. Olins
Original assignee: Somatogen, Inc.
Priority date: 1997-02-28
Filing date: 1998-02-27
Publication date: 1998-09-03
Also published as: WO1998038211A3; AU6340998A

Abstract

The present invention provides novel hemoglobin-like proteins that have permuted termini such that novel N- and C-termini are created. The new termini can be attached to functional polypeptides or other hemoglobin molecules to form multimeric hemoglobins.

Description

PERMUTED HEMOGLOBIN-LIKE PROTEINS

Field of the Invention This invention relates to permuted hemoglobin-like proteins or

"permuteins" and more particularly to multimeric hemoglobin-like proteins composed of two or more globin subunits linked together by genetic fusion to form permuteins.

Background of the Invention

Hemoglobin ("Hb") is the oxygen-carrying component of blood that circulates through the bloodstream inside small enucleate cells called erythrocytes or red blood cells ("RBCs")- Hb is a protein consisting of four associated polypeptide chains (two α-globins and two β-globins) and prosthetic groups known as hemes. The erythrocyte helps maintain Hb in its reduced, functional form.

About 92% of the normal adult human hemolysate is HbA, which is comprised of two α and two β globin chains. The structure of HbA and various naturally-occurring variants are well known (Bunn and Forget, eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W.B. Saunders Co., Philadelphia, PA, 1986) and Fermi and Perutz "Hemoglobin and Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in Biology (Clarendon Press, 1981)).

Segments of the globin chains are stabilized by folding into one of two common conformations, the alpha helix and the beta pleated sheet. In its native state, about 75% of the Hb molecule is alpha-helical. The alpha-helical segments are separated by non-helical regions or segments of the chain that are less constrained than the helical regions. The alpha-helical segments of each chain are commonly identified by letters and each amino acid within a helix additionally by a number. For example, the proximal histidine of the alpha chain is F8 (i.e., residue 8 of the F helix). The non-helical segments are identified by letter pairs, indicating which helical segments they connect. For example, non-helical segment BC lies between the B helix and the C helix. For the amino acid sequence and helical residue notation for the alpha and beta chains of normal human Hb A, see Bunn and Forget, supra.

The tertiary structure of the Hb molecule refers to the steric relationships of amino acid residues that are not proximal in the linear sequence of each chain, while the quaternary structure refers to the way in which the subunits (chains) are packed together. The tertiary and quarternary structure of the Hb molecule have been determined using X-ray diffraction analysis of Hb crystals. This determination allows one to calculate the three-dimensional positions of the atoms of the molecule and to determine the relative proximity of various amino acids to one another in the globin subunit chains. The tertiary and quaternary structures of native oxyhemoglobin and deoxyhemoglobin are sufficiently well known that almost all of the non-hydrogen atoms can be positioned with an accuracy of 0.5 A or better. For human deoxyhemoglobin, see Fermi, et al., T. Mol. Biol., 175: 159 (1984), and for human oxyhemoglobin, see Shaanan, T. Mol. Biol., 171: 31 (1983).

In its unoxygenated ("deoxy," or "T" for tense) form, the subunits of Hb A form a tetrahedron having a two-fold axis of symmetry. The axis runs down a water-filled "central cavity." The subunits interact with one another by means of Nan der Waals forces, hydrogen bonds and by ionic interactions (or "salt bridges"). The αiβi and ct2β2 interfaces remain relatively fixed during oxygenation. In contrast, there is considerable flux at the αχβ2 and ct2βi interfaces. In the oxygenated ("oxy," or "R" for relaxed) form, the Hb intersubunit distances are increased. The human α and β globin genes are located on chromosomes 16 and 11, respectively. Both genes have been cloned and sequenced (Liebhaber et al., PΝAS 77:7054-58 (1980); Marotta et al., T. Biol. Chem.. 252:5040-53 (1977); Lawn et al, Cel 21:647 (1980)).

It is not always practical to transfuse a patient with donated blood. In these situations, use of a red blood cell "substitute" is desirable. The substitute must effectively transport oxygen, preferably but not necessarily as well as or better than RBCs. The two types of such oxygen-delivering therapeutics that have been studied most extensively are Hb solutions and fluorocarbon emulsions.

Native HbA, injected directly into the blood stream, does not provide efficient oxygen transport about the body. The essential allosteric regulator 2,3- DPG is not present in sufficient concentration in the plasma to allow free Fib to release as much oxygen at venous oxygen tension as hemoglobin does in RBCs. By encapsulating Hb with 2,3-DPG, RBCs maintain Hb in its reduced, functional form. Moreover, free Hb in the bloodstream dimerizes and is rapidly removed from the blood.

Nonetheless, solutions of Hb obtained from RBCs have been tested for use as oxygen-delivering therapeutics. Briefly, the RBCs are lysed and cellular debris is removed, leaving what is termed "stromal-free hemoglobin" (SFH) which may then be administered.

Several basic problems have been observed with this approach. First, the solution must be freed of any toxic components of the RBC membrane without resorting to cumbersome and tedious procedures which would make large-scale production difficult. Second, the oxygen affinity of the SFH is too high to efficiently unload oxygen into the tissues. Benesch and Benesch, Biochem. Biophys. Res. Comm.. 26:162-67 (1967). Third, SFH has only a limited half-life in the circulatory system because, as set forth above, oxy Hb partially dissociates into a dimer (αβ). These dimers are rapidly cleared from the blood by binding to circulating haptoglobin, which are then subjected to subsequent glomerular filtration. If large amounts of soluble hemoglobin are introduced into the circulation, glomerular filtration of the dimers may cause renal damage.

In order to overcome some of these problems with SFH, various modifications have been made to the Hb molecule. For example, certain properties of Hb have been altered by chemically cross-linking the alpha chains. (U.S. patent Nos. 4,600,531 and 4,598,064; Snyder et al, PNAS (USA) 84:7280-84 (1987); Chaterjee et al., T. Biol. Chem.. 261:9927-37 (1986)). The beta chains have also been chemically cross-linked as described, for example, in Kavanaugh et al., Biochemistry. 27: 1804-08 (1988).

Hoffman and Nagai, U.S. Patent No. 5,028,588 describe the stabilization of the T state of Hb by intersubunit (but intratetrameric) disulfide cross-links resulting from substitution of cysteine residues for other residues. A preferred cross-link connects beta Gly Cys with either alpha G17 (Ala→Cys) or G18 (Ala→Cys).

Genes encoding various polypeptides can be fused together by removing the stop codon of a first gene, and joining it in phase to a second gene. Parts of genes may also be fused, and spacer DNAs, which maintain phase, may be interposed between the fused sequences. The product of a fused gene is a single polypeptide, not a number of polypeptides as expressed by a polycistronic operon. Different genes have been fused together for a variety of purposes. For example, Gilbert, U.S. Patent No. 4,338,397 inserted a rate preproinsulin gene behind a fragment of the E. coli penicillinase gene.

Hoffman et al, U.S. Patent No. 5,545,727, incorporated herein by reference, describe the production of Hb and analogs thereof in bacteria and yeast. The disclosed analogs include Hb proteins in which one of the component polypeptide chains consists of two alpha or two beta globin amino acid sequences covalently connected, or fused, by peptide bonds, preferably through an intermediate linker of one or more amino acids. In rHbl.l, for example, a glycine bridges the normal C terminus of one alpha subunit with the N terminus of the other alpha subunit. The resulting di-alpha polypeptide binds 2 beta globins and is capable of delivering oxygen. In normal Hb, the alpha and beta globin subunits are non-covalently bound.

Anderson et al., U.S. Patent No. 5,599,907, incorporated herein by reference, disclose a fusion of alpha and beta globins. These Hbs are functional, have oxygen binding properties that make them suitable for use as oxygen- carrying therapeutics, and do not dissociate into αβ dimers or filter through the kidney as do non-crosslinked or non-fused Hb.

Fusing the N- and C-termini in a protein can also increase the performance of the protein in a number of other ways, including increasing its stability and/or soluble accumulation when synthesized in E. coli, by increasing stability during formulation, and by increasing the circulating half-life in vivo by sequestration of site(s) within the protein that would otherwise be involved in clearance (e.g., haptoglobin or a clearance receptor). On the other hand, fusion may lead to limited destabilization or proteolysis. Creation of new N- and C- termini and, optionally, fusion at sites other than the normal N- and C-termini can result in greater stability and higher expression of intact molecules and /or can result in reduced binding to proteins and receptors involved in clearance.

Therefore, a need exists for additional Hb proteins that overcome the problems discussed above relating to blood-derived hemoglobin. The present invention satisfies this need by providing various Hb permuteins. Such Hb permuteins contain novel N- and C-termini that can be used for fusion of other protein domains, for example, oligomerization domains or more Hb subunits as described in PCT/US96/20632 and U.S. Patent No. 5,599,907, both incorporated herein by reference. Larger Hb molecules may also advantageously have low oncotic pressure.

Summary of the Invention

The present invention relates to permuted hemoglobin-like proteins or "permuteins" having non-naturally occurring N- and C-termini. The new termini are created at a predetermined site of an unaltered globin having at least one naturally occurring terminus prior to the creation of the new termini. The termini of the unaltered globin is referred to herein as the "former termini." Preferably, the predetermined site is between two adjacent surface residues, non- helical residues, non-interface residues, non-heme pocket residues or other residues that will not substantially affect a desired hemoglobin function. The former termini can be linked to at least one other globin by a peptide bond or a polypeptide linker to produce a hemoglobin permutein. Such linkers can preferably contain alanine, glycine, serine or threonine. Particularly suitable linkers are polyglycine linkers or linkers containing a "ser-gly-gly" motif.

The novel hemoglobin permuteins can also be attached to a functional polypeptide through at least one of the new, non-naturally occurring termini.

Such functional polypeptides include, for example, an oligomerizing domain, a therapeutically-active peptide or one or more hemoglobin globins or hemoglobin tetramers to form a multimeric hemoglobin molecule.

The present invention is further directed to novel nucleic acids coding for a hemoglobin-like globin having non-naturally occurring N- and C-termini and to pharmaceutical compositions containing the ;novel hemoglobin permuteins..

Brief Description of the Figures

Figure 1 schematically shows a circularly permuted di-alpha Hb permutein. Alpha 1 is shown as D and Alpha 2 is shown as D, with the Gly-Gly linker between alpha 1 and alpha 2 shown as ••••. The ■ areas depict the potential new C- and N-termini.

Figure 2 shows a strategy for constructing a Hb permutein. PCR Product #1 is from mono-alpha, while PCR Product #2 is from mono-alpha or annealed oligos.

Detailed Description of the Invention

The present invention relates to Hb-like proteins wherein two or more subunits are covalently bonded (genetically fused) and new N- and C-termini are produced. The various polypeptides so-produced are termed "permuteins." Between any pair of covalently linked subunits, either or both of which may contain permuted amino acid sequences conferring new N- and C-termini, the covalent linkage can be a peptide bond or peptide linker connecting the new carboxy-most residue of a globin-like domain with the new amino-most residue of a second globin-like domain. Also provided are multimeric Hb-like molecules which are oligomerized through the new N- and C-termini. The preparation of Hbs with a genetically-fused backbone avoids the disadvantages of chemical cross-linking. Conventional chemical cross-linking methods are inefficient and result in a heterogeneous population of molecules. Anderson et al, U.S. Patent No. 5,599,907 and WO 96/40920 disclose various "pseudomeric hemoglobin-like proteins" and "genetically-fused hemoglobins" and are incorporated herein by reference.

As discussed above, haptoglobin appears to play a role in Hb catabolism. If so, intravascular retention of Hb might be enhanced by mutations that inhibit haptoglobin binding. Oxyhemoglobin dissociates into alpha-beta dimers, which are then bound by haptoglobin. While much of the binding energy is associated with binding to residues that are buried in the tetramer but exposed in the dimer, it appears that there are also secondary binding sites on the surface of the tetramer.

It is known that even covalently cross-linked ibs can be processed by haptoglobin. It is believed that this haptoglobin processing can occur as the result of the "breathing" of the tetramer in its oxy form sufficiently to allow the haptoglobin access to the normally buried residues of the subunit interfaces. This processing can be prevented by tightly cross-linking the globin subunits so dissociation will not occur within the time span of interest.

In the liganded form, native Hb dissociates into αβ dimers which are small enough to pass through the renal glomeruli and are rapidly removed from the circulatory system. Intravenous administration of Hb in amounts less than that needed to support oxygen transport can result in long term kidney damage or failure. If dimerization is prevented, there is an increase in intravascular half life and a substantial reduction or elimination of renal toxicity. Some of the Hb- like proteins of the present invention cannot dissociate into αβ dimers without the breakage of a peptide bond and, consequently, have the advantages of a longer intravascular half life and reduced renal toxicity. Accordingly, the proteins of the present invention are believed to prolong the half-life of recombinant Hb by reducing glomerular filtration of dissociated subunits in v ivo compared to native human Hb.

The globin-like domains of the Hb-like proteins of the invention may be, but need not be, one per polypeptide chain, and they need not correspond exactly in sequence to the alpha and beta globins of native human Hb. Rather, mutations can be introduced to alter oxygen and /or nitric oxide affinity and other characteristics, including heme binding, stability of the Hb, to facilitate genetic fusion or cross-linking, or to increase the ease of expression and assembly of the individual chains. Certain types of desired mutations are disclosed, for example, in US Patent Nos. 5,028,588, 5,599,907, and WO 96/40920. In mammals, the alpha globin of native Hb contains 50 invariant amino acids out of 141, while the beta globin contains 51 invariant amino acids out of 146. Invariant positions cluster around the centers of activity of the molecule: the heme crevice and the intersubunit contacts. Consideration should be given to the locations of these invariant amino acids when deciding where to make a "break point" when constructing the permuteins of the present invention.

In making the molecules of the instant invention, functionality is less likely to be affected by changes, including break points, at surface residues and at other residues not involved in either the heme crevice or the subunit contacts, which are also referred to herein as "interfaces." In addition, "loops" connecting helical regions, as well as free amino or carboxy termini, are expected to be more tolerant.

Linkage of the normal C- and N-termini of two alpha globins can be by a direct peptide bond or by a polypeptide linker. Preferably a linker is used to connect the α subunits and is 1-5 amino acids which may tbe the same or different. Beta to beta linkers between normal N- and C-termini are preferably longer. A poly-Gly linker is preferred. In designing such a linker, it is desirable to use one or more amino acids that will flexibly connect the subunits to produce a single polypeptide containing multiple subunits. When deciding on linker length, consideration should be given to the desired distance between the subunits in the quaternary structure. Those skilled in the art can readily make the determination, for example, by following the guidance provided in U.S. Patent No. 5,545,727, incorporated herein by reference.

The preparation of "di-alpha," "di-beta" and alpha/beta fusion polypeptides are also contemplated. For example, one such molecule comprises a fusion of the alpha and beta globin domains. The alpha and beta globins can be fused, for example, by attaching the alphai C-terminal residue to the N-terminal residue of the beta₂ C helix, creating a new C-terminus at the end of the beta₂ B helix. The original beta N-terminus, Vail, would be fused to the original beta subunit C-terminal residue, thus creating a continuous polypeptide chain comprising the alpha and beta subunits of different dimers. (See Anderson et al., U.S. Patent No. 5,599,907.)

As set forth in U.S. Patent No. 5,599,907, inspection of the structure of human deoxyhemoglobin using a molecular graphics computer can be used to determine relevant distances between amino acids to be linked. For example, the distance between the alphai Argl41 carboxyl carbon and beta₂ Tyr35 N atoms is approximately 8.6 Angstroms. A fully extended linear triglycine peptide measures approximately 10.1 Angstroms from the N- to C-terminal residues. This suggests that three glycine residues could be used to span the distance between the Argl41 and Tyr35 residues with a minimum of unfavorable steric interactions and maximum conformational freedom. The distance requirements could be different in oxyhemoglobin, and if so, the sequence of the fusion peptide could be altered to best accommodate the requirements of both structures.

Glycine is the preferred amino acid in the linkers because it is flexible. However, the residues comprising the linker are not limited to glycines. Other residues may be included instead or in addition to glycine, including alanine, serine, and threonine. Because these amino acids have a more restricted conformational space in a protein, they will likely produce a more rigid linking chain.

The minimum and maximum number of amino acids in the linker depends on the distance to be spanned in both the molecules, the amino acids used, and the propensity of the particular amino acid sequence to form a secondary structure. While a random coil is usually preferred, it is not required and a linker with a large number of amino acids in a secondary structure may have the same span as a random linker with fewer amino acids. A linker may comprise, e.g., 1-3 glycines, followed by a sequence having a secondary structure, followed by 1-3 more glycines. Linkers consisting of (ser gly gly)_n are particularly useful to join globin chains in adjacent Hb tetramers as described, for example, in WO 96/40920, incorporated herein by reference. There is no fixed upper limit on the length of the linker, however, the longer the linker, the more susceptible it is to protease cleavage, and if the molecule is large enough, it may be phagocytosed by macrophages of the reticuloendothelial system.

In a preferred embodiment, the di-alpha permutein and beta globin genes are combined into a single polycistronic operon. The use of a polycistronic operon is not, however, necessary to practice the invention, and the alpha (or di- alpha) and beta (or di-beta) globin genes may be expressed from separate promoters which may be the same or different.

While the preferred "genetically-fused hemoglobin" of the present invention comprises a permuted di-alpha globin, other globin chains may be genetically fused and, furthermore, used to make Hb multimers. To make multimers, the new N- and C-termini may be fused via polypeptide linkers to produce poly (globin) chains, such as those described in WO 96/40920. The new N- and C-termini may also be used as points of attachment for other functional polypeptides and other molecules, including, for example, oligomerizing domains described in PCT/US96/20632, incorporated herein by reference, and peptide drugs as described in WO 93/08842. Such peptide drugs are also referred to as polypeptides that are "therapeutically active."

A preferred embodiment of a human Hb permutein may be made by fusing the C-terminus of the alphai globin with the alpha2 N-terminus using a flexible peptide linker as disclosed above, of, for example, 1-5 glycine residues, together with fusing the alphai N-terminus with the alpha2 C-terminus using a similar flexible peptide linker. Because it is not possible to make a circular Hb molecule, it is necessary to make new N- and C-termini. These new termini preferably consist of a break in one of the alpha subunits, at a point in the polypeptide sequence which does not sustantially affect hemoglobin structure or function. The "break point" preferably selected to have at least one of the following criteria:

(i) a surface residue (e.g., whose amino acid side-chain has a high degree of solvent exposure); (ii) in a non-helical region of the protein (or, if the non- helical region is a single amino acid, within one or two amino acids of the non-helical region); and (iii) between residues that are not involved in electrostatic interactions, based on known crystal structures for Hb.

An example of such a "circularly permuted " di-alpha hemoglobin-like molecule is set forth in Figure 1. The molecule consists of two alpha globin molecules (alphai and alpha2) that are genetically fused. The blackened areas represent preferred potential C- and N-termini. These new termini are presented in more detail in Table 1. The single letters indicate the helical areas of each of the subunits; double letters indicate non-helical regions. The preferred break points for new N- and C-termini are as indicated in Table 1. Each of the specific sequences set forth in Table 1 corresponds to an indicated blackened area in Figure 1 (i.e., the AB, CE, and EF corners). The particular sequences for making a break to produce new N- and C-termini were selected in accordance with the criteria set forth above, i.e., they are preferably surface-exposed, non- helical regions. Referring again to Figure 1, the areas highlighted with a dotted line indicate where the glycine linkers are added. Table 1

CIRCULAR PERMUTATION OF AB "CORNER" OF ALPHA 1 HELIX

A12 A13 A14 A15 A16 AB1 Bl B2 B3 B4 B5 B6 B7 Trp Gly Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala (SEQ.ID.No: 1)

CIRCULAR PERMUTATION OF CE "CORNER' ALPHAI HELIX

C3 C4 C5 C6 C7 CE1 CE2 CE3 CE4 CE5 CE6 CE7 CE8 CE9 El E2 Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser Ala

E3 E4 E5 Gin Val Lys (SEQ.ID.NO: 2)

CIRCULAR PERMUTATION OF EF "CORNER" ALPHAI HELIX

*

E16 E17 E18 E19 E20 EFl EF2 EF3 EF4 EF5 EF6 EF7 EF8 FI F2 F3 Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu Ser Ala F4 F5 F6

Leu Ser Asp (SEQ.ID.NO: 3)

* symbol indicates "break" point for new N- and C- termini

Figure 2 sets forth the strategy for constructing such a permuted di-alpha hemoglobin-like molecule. The gene encoding the di-alpha Hb may be obtained as set forth in U.S. Patent No. 5,545,727, U.S. Patent No. 5,599,907, WO 96/40920 and PCT/US96/20632, which are incorporated herein by reference. The di-alpha encoding sequence is then digested with BstBl to obtain the "cloned fragment from Di-alpha" as indicated in Figure 2. PCR Products #1 and #2 are produced by standard PCR methods from a mono-alpha-containing gene obtained from any source (see, e.g., U.S. Patent No. 5,545,727; Dieffenbach and Dreksler, eds., 1995. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The three fragments so-produced are then annealed by standard methods known to those skilled in the art to produce the gene encoding the permuted di-alpha Hb permutein.

Di-beta permuteins can be made similarly using similar considerations. Additionally, the D-helix of the β subunit (or the corresponding site in the α subunit where a D helix may be inserted) is a likely site for various genetic manipulations (see Whitaker, et al, Biochemistry, 34:8221-26 (1995) and Komiyama, et al., Nature, 352:349-51 (1991)).

It is also possible to link alpha globin with beta globin to produce a Hb permutein. The criteria for selecting new N- and C-termini are the same as those set forth above for alpha globin permuteins. Also, as set forth above, the choice of linker will depend, in part, on the distance between the residues used for fusion. This distance can be readily ascertained from the known crystal structure of Hb, and preferred peptide linkers would consist of peptides consisting primarily of amino acids such as glycine and serine.

In general, those skilled in the art can construct genes encoding permuted proteins by techniques well-known in the art, including by complete DNA synthesis according to methods known in the art or by standard recombinant DNA methods (see, e.g., Sambrook, et al., 1989. Molecular Cloning: A Laboratory Manual, 2^nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.)

Recombinant systems for producing such heterologous proteins or polypeptides, including hemoglobin-related proteins, are well known in the art. The genes encoding the target protein can be placed in a suitable expression vector and inserted into a microorganism, animal, plant, insect or other organism, or inserted into cultured animal or plant cells or tissues. These host cells, organisms or tissues may be produced using standard recombinant DNA techniques, and may be grown in cell culture or in fermentations. For example, human alpha and beta globin genes have been cloned and sequenced by Liebhaber et al., Proc. Natl. Acad. Sci. USA. 77:7054-58 (1980) and Marotta et al., L Biol. Chem., 252:5040-53 (1977) respectively. Techniques for expression of both native and mutant alpha and beta globins and their assembly into hemoglobin are described, for example, in U.S. Patent No. 5,545,727 and PCT application nos. PCT/US90/02654, PCT/US91/09624, PCT/US91/02568 and PCT/US91/08108, all incorporated herein by reference.

After the protein has been expressed, it generally should be released from the cell to create a crude protein solution. The release can usually be accomplished by breaking open the cells, e.g., by sonication, homogenization, enzymatic lysis or any other cell breakage technique known in the art. The proteins can also be released from cells by dilution at a controlled rate with a hypotonic buffer so that some contamination with cellular components can be avoided (U.S. Patent No. 5,264,555). If refolding is required, techniques known in the art, including those taught in U.S. Patent No. 5,028,588, incorporated herein by reference, can be used.

After breakage of the cells or secretion into the media, the target protein is contained in a crude cell lysate or crude cell broth or solution. The protein may be purified according to methods well known in the art. For example, methods for purifying hemoglobin-like proteins are taught in PCT publication WO 96/15151, incorporated herein by reference.

Appropriate recombinant host cells can be produced according to conventional methods. Any suitable host cell can be used to express heterologous polypeptides, including, for example bacterial, plant, yeast, mammalian, and insect cells. E. coli cells are particularly useful for expressing heterologous hemoglobin-like proteins.

The proteins, so-produced, can be used for their known purposes. For example, the hemoglobin-like proteins, and compositions containing them, can be used for in vitro applications, including delivery of oxygen for the enhancement of cell growth (PCT publication WO 94/22482), removing oxygen from solutions requiring the removal of oxygen (U.S. patent No. 4,343,715) and as reference standards for analytical assays and instrumentation (U.S. patent No. 5,320,965).

In a further embodiment, the permuteins of the present invention can be formulated for use in therapeutic applications. Example formulations suitable for the hemoglobin permuteins of the instant invention are described in Milne, et al., WO 95/14038 and Gerber et al, WO 96/27388, both herein incorporated by reference. Pharmaceutical compositions of the invention can be administered by, for example, subcutaneous, intravenous, or intramuscular injection, topical or oral administration, large volume parenteral solutions useful as blood substitutes, etc. Pharmaceutical compositions of the invention can be administered by any conventional means such as by oral or aerosol administration, by transdermal or mucus membrane adsorption, or by injection. For example, the permuted hemoglobins of the present invention can be used in compositions useful as substitutes for red blood cells in any application that red blood cells are used or for any application in which oxygen delivery is desired. Such permuted hemoglobins of the instant invention formulated as red blood cell substitutes can be used for the treatment of hemorrhages, traumas and surgeries where blood volume is lost and either fluid volume or oxygen carrying capacity or both must be replaced. Moreover, because the permuted hemoglobins of the instant invention can be made pharmaceutically acceptable, they can be used not only as blood substitutes that deliver oxygen but also as simple volume expanders that provide oncotic pressure due to the presence of the large hemoglobin protein molecule. In a further embodiment, the permuteins of the instant invention can be crosslinked by methods known in the art and used in situations where it is desirable to limit the extravasation or reduce the colloid osmotic pressure of the hemoglobin-based blood substitute. Thus, the permuted hemoglobins can act to transport oxygen as a red blood cell substitute, while reducing the adverse effects that can be associated with excessive extravasation.

A typical dose of the permuted hemoglobins of the instant invention as an oxygen delivery agent can be from 2 mg to 5 grams or more of extracellular hemoglobin per kilogram of patient body weight. Thus, a typical dose for a human patient might be from a few grams to over 350 grams. It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount could be reached by administration of a plurality of administrations as injections, etc. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of the skilled artisan in the field.

Administration of the permuted hemoglobins of the instant invention can occur for a period of seconds to hours depending on the purpose of the hemoglobin usage. For example, as an oxygen delivery vehicle, the usual time course of administration is as rapid as possible. Typical infusion rates for hemoglobin solutions as blood replacements can be from about 100 ml to 3000 ml/hour.

In a further embodiment, the hemoglobins of the instant invention can be used to treat anemia, both by providing additional oxygen carrying capacity in a patient that is suffering from anemia, and/or by stimulating hematopoiesis as described in PCT publication WO 95/24213, incorporated herein by reference. When used to stimulate hematopoiesis, administration rates can be slow because the dosage of hemoglobin is much smaller than dosages that can be required to treat hemorrhage. Therefore the permuteins of the instant invention can be used for applications requiring administration to a patient of high volumes of hemoglobin as well as in situations where only a small volume of the hemoglobin of the instant invention is administered.

Because the distribution in the vasculature of the permuted hemoglobins of the instant invention is not limited by the size of the red blood cells, the permuted hemoglobins of the present invention can be used to deliver oxygen to areas that red blood cells cannot penetrate. These areas can include any tissue areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of thrombi, sickle cell occlusions, arterial occlusions, angioplasty balloons, surgical instrumentation, any tissues that are suffering from oxygen starvation or are hypoxic, and the like. Additionally, any types of tissue ischemia can be treated using the permuted hemoglobins of the instant invention. Such tissue ischemias include, for example, stroke, emerging stroke, transient ischemic attacks, myocardial stunning and hibernation, acute or unstable angina, emerging angina, infarct, and the like. Because of the broad distribution in the body, the permuted hemoglobins of the instant invention can also be used to deliver drugs and for in vivo imaging as described in WO 93/08842, incorporated herein by reference.

The permuted hemoglobins of the instant invention can also be used as replacement for blood that is removed during surgical procedures where the patient's blood is removed and saved for reinfusion at the end of surgery or during recovery (acute normovolemic hemodilution or hemoaugmentation). In addition, the permuted hemoglobins of the instant invention can be used to increase the amount of blood that can be predonated prior to surgery, by acting to replace some of the oxygen carrying capacity that is donated. The foregoing description of the invention is exemplary for purposes of illustration and explanation. It will be apparent to those skilled in the art that changes and modifications are possible without departing from the spirit and scope of the invention. It is intended that the following claims be interpreted to embrace all such changes and modifications.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANTS: Best, Elaine A. Olins, Peter 0.

(ii) TITLE OF INVENTION: Permuted Hemoglobin-Like

Proteins (iϋ) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Somatogen, Inc.

(B) STREET: 2545 Central Avenue, Suite FDl (C) CITY: Boulder

(D) STATE: Colorado

(E) ZIP: 80301

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.50 inch, 1.4 Mb storage

(B) COMPUTER: Apple Macintosh

(C) OPERATING SYSTEM: System 7.5

(D) SOFTWARE: Microsoft Word 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown

(B) FILING DATE: 27 FEB. 1998

(C) CLASSIFICATION: not known (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/039,657

(B) FILING DATE: 28 Feb. 1997

(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Brown, Theresa A.

(B) REGISTRATION NUMBER: 32,547

(C) REFERENCE/DOCKET NUMBER: 271/PCT

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 303-541-3356

(B) TELEFAX: 303-444-3013

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13

(B) TYPE: amino acid

(D) TOPOLOGY: unknown to applicant (ii) MOLECULE TYPE: polypeptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : Trp Gly Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala

5 10

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid

(D) TOPOLOGY: unknown to applicant (ii) MOLECULE TYPE: polypeptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Thr Thr Lys Thr Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser 5 10 15

Ala Gin Val Lys

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19

(B) TYPE: amino acid (D) TOPOLOGY: unknown to applicant

(ii) MOLECULE TYPE: polypeptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala Leu Ser

5 10 15

Ala Leu Ser Asp

Claims

Claims:

1. A hemoglobin permutein comprising at least one globin having non-naturally occurring N- and C-termini.

2. The hemoglobin permutein of claim 1, wherein the non-naturally occurring N- and C-termini are created at a predetermined site of an unaltered globin having at least one naturally occurring terminus.

3. The hemoglobin permutein of claim 2, wherein said predetermined site does not substantially affect a desired hemoglobin function.

4. The hemoglobin permutein of claim 2, wherein the predetermined site is between two adjacent (a) surface residues, (b) non-helical residues, (c) non- interface residues, or (d) non-heme pocket residues.

5. The hemoglobin permutein of claim 2, wherein a former terminus is linked to a second globin by a peptide bond or a polypeptide linker.

6. The hemoglobin permutein of claim 4, wherein said linker contains alanine, glycine, serine or threonine.

7. The hemoglobin permutein of claim 4, wherein the linker has 0 to 5 amino acids.

8. The hemoglobin permutein of claim 4, wherein the linker is a polyglycine linker.

9. The hemoglobin permutein of claim 4, wherein the linker comprises serine-glycine-glycine.

10. The hemoglobin permutein of claim 1, wherein at least one of said non-naturally occurring N- or C-termini is attached to a functional polypeptide.

11. The hemoglobin permutein of claim 10, whererin the functional polypeptide is an oligomerizing domain.

12. The hemoglobin permutein of claim 10, wherein the functional polypeptide is therapeutically-active.

13. The hemoglobin permutein of claim 10, wherein the functional polypeptide is a second hemogloibn molecule.

14. A nucleic acid encoding the hemoglobin permutein of claim 1.

15. A pharmaceutical composition comprising the hemoglobin permutein of claim 1.