# Patent

## Title
Blood substitutes

## Abstract

The functional characteristics of heme proteins can be modified to produce hemoglobins that can be used as blood substitutes in different therapeutic applications. Stable polymers of tetrameric hemoglobin, and of myoglobin molecules, are provided for use in the blood substitutes.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of co-pending U.S. provisional patent application Ser. No. 60/515,759, filed on Oct. 30, 2003, the entire disclosure of which is herein incorporated by reference.


REFERENCE TO GOVERNMENT GRANT
The present invention was made at least partly with government funds under grant nos. HL 48517 and NS 38684 from the National Institute of Health. The U.S. government may have certain rights in the invention.


FIELD OF INVENTION
This invention relates to novel modified heme protein compositions useful as blood substitutes, and to methods of preparing the blood substitutes and the modified heme proteins.
BACKGROUND OF THE INVENTION
In medicine, the need for transfusional fluids is continually increasing. The use of stroma-free hemoglobin (Hb) solutions as a red cell replacement has been considered. Solutions of stroma-free Hb contain tetrameric (MW 64 kDa) and dimeric (MW 32 kDa) Hb molecules at equilibrium. Due to the rapid filtration of the dimers through the kidneys, the retention time of infused Hb solutions is short. In addition, Hb molecules extravasate through the endothelium, scavenging the NO from the interstitial fluid. The latter is believed to be the main reason for the increase in mean arterial pressure observed upon administration of a stroma-free Hb solution.
Chemical modifications have been used to transform mammalian Hbs into efficient blood substitutes, but the introduction of such chemical modifications can result in toxicity and/or an immunogenic response. For example, much work has been devoted to the preparation of Hb solutions with oxygen affinities similar to that of whole blood. Recent studies, however, indicate that Hb with high oxygen affinity can efficiently deliver O2 to the tissues. Thus, the question of optimal oxygen affinity for blood substitutes has not been resolved.
Adult Hb is a tetrameric protein comprised of two structurally similar subunits, α and β, assembled through two different interfaces. Each subunit contains eight α-helices (labeled A-H) that form a pocket containing the heme. The heme pocket of each subunit is lined by hydrophobic residues, except for the proximal (F8) and distal (E7) histidines, which are critical to the functional properties of Hb. The functional properties of the heme pocket are also greatly sensitive to the polar character of the amino acid side chains lining the pocket.
As previously mentioned, problems related to the use of Hb solutions for transfusion include the rapid loss of Hb through the kidneys and vasoconstriction with an increase in arterial blood pressure, which is thought to be due to scavenging of NO released from the endothelium. Moreover, at the physiological colloid-osmotic pressure of human plasma, only a limited amount of Hb may safely be infused, and thus the oxygen-carrying capacity of blood cannot be fully restored. In an effort to prevent these effects, tetramers of Hb molecules that resist dissociation into dimers in serum have been produced by using bifunctional reagents to effect intramolecular crosslinking. In the absence of further intermolecular crosslinking, stabilized tetrameric Hbs still extravasated across the endothelium and failed clinical tests. See, e.g., Saxena et al., 1999, Stroke. 30: 993-996 and Sloan et al., 1999, JAMA 282: 1857-1864.
Myoglobin (Mb) is a 17.5 kilodalton monomeric heme protein found mainly in muscle tissue, where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation, oxymyoglobin releases its bound oxygen which is then used for metabolic purposes.
The tertiary structure of Mb is that of a typical water soluble globular protein, and contains approximately 75% α-helical secondary structure. A Mb polypeptide is comprised of eight separate right handed α-helices, designated A through H, that are connected by short non-helical regions. Amino acid R-groups oriented towards the interior of the molecule are predominantly hydrophobic in character, and those on the surface of the molecule are generally hydrophilic, thus making the molecule relatively water soluble. Each Mb molecule contains one heme prosthetic group inserted into a hydrophobic cleft in the protein. To date, an adequate blood substitute which utilizes Mb is not available.
The curve of oxygen binding to Hb is sigmoidal, which is typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector. When oxygen binds to the first subunit of deoxyhemoglobin, the first oxygen molecule increases the affinity of the remaining subunits for additional oxygen molecules. As additional oxygen is bound to the other Hb subunits, oxygen binding is incrementally strengthened, so that Hb is fully oxygen-saturated at the oxygen tension of lung alveoli. Likewise, oxygen is incrementally unloaded and the affinity of Hb for oxygen is reduced as oxyhemoglobin circulates to deoxygenated tissue.
In contrast, the oxygen binding curve for Mb is hyperbolic in character, indicating the absence of allosteric interactions in this process. Mb therefore has a higher oxygen affinity and lower cooperativity than Hb. A large array of Mb heme pocket mutants have been constructed and investigated (Springer et al., Chem Rev 1994; 94: 699-714), and information is thus available on the molecular control of the conformational and functional properties of Mb. While these studies are certainly useful in the design of Hb-based oxygen carriers (Dou et al., Biophys Chem 2002; 98: 127-148), heme pocket differences exist between Mb and Hb. The effect of a particular mutation in Mb is therefore not necessarily predictive of the effect the analogous mutation would have in Hb.
Abbreviations
Hb—hemoglobin
HbA—human Hb
PolyHb—polymerized Hb
Mb—Myoglobin
PolyMb—polymerized Mb
ZL-HbBv—chemically cross-linked bovine Hb
βL28(B10)N—recombinant HbA, in which βLys28(B10) was replaced by Asn
βV67(E11)T—recombinant HbA, in which βVa67(E11) was replaced by Thr
αV63(E11)T—recombinant HbA, in which αVal63(E11) was replaced by Thr
PB4—recombinant HbA with mutations β(V1M+H2deleted+T4I+P5A)
PB5—recombinant HbA with mutations β(V1M+H2deleted+T4I+P5A+A76K)
Hb Prisca—recombinant HbA with mutations β(S9C+C93A+C112G)
Hb Minotaur (αHβBv)—Hybrid Hb containing α-human and β-bovine chains
Hb Polytaur—polymerized Hb Minotaur with mutations (αHC104SβBvA9C+C93A)
Hb (Polytaur)n—polymerized Hb Minotaur with mutations (αHβBvA9C)



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the oxygen affinity of βL28 (B10)N (-.-.); HbA ( . . . ); βV67 (E11)T (- -); αV63 (E11)T (-). Measurements were carried out using the Gill cell with an AVIV 14DS spectrophotometer. Protein concentration was 1.5 mM in heme. Buffer (pH 7.4, 50 mM Hepes+100 mM NaCl): Temperature 25° C.
FIG. 2 is a graph showing the fractional ionization of water in the aquomet derivatives of HbA (▴); βV67 (E11)T (▪); αV63 (E11)T (●). Fractional changes were calculated from the deconvolution of absorption spectra recorded from 480-700 nm using aquomet HbA standards.
FIG. 3 is a histogram showing the partial pressure of oxygen at 50% saturation (P50) of HbA, PB4, and PB5 at pH 7.4 in 50 mM Hepes buffer, and of PB5 at pH7.4 in Hepes buffer+100 mM NaCl. Other conditions as in FIG. 1.
FIG. 4 is a graph showing the size exclusion chromatography of Hb Polytaur (--) and Hb (Polytaur)n (-). Hb Polytaur was eluted as a homogeneous peak as for the polymerization of  ̃7 tetrameric Hb molecules (MW=500 kDa). The elution pattern of Hb (Polytaur)n was heterogeneous with a large fraction eluted with the void volume of the column (35 ml). (MW=>1,000 kDa). Measurements were carried out at 4° C., on a fractoget EMD BioSec column. Buffer: 20 mM phosphate with 300 mM NaCl, pH 7.2.
FIG. 5 is a histogram showing the infarct volume (±SE) after 2 h focal cerebral ischemia in control mice with no transfusion, and in mice transfused with either 5% albumin, 3% Hb Polytaur (P50=16.0 torr, n=1.7), 3% Hb (Polytaur)n (P50=2.0 torr, n=1.0) 0, or 6% ZL-HbBv (P50=4.0 torr, n=1.0). *P<0.05 from control and albumin groups by ANOVA and Newman-Keuls test.
FIG. 6 is a graph showing the fractional saturation with oxygen binding of Hb Polytaur(●) and Hb (Polytaur)n (▪) in 50 mM Hepes buffer+100 mM NaCl at pH 7.4. T=37° C.
FIG. 7 is a graph showing the size exclusion chromatography of Mb (--) and PolyMb (-). Most of the PolyMb is eluted with the void volume of the column (MW 1,000 kDa). Experimental conditions as in FIG. 4.
FIG. 8 shows the normalized time courses of oxygen dissociation from Mb and from a combination with Mb and PolyMb. A: Normalized time course of oxygen dissociation from MbO2 (asterisks) and PolyMbO2 (circles). Continuous lines represent the best fit of the experimental data to a single exponential. Observation wavelength: 436 nm; Buffer: 0.05 M Tris with 0.1 M NaCl. PH 7.2; temperature 23° C. B: Normalized time course of combination to Mb and PolyMb as observed after laser flash photolysis. Symbols and experimental conditions as in FIG. 8A.
SUMMARY OF THE INVENTION
The functional characteristics of heme proteins have been modified to produce Hbs that can be used as resuscitating fluids (i.e., blood substitutes) in different therapeutic applications. For example, modification at the heme pocket can produce Hbs with a range of oxygen affinities. Stable polymers of tetrameric Hb molecules of different sizes and with different oxygen affinities have therefore been obtained. Modifications on the protein surface produced a human Hb with decreased oxygen affinity, regulated by the concentration of chlorides in the plasma. Blood substitutes comprising modified Mb can also be produced. For example, modified Mb can be induced to form stable polymers. The polymerization of such modified Mb does not alter the functional characteristics of the component monomeric Mb. Polymeric Mbs can therefore be constructed with additional mutations to engineer functional characteristics tailored to different clinical applications.
The invention thus provides an isolated Hb comprising a first and a second polypeptide having the amino acid sequence of the normal human Hb alpha chain, and a third and fourth polypeptide having the sequence of normal bovine Hb beta chain. At least one polypeptide of the isolated Hb comprises at least one mutation which introduces a polymerization site. In one embodiment, the at least one mutation comprises the substitution of the alanine at position 9 for cysteine on at least one of the normal bovine Hb beta chains. In bovine Hb beta chains, the histidine corresponding to position beta2 in HbA is missing. For the purposes of the present invention, a number has been assigned to this position in bovine Hb for consistency in numbering the amino acid residues. In another embodiment, the at least one mutation comprises a mutation of residue 104 in the normal human Hb alpha chain from cysteine to serine, and a mutation of residue 9 from alanine to cysteine and a mutation of residue 93 from cysteine to alanine in the normal bovine Hb beta chain.
The invention also provides an isolated hemoglobin molecule comprising at least one human hemoglobin subunit polypeptide and at least one non-human mammalian hemoglobin subunit polypeptide.
The invention further provides a polymer comprising an isolated modified Hb of the invention.
The invention further provides a polypeptide comprising a subunit of the modified Hb of the invention, and a nucleic acid comprising a nucleotide sequence encoding the polypeptide subunit.
The invention further provides a method of producing the isolated modified Hb of the invention, comprising modifying at least two polypeptide subunits of the Hb with at least one mutation that introduces a polymerization site.
The invention also provides a polymer comprising a plurality of modified Mb monomers, in which the Mb monomers comprise at least one modification which introduces a polymerization site. The modification which introduces a polymerization site in the Mb monomers can comprise a chemical modification or a mutation in the Mb monomer.
The invention also provides a Mb monomer modified to include a polymerization site, and a nucleic acid comprising a nucleotide sequence encoding the Mb monomer.
The invention further provides a method of producing the Mb monomer of the invention, comprising modifying at least one Mb monomer to introduce a polymerization site in the monomer.
The invention still further provides a method of producing an isolated Mb polymer of the invention, comprising modifying at least one Mb monomer to introduce a polymerization site, and subjecting the modified Mb monomer to conditions which cause the monomers to polymerize.
The invention still further provides a blood substitute comprising a modified heme protein of the invention.
The invention still further provides a method of supplementing the oxygen-carrying capacity of a subject's blood, comprising administering to the patient an effective amount of the blood substitute comprising an isolated modified heme protein of the invention, or a polymer thereof. Where the isolated modified heme protein of the invention is a Hb, the Hb comprises a first and a second polypeptide having the amino acid sequence of the normal human Hb alpha chain, and a third and fourth polypeptide having the sequence of normal bovine Hb beta chain, and wherein at least one polypeptide comprises at least one mutation which introduces a polymerization site. Where the isolated modified heme protein of the invention is a Mb, the Mb comprises at least polymerization site.



DETAILED DESCRIPTION OF THE INVENTION
Blood substitutes can be constructed which comprise modified oxygen-binding heme proteins, such as modified hemoglobin (Hb) or modified myoglobin (Mb) or polymers thereof.
Human adult Hb, designated Hb A, comprises two alpha and two beta polypeptide subunits. The alpha subunit consists of 141 amino acids. The iron atom of the heme (ferroprotoporphyrin IX) group is bound covalently to the imidazole of His87 (the “proximal histidine”) of the alpha subunit. The beta subunit is 146 residues long, and the heme group is bound to this subunit at His 92.
The primary amino acid structure of the human adult Hb (HbA) alpha and beta subunits, and the nucleic acid sequences which encode them, are known in the art (see Wilson et al., J. Biol. Chem., 1980, 255(7), 2807-2815, the entire disclosure of which is herein incorporated by reference. The HbA subunit amino acid sequences are presented herein as SEQ ID NO: 1 (alpha subunit) and SEQ ID NO: 2 (beta subunit). The nucleotide sequences encoding the HbA alpha and beta subunits are presented herein as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Likewise, the bovine Hb (HbBv) beta subunit amino acid sequence is known in the art (see, e.g., Schimenti et al., Nucleic Acids Res., 1984, 12(3), 1641-1655 and Bobofchak et al., Am J Physiol Heart Circ Physiol 2003; 285: H549-61, the entire disclosures of which are herein incorporated by reference and are provided herein as SEQ ID NO: 5. The nucleic acid sequence encoding the HbBv beta subunit represented by SEQ ID NO: 5 is presented herein as SEQ ID NO: 6.
Various mutations have been introduced into the alpha and beta subunits of HbA which have altered the oxygen affinity of the modified Hbs. Such modified HbA have been previously described, and their functional characteristics are presented in Table 2 below. However, it was not always possible to obtain sufficient amounts of recombinant HbA. Therefore, a tetrameric hybrid Hb comprising two human alpha subunits and two bovine subunits (called “Hb Minotaur”) was constructed; see Bobofchak et al., supra. Hb Minotaur can be expressed in Escherichia coli at considerably higher levels than all-human Hbs, and has the same oxygen affinity as HbA. Hb Minotaur can be used as the starting point for construction of modified Hb of the invention, in which at least one polymerization site is introduced into at least one Hb subunit. An isolated molecule comprising, or consisting essentially of, Hb Minotaur is therefore considered to be part of the present invention.
The invention also provides an isolated hemoglobin molecule comprising, or consisting essentially of, at least one human hemoglobin subunit polypeptide and at least one non-human mammalian hemoglobin subunit polypeptide. Such isolated hemoglobin molecules are referred to herein as “hybrid hemoglobin molecules.” Preferably, a hybrid hemoglobin molecule comprises two of the same human hemoglobin subunit polypeptides (e.g., two human alpha or beta subunit polypeptides) and two of the same non-human mammalian hemoglobin subunit polypeptides (e.g., two non-human mammalian alpha or beta subunit polypeptides). However, a hybrid hemoglobin molecule can comprise at two different human or non-human hemoglobin subunit polypeptides; for example, a hybrid hemoglobin can comprise one human and alpha and one human beta subunit polypeptide, or one non-human mammalian alpha and one non-human mammalian beta subunit polypeptide. Preferably, the hybrid hemoglobin comprises at least two of the same hemoglobin subunit polypeptides from a given human or non-human mammalian species.
The primary amino acid sequences (and the corresponding nucleic acid sequences which encode them) for mammalian hemoglobin subunit polypeptides are known in the art, and can be readily obtained by one skilled in the art. In addition to the bovine hemoglobin beta subunit polypeptide sequences described above, representative non-human mammalian hemoglobin subunit polypeptides are given in SEQ ID NOS: 18 to 35, as indicated in Table 1.










TABLE 1





Organism
Hb subunit
Sequence Type
SEQ ID NO.


















Rattus norvegicus
alpha
protein
18


Rattus norvegicus
alpha
nucleic acid
19


Rattus norvegicus
beta
protein
20


Rattus norvegicus
beta
nucleic acid
21


Mus musculus
alpha
protein
22


Mus musculus
alpha
nucleic acid
23


Mus musculus
beta (major)
protein
24


Mus musculus
beta (major)
nucleic acid
25


Mus musculus
beta (minor)
protein
26


Mus musculus
beta (minor)
nucleic acid
27


Bos taurus
alpha (allele Y)
protein
28


Bos taurus
alpha (allele Y)
nucleic acid
29


Bos taurus
alpha (allele S)
protein
30


Bos taurus
alpha (allele S)
nucleic acid
31


Bos taurus
alpha (allele N)
protein
32


Bos taurus
alpha (allele N)
nucleic acid
33


Sus scrofa
alpha
protein
34


Sus scrofa
beta
nucleic acid
35









As used herein, “isolated” refers to a molecule which is wholly or partially synthetic, or which is altered or removed from the natural state through human intervention. For example, a heme protein, such as a Hb or Mb (or subunit thereof) which is partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated molecule of the invention can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell in which the molecule is produced or introduced, or a structure (such as the vasculature) into which the molecule has been delivered.
As used herein, a “polymerization site” on a heme protein polypeptide, such as a Hb subunit or Mb monomer, means any residue or region of the subunit polypeptide which has, or can be induced to have, the ability to form an intermolecular bond with another subunit. For example, a polymerization site can comprise a modification to an amino acid residue which provides that residue with a chemical group which can react with at least one amino acid residue (or chemical group thereon) on another subunit. Preferred polymerization sites comprise a naturally-occurring or non naturally-occurring cysteine. As used herein, a polymerization site can be “introduced” into a heme protein subunit by chemical techniques, or by mutating the amino acid sequence of the subunit to substitute one amino acid residue (e.g., a cysteine) for another. Techniques for mutating the amino acid sequence of a heme protein subunit are discussed in more detail below.
Isolated Hb subunits of the invention can therefore be produced by introducing a polymerization site into the molecule. These modified Hb subunits can then be assembled into functional Hb α2β2 tetramers, which in turn can be polymerized by subjecting the Hb α2β2 tetramers comprising the modified subunits to conditions which activate the polymerization sites. A suitable technique for activating polymerization sites is described in the Examples below.
In one embodiment, the amino acid sequence of at least one Hb subunit (e.g., at least one alpha or at least one beta subunit, preferably both alpha and both beta subunits) was mutated by substituting at least one amino acid residue located on the surface of the folded subunit polypeptide with a cysteine. Optionally, one or more naturally-occurring cysteines in a Hb subunit (if any) are substituted with amino acid residues which do not form intermolecular bonds.
For example, subunits of Hb Minotaur were modified to introduce polymerization sites by substituting a cysteine for alanine at position β9, and by substituting certain naturally occurring cysteines with amino acid residues that do not form intermolecular bonds. Preferably, the alpha subunit of Hb Minotaur is modified by substituting a serine for the cysteine at position α104, and beta subunit is modified by substituting a alanine for the cysteine at β93. The two modified subunits are then combined to form the isolated mutant Hb αHC104SβBvA9C+C93A, which is designated “Hb Polytaur” after polymerization. The biochemical characterization of Hb Polytaur is given in Table 2.
As can be seen from Table 2, intermolecular polymerization through S—S disulfide bonds at position β9 does not modify the oxygen affinity of Hb Polytaur, which remained similar to that of HbA (about 17.0 torr at 37° C.). However, the affinity of Hb Polytaur for heme was increased upon polymerization. Measurements of autoxidation rates under in vivo conditions (e.g., in whole blood) indicated the protective effect of blood components toward heme oxidation in Hb Polytaur. As can be seen from Table 2, the half-time of autoxidation of Hb Polytaur heme in blood is 46 h, which is about 15-fold longer than Hb Polytaur heme oxidation under in vitro conditions. This value indicates that within the approximately 20 h retention time in circulation measured in humans with other polymerized Hbs, only 25% of infused Hb Polytaur would be oxidized, while the remaining 75% would remain reduced and functionally active as an oxygen carrier.
In another embodiment, a modified Hb of the invention is produced by introducing a polymerization site in the beta subunits of Hb Minotaur by substituting a cysteine for the alanine at position β9. In this embodiment, there is no substitution of the naturally-occurring cysteines present at α104 and β93. The isolated Hb formed with the modified beta subunits (αHβBvA9C) is called Hb (Polytaur)n. Hb (Polytaur)n polymerizes more rapidly than Hb Polytaur.
Hb (Polytaur)n is a heterogeneous polymer with a molecular weight of about 1000 kilodaltons (kDa) or higher, as measured by size exclusion chromatography as shown in FIG. 4, where the elution patterns of Hb Polytaur and Hb (Polytaur)n are compared. Oxygen-binding measurements indicated that Hb (Polytaur)n had oxygen-binding characteristics similar to Mb; i.e., high oxygen affinity (P50=about 2.0 torr) and loss of cooperativity (n=1). Without wishing to be bound to any theory, it is believed that Hb (Polytaur)n has similar stability and heme affinity as that measured for Hb Polytaur.
As shown in the Examples below, vasoactivity was not observed following infusion of polymerized isolated Hb of the invention, from which any residual unpolymerized Hb molecules had been removed.
The biochemical characteristics of the polymerized isolated Hb of the invention can be compared to the biochemical characteristics of a known polymerized Hb (Hb Prisca), with normal HbA, and with various non-polymerized Hb molecules with modifications on surface or heme-pocket amino acids. These comparative biochemical characteristics are shown in Table 2.











TABLE 2










Heme transfer




Autoxidation
T1/2, h
Brain















P50

NO release
T1/2, h
α-
β-
Infarct

















Mutations
(torr)
nmax
T1/2, h
In vitro
In blood
chains
chains
Reduction




















Heme
HbA
6.5*
2.9
260.0
36.0 (1.0)ν

1.38
0.06



















pocket



α-chains
β-chains
α-chains
β-chains







βV63(E11)T
12.0*
2.2
340
 30
29.0 (0.5)
0.06 (0.5)ν
—
1.15
0.35



αV58(E11)T
25.0*
<~1.1
 15
380
0.03 (0.5)
29.0 (0.5)ν
—
—
—



βL28(B10)N
0.64*
1.8
—
—
—
—

—
—
















Protein
HbA
1.6&
2.8

33.0 (1.0)ν

4.6
0.4



surface
PB4
4.0&
2.1

10.0 (0.4), 138(0.6)ν

4.2
0.2



[βV1M+H2Δ+T4I+P5A)]



PB5
12.0*
2.4

6.9 (0.3), 115 (0.7)ν

2.3
0.1



[βV1M+H2Δ+T4I+P5A+A76K)]


Polymerization
HbA
17.0°
2.8

63.0Λ


HbA αHβH
HbPrisca
17.0°
2.3

54.0Λ



[αHβH(S9C+C93A+C112G)


Polymerization
HbA
18.0°
2.4

33ν
160
3.4
0.32















HbA αHβBv
HbPolytaur
16.0°
1.7

0.7 (0.25), 4.0 (0.75)ν
 46
0.7 (30%),
20%



[αH(C104S)βBv(S9C+C93A]





5.5 (30%),



Hb(Polytaur)n
~2-3°
1.0



0.0 (40%)
40%



[αHβBvS9C)]


Polymerization
Mb
1.1
1.0


Myoglobin
PolyMb
1.1
1.0



[N8C + K50C + K76C]









Regarding Table 2, due to differences in experimental conditions, comparisons can only be made within each study. *Gill cell33 (pH 7.4, 25° C. 50 mM Hepes+100 mM NaCl): &Gill cell (pH 7.4, 25° C. 50 mM Hepes); ∘Hemox analyzer (TES Medical Products) (pH 7.4, 37° C. 50 mM Hepes+100 mM NaCl); νpH7.0, 37° C.37: ΛpH 8.5, 37° C.
Hb Prisca is a recombinant HbA with mutations β(S9C+C93A+C112G); see Fronticelli et al., Proteins, 2001; 44: 212-222, the entire disclosure of which is herein incorporated by reference. The polymerization of Hb Prisca is obtained through the formation of intermolecular S—S bonds between cysteine residues introduced at position β9, based on the model of Hb Porto Alegre (β9Ser to Cys; see Bonaventura and Riggs, Science 1967; 155: 800-802). The cysteines at β93 and β112 were replaced in order to prevent formation of spurious S—S bonds during the expression, assembly, and polymerization of the modified subunits. The final polymerization product (i.e., HB Prisca) is mainly formed by 6 to 8 tetrameric Hb molecules.
Three of the modified, non-polymerized Hb molecules against which the polymerized Hb of the invention were compared have modifications in the amino acid residues of the heme pocket. These modified, non-polymerized Hb molecules are: βL28(B10)N, which is a recombinant HbA in which βLys28(B10) was replaced by Asn; βV67(E11)T, which is a recombinant HbA in which βVal67(E11) was replaced by Thr; and αV63(E11)T, which is a recombinant HbA in which αVal63(E11) was replaced by Thr. See, e.g., Fronticelli et al., Biochemistry, 1993; 32: 1235-1242 and Pechik et al., Biochemistry, 1996; 35: 1935-1945, the entire disclosures of which are herein incorporated by reference. These three mutant Hb molecules have a range of oxygen affinities, as can be seen in FIG. 1.
For βL28(B10)N, Leu28(B10) in the B-helix of the β-chains (which is a highly conserved residue in the hydrophobic cluster on the heme distal side) was replaced with the isosteric, but more polar, asparagine. The modified residue interacts with bound O2, destabilizing the T-state and increasing the oxygen affinity in this mutant Hb. Comparison of oxygen-binding curves indicated that βL28(B10)N has a high oxygen affinity (P50=0.64 torr) and that, in the liganded form (R-state), the affinity βL28(B10)N for O2 of is similar to that of HbA (12.5 and 13.8 torr, respectively). In the unliganded form (T-state), the O2 affinity is about one order of magnitude larger in βL28(B10)N than in HbA (0.2 and 0.03 respectively).
For βV67(E11)T, the valine at position E11 in the E-helix of the α or β chains was replaced with the isosteric, but polar, threonine. βV67(E11)T has a two-fold decrease in oxygen affinity (P50=12 torr) with respect to HbA (P50=6.5 torr), and retains a high level of cooperativity (n=2.2); see Fronticelli et al, 1993, supra. Crystallographic analysis indicated the presence of only subtle changes in the local geometry, with the presence of an H bond between the Oγ atom of βThr67(E11) and the backbone carbonyl of βHis63(E7). A water molecule was not introduced into the β-heme pocket as result of this mutation; see Pechik et al., 1996, supra. The same mutation was introduced at the same E11 site of the α-chains with different functional effects: the oxygen affinity of αV62 (E11)T was decreased four-fold (P50=25 torr) and cooperativity was practically absent. The significantly decreased oxygen affinity measured in αV62(E11)T was attributed to stabilization of the water molecule present in the distal heme pocket, which becomes H-bonded to Nepsilon of βHis63 and Oγ of βThr67. This water molecule must be dissociated prior to O2 bonding to the Fe-heme; see Karavitis et al., J. Biol. Chem. 1998; 237: 23740-23749, the entire disclosure of which is herein incorporated by reference.
Inspection of the data in Table 2 indicates that increasing the heme pocket polarity increases the rate of autoxidation, as measured for the V(E11)T mutants. The stability and toxicity of a Hb-based oxygen carrier is dependent on the rate of heme loss. Thus, placing a polar residue at position E11 causes a decrease in the rate of heme transfer in βV67(E11)T with respect to HbA; this indicates an increased heme affinity. This property should be considerably enhanced in αV63(E11)T; see, e.g., the titration curves of the aquomet derivatives as shown in FIG. 2. These titration curves, while indicating a similar pKa of ionization of the Fe-coordinated water in HbA and βV67(E11)T (8.2 and 8.35, respectively), show a shift to 8.7 in the pKa of αV63(E11)T. This is consistent with the presence, in αV63(E11)T, of a H-bond between the water molecule and Oγ of Thr(E11), which helps to retain the heme in place. Introducing a polar residue at position E11 also causes the decrease in the half-time of NO dissociation from the heme of the mutant heme-pocket; this indicates a reduction in NO affinity in the V(E11)T mutants and a potential decrease in in vivo vasoactivity.
Three of the modified, non-polymerized Hb molecules against which the polymerized Hb of the invention were compared have modifications in the amino acid residues on the surface of the polypeptide subunits. These modified, non-polymerized Hb molecules have an intrinsically low oxygen affinity in the absence of heme pocket modifications, which is achieved through mutations that increase the hydrophobic interactions between the A-helix and the hydrophobic core of the β-subunits.
These surface-modified, non-polymerized Hb molecules are: PB4, which is a recombinant HbA with mutations β(V1M+H2deleted+T4I+P5A); and PB5, which is a recombinant HbA with mutations β(V1M+H2deleted+T4I+P5A+A76K). See Fronticelli et al., J. Biol. Chem. 1995; 270: 30588-30592 and Fronticelli et al., Biophys. Chem. 2002; 98: 115-126, the entire disclosures of which are herein incorporated by reference.
PB4 has an N-terminal end similar to that of bovine Hb, and exhibits a three-fold decrease in oxygen affinity with respect to HbA (see FIG. 3 and Table 2). A regulatory mechanism whereby Cl— ions are the principal effectors stabilizing the T-state was engineered into PB4, by replacing alanine at β76 with lysine (which is the residue present at this site in bovine Hb). This mutant is designated PB5, and has an additional three-fold decrease in oxygen affinity compared to PB4 in the presence of the Cl— concentration present in the blood plasma (100 mM) (see FIG. 3 and Table 2).
The comparison of the mutant, non-polymeric Hb, normal HbA, and Hb Prisca with the polymeric Hb of the invention shown in Table 2 indicate that low oxygen affinity and high cooperativity are disadvantageous for some applications of blood substitutes. Polymers of Mb were therefore constructed, which can be used to efficiently deliver oxygen to tissues with a restricted blood flow. Moreover, a large array of Mb heme pocket mutants have been constructed and are known in the art; see, e.g., Springer et al., Chem Rev 1994; 94:699-714, the entire disclosure of which are herein incorporated by reference. The PolyMb of the invention, and its component monomers, can comprise any of the known Mb heme pocket mutations.
The primary amino acid structure of Mb is known. For example, the primary amino acid structure of human Mb is described in Kunishige et al., Muscle Nerve, 2003, 28(4), 484-492, and is presented herein as SEQ ID NO: 7. Three splice variants of the human mRNA encoding SEQ ID NO: 7 have been identified, see Kunishige et al., supra, and are provided herein as SEQ ID NO: 8 (splice variant 1), SEQ ID NO: 9 (splice variant 2) and SEQ ID NO: 10 (splice variant 3). The primary amino acid sequence of Mb from other species are also known. For example, sperm whale (Physeter macrocephalus) Mb amino acid sequence is described in Springer et al., Proc. Natl. Acad. Sci. U.S.A., 1987, 84(24), 8961-8965, the entire disclosure of which is herein incorporated by reference, and is provided herein as SEQ ID NO: 11. The nucleic acid sequence encoding sperm whale Mb is provided herein as SEQ ID NO: 12. Domestic pig (Sus scrofa) Mb amino acid sequence is described in Akaboshi, Gene, 1985, 40(1): 137-140, the entire disclosure of which is herein incorporated by reference, and is provided herein as SEQ ID NO: 13. The nucleic acid sequence encoding pig Mb is provided herein as SEQ ID NO: 14. Bovine (Bos taurus) Mb amino acid sequence is described in Shimada et al., J. Biochem., 1989, 105(3): 417-422, the entire disclosure of which is herein incorporated by reference, and is provided herein as SEQ ID NO: 15. The nucleic acid sequence encoding bovine Mb is provided herein as SEQ ID NO: 16.
Isolated Mb monomers of the invention can be produced by introducing polymerization sites into the molecule. These modified Mb monomers can then be polymerized by subjecting the monomers to conditions which activate the polymerization sites. A suitable technique for introducing and then activating polymerization sites on Mb monomers is described in the Examples below.
Thus, the invention provides an isolated, modified Mb monomer, wherein polymerization sites are introduced into the molecule. In a preferred embodiment, the polymerization sites are introduced into the Mb monomer by substitution of at least one, for example two or more amino acid residue with a cysteine. However, polymerization sites can also be introduced chemically; for example, with bi-functional reagents, as is known in the art. For instance, non-specific bifunctional agents can be used to cross-link the reactive amino groups of Mb monomers. Any agent that produces an intermolecular cross-link between two polypeptides such as are known in the art can be used. Suitable non-specific bi-functional agents include sebacyl chloride, aldehydes such as glutaraldehyde or polyaldehydes, diasprin derivatives, and other bifunctional polymers such as such as PEG derivatives, inulin or dextran. See, e.g., U.S. Pat. No. 6,747,132 to Privalle et al., the entire disclosure of which is herein incorporated by reference.
Alternatively, “zero-link” technology can be used to form intermolecular cross-links between Mb monomers. For example, COOH groups on the surface of the Mb monomer can be activated, followed by a reaction of the activated COOH group with an available NH2 group on the surface of another Mb monomer. See, e.g., U.S. Pat. No. 5,998,361 to Bucci et al., the entire disclosure of which is herein incorporated by reference.
In a preferred embodiment, the amino acid sequence of Mb monomers is mutated by substituting at least one, preferably two or more amino acid residues located on the surface of the folded polypeptide with a cysteine. Optionally, one or more naturally-occurring cysteines in a Mb monomer (if any) are substituted with amino acid residues which do not form intermolecular bonds. Techniques for mutating the amino acid sequence of a given polypeptide are known in the art, and are discussed in more detail below.
Any Mb molecule can be modified by introducing polymerization sites. Preferably, the modified Mb molecule comprises a sperm whale Mb molecule, although human, pig or bovine Mb molecules can be used.
One skilled in the art would understand which surface residues of a given Mb molecule can be modified to introduce a polymerization site. For example, positively charged amino acid residues such as His and/or Lys residues on the Mb monomer surface can be substituted with cysteines. Suitable residues on the surface of sperm whale Mb which can be replaced by cysteines include: Val1, Gln8, Lys50, Lys76, His12, His36, His48, His81, His113, His116, and His119. See, e.g., Yung-Hsiang et al., Biophys. J., 2000, 79: 1637-1654, the entire disclosure of which is herein incorporated by reference. One skilled in the art can readily identify the analogous amino acid residues in Mb monomers from other species, for example in human, pig and bovine Mb monomers. See, e.g., Springer, Chem. Rev., 1994, 94: 699-714, the entire disclosure of which is herein incorporated by reference, which states that there is qualitative agreement between analogous pig, human and sperm whale Mb heme-pocket mutants, indicating that generality of results between Mb mutants of different species can be expected.
In one embodiment, polymerization of sperm whale Mb monomers can be achieved by introducing the substitutions Gln8 to Cys, Lys50 to Cys and Lys76 to Cys. These residues are external (i.e., on the surface) of folded Mb monomers, and are in favorable reactive positions. The replacement of the lysine residues with cysteines decreases the Mb net charge, favoring intermolecular interaction and S—S bond formation. FIG. 7 shows the gel filtration chromatography of such a modified sperm whale Mb and of polymerized Mb (polyMb) made with the modified Mb monomers.
As stated above, Mb monomers of the invention can further comprise heme-pocket mutations or other which alter the oxygen affinity of the monomers. Such heme-pocket mutations and techniques for introducing them into Mb monomers are known in the art; for example, mutations to the amino acids at positions E7 (His to Gly, Gln, Ala, Val, Thr, Ile, Met, or Phe), E11 (Val to Ala, Leu, Ile, Phe, Ser, Thr, Asn, or Gln), CD1 (Phe to Val or Trp), CD4 (Phe to Leu or Val) and B10 (Leu to Ala, Val, Ile, Phe or Trp). See, e.g., Springer, 1994, supra; Carver et al., J. Biol. Chem., 1994, 267, 14443-14450, and La Mar et al., J. Biol. Chem., 1994, 269: 29629-29635, the entire disclosures are herein incorporated by reference.
The invention thus provides a polymer comprising polymerized Mb monomers of the invention. Preferably, the Mb polymers of the invention (also called “PolyMb”) have a molecular weight of about 500 to about 1000 kilodaltons (kDa) or higher, e.g., about 1500 kDa, about 2000 kDa, about 2500 kDa or about 3000 kDa, as measured for example by size exclusion chromatography.
Polymerization does not appear to affect the ligand binding properties of Mb, as below indicated, and therefore, the vast amount of data correlating the effects of mutations on functional properties of Mbs can be utilized to tailor Mb polymers for specific clinical situations. For example, patients in septic shock can become hypertensive, in spite of massive fluid therapy and treatment with vasoconstrictor agents. In this instance, the overproduction of nitric oxide (NO) results in the lowered blood pressure. Therefore, in one embodiment, a Mb polymer of the invention (which has NO scavenging activity) can be used to treat septic shock.
In cancer, delivery of O2 to the hypoxic inner core of a tumor mass increases its sensitivity to treatments such as radiotherapy and chemotherapy. Because the microvasculature of a tumor is unlike that of other tissues, sensitization through increasing O2 levels requires O2 be unloaded within the hypoxic core. Thus, a PolyMb of the invention that has a high oxygen affinity (denoted by a low P50 value) can be used to treat cancer, because such a PolyMb would not unload all of the bound O2 before reaching the hypoxic tumor core.
As used herein, the term “oxygen affinity” refers to the avidity with which an oxygen carrier such as a heme protein binds molecular oxygen. This characteristic is defined by the oxygen equilibrium curve which relates the degree of saturation of heme protein molecules with oxygen (Y axis) with the partial pressure of oxygen (X axis). The position of this curve is denoted by the value, or “P50”, which is the partial pressure of oxygen at which the oxygen carrier is half-saturated with oxygen. P50 is inversely related to oxygen affinity. The oxygen affinity of a heme protein can be measured by a variety of methods known in the art. (See, e.g., Winslow et al., J. Biol. Chem. 252(7):2331-37 (1977), the entire disclosure of which is herein incorporated by reference).
The polypeptides comprising the heme protein subunits of the invention can comprise natural or synthetic peptides produced by any known means, including synthesis by biological systems and chemical methods.
Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic gene encoding the heme protein subunits, as described in more detail below. Chemical peptide synthesis includes manual and automated techniques well known to those skilled in the art.
Techniques to synthesize or otherwise obtain heme protein subunits are well known in the art. For example, automated peptide synthesis can be performed with commercially available peptide synthesizers, using conventional solid phase synthesis methods. In such methods, the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group; various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldimidazole; various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and the various cleavage reagents, e.g., trifluoroacetic acid (TFA), HCl in dioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reaction in solution with isolation and purification of intermediates, are well-known to those of ordinary skill in the art.
A preferred peptide synthesis method follows conventional Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W. H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzymology 32:221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins 2:61-64 (1990), the entire disclosures of which are herein incorporated by reference. Crude peptide preparations resulting from solid phase syntheses can be purified by methods well known in the art, such as preparative HPLC. The amino-terminus can be protected according to the methods described for example by Yang et al., FEBS Lett. 272:61-64 (1990), the entire disclosure of which is herein incorporated by reference.
Biological methods for synthesizing polypeptides comprising heme protein subunits of the invention are also within the skill in the art. For example, a nucleic acid sequence encoding the heme protein subunit can be synthesized de novo and subcloned into the appropriate expression vector for propagation in an appropriate host.
The subcloned nucleic acid sequences can be expressed directly to generate the heme protein subunit, or subjected to site-directed mutagenesis to introduce sequences coding for polymerization sites or other mutations.
Intracellularly produced heme protein subunits can be obtained from the host cell by cell lysis, or by using heterologous signal sequences fused to the protein which cause secretion of the protein into the surrounding medium. Preferably, the signal sequence is designed so that it can be removed by chemical or enzymatic cleavage. The proteins thus produced can then be purified by affinity chromatography.
The techniques used to transform cells, construct vectors, construct oligonucleotides, and perform site-specific mutagenesis are widely practiced in the art, and most practitioners are familiar with the standard resource materials which describe specific conditions and procedures. However, the following discussion is presented as a guideline for generating heme protein subunits with mutations introducing polymerization sites or other desirable characteristics, according to the present invention.
The mutant heme protein subunits of the present invention can be prepared utilizing well-known expression systems for producing recombinant heme proteins in suitable microbial hosts, such as E. coli. See Fronticelli et al., J. Prot. Chem. 10:495-501(1991), Sanna et al., J. Biol. Chem. 272: 3478-3486 (1996) and U.S. Pat. No. 5,239,061 to Fronticelli et al., the entire disclosures of which are incorporated herein by reference. Site-specific mutagenesis is used to introduce appropriate mutations into the native heme protein subunit nucleotide coding sequences, to provide a mutant DNA encoding the desired mutant polypeptide subunit.
Techniques of site-specific mutagenesis are within the skill in the art. The two principal techniques are the gapped duplex (A. A., Kruse, K. B., Brown, J. L. BioTechniques 6, 338-339, 1988) and M-13 (Zoller, M. J. and Smith, M. Meth. Enz. 100, 468-500, 1987) methods, the entire disclosures of which are herein incorporated by reference. Such site-directed mutageneses can be carried out using standard reagents such as the “Muta-Gene M-13 In Vitro Mutagenesis Kit” available from Bio-Rad Laboratories and used according to the manufacturer's instructions.
The modified heme proteins of the present invention can also be produced in transgenic animals. See, e.g., U.S. Pat. No. 5,922,854 to Kumar et al., the entire disclosure of which is herein incorporated by reference.
According to one embodiment of the invention, the heme protein polypeptides of the invention, including mutants thereof, are advantageously expressed as fusion proteins. The normal heme protein subunit polypeptides can be expressed, for example, as the fusion protein NS1-FX-(heme protein subunit). This fusion protein comprises 81 residues of the flu virus protein NS1, the Factor Xa recognition sequence Ile-Glu-Gly-Arg (SEQ ID NO:17), and the sequence of the heme protein subunit or mutant thereof. The fusion protein can be expressed in any suitable host, for example in E. coli AR58 by transformation with plasmid pJKO5, the construction of which is described by Fronticelli et al., J. Prot. Chem, 1991, supra, the entire disclosure of which is incorporated herein by reference.
Mutants of heme protein subunits can also be prepared using the expression plasmid pNF.alpha, which is structurally analogous to pJKO5. The construction of pNF.alpha is described by Sanna et al., J. Biol. Chem., 1997, supra. Expression of the plasmid in any suitable host, for example, E. coli strain AR120 yields large amounts of the NS1-FX-heme protein subunit fusion protein. Mutagenesis can be carried out with the appropriate mutagenizing oligonucleotides, as with pJKO5, to yield the desired heme protein subunit mutants.
The modified heme proteins of the present invention, in particular the polymerized Hb and Mb proteins, can be incorporated into physiologically acceptable blood substitute solutions, according to techniques within the skill in the art, for example as described in U.S. Pat. No. 5,028,588 to Hoffmann et al, the entire disclosure of which is herein incorporated by reference. The blood substitutes of the invention comprise at least one heme protein of the invention, for example a PolyMb, and a physiologically acceptable carrier. Suitable physiologically acceptable carriers are characterized as being sterile and non-toxic, and include water, balanced saline solution, physiologic saline solutions (e.g., Lactated Ringer's solution, Hartman's solution), dextrose solution and the like. Additional agents such as albumin, dextran and polyethylene glycol, for example in the amounts suggested in U.S. Pat. No. 5,028,588, the entire disclosure of which is herein incorporated by reference, can be added to increase oncotic pressure. Antioxidants and/or free radical scavengers such as mannitol, glutathione, ascorbic acid or vitamin E can also be included.
In one embodiment, a blood substitute solution of the invention contains from about 6 to about 120 g/L heme protein polymer, from about 135 to about 145 mEq/L sodium, from about 3.5 to about 4.5 mEq/L potassium, and from about 90 to about 100 mEq/L chloride. Preferably, the solution has a pH of about 7.3 to about 7.5, an osmolarity of about 280 to about 310, and an oncotic pressure of about 20 to about 30 mm Hg. Glucose can optionally be added to adjust the osmolarity.
The blood substitutes of the invention can be administered to a subject for any condition requiring supplementation of the oxygen-carrying capacity of the subject's blood. The amount of the blood substitute administered to a given subject will depend on the size, weight and age of the subject, the clinical condition of the subject, and the degree of impairment of the natural oxygen-carrying capacity of the subject's blood. Thus, the invention provides a method of enhancing the oxygen-carrying capacity of a subject's blood, comprising administering an effective amount of a blood substitute of the invention. As used herein, an “effective amount” of blood substitute is any amount which brings about a clinically relevant enhancement of the oxygen-carrying capacity of the subject's blood over a clinically meaningful time interval, as can be determined by the ordinarily-skilled physician. As used herein, a “subject” includes human and non-human animals.
Conditions which require supplementation of a subject's blood include trauma causing acute loss of whole blood, ischemia, hemodilution, septic shock, cancers, chronic anemia, sickle cell anemia, cardioplegia, and hypoxia.
The present blood substitutes can also be used in non-human animals, for example domestic animals such as livestock and companion animals (e.g., dogs, cats, horses, birds, reptiles), as well as other animals in aquaria, zoos, oceanaria, and other facilities that house animals. It is contemplated that the present invention finds utility in the emergency treatment of domestic and wild animals suffering a loss of blood due to injury, hemolytic anemias, and the like. For example, it is contemplated that embodiments of the present invention are useful in conditions such as equine infectious anemia, feline infectious anemia, hemolytic anemia due to chemicals and other physical agents, bacterial infection, Factor IV fragmentation, hypersplenation and splenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia, aplastic anemia, idiopathic immune hemolytic conditions, iron deficiency, isoimmune hemolytic anemia, microangiopathic hemolytic, parasitism, etc. In particular, the present invention finds use in areas where blood donors for animals of rare and/or exotic species are difficult to find.
The invention will now be illustrated with the following non-limiting examples.
EXAMPLES
Methods
For the studies of the heme pocket mutants, protein surface modifications, and HbA polymerization (Hb Prisca), the fusion protein expression systems pNFα and pJK05 were used for the α- and β-globins, respectively. See Sanna et al., J. Biol. Chem., 1997, 272:3478-86 and Fronticelli et al., J Protein Chem 1991, 10:495-501, the entire disclosures of which are herein incorporated by reference. The advantage of these systems is that the reconstituted Hbs have only one type of chain that is recombinant, the other being isolated from native human Hb. This permits the unambiguous assignment of any modified behavior to the substitutions made in the recombinant chains. The hybrid Hb Minotaur (αH βBv) and the polymerization derivatives Hb Polytaur and Hb (Polytaur)n were expressed in a soluble form in pDLαHβBv and purified as previously described (Bobofchak et al., Am J Physiol Heart Circ Physiol, 2003, 285:H549-61, the entire disclosure of which is herein incorporated by reference. Sperm whale Mb was expressed in pMb413, as described in Springer et al., Proc Natl Acad Sci USA, 1978, 84:8961-65, the entire disclosure of which is herein incorporated by reference, and purified according to the method described in Piro et al., Biochemistry 2001, 40:11841-50, the entire disclosure of which is herein incorporated by reference. In all of the systems used, the mutations introduced were verified by DNA sequencing. The rate constant O2 dissociation from Mb and PolyMb was determined using the oxygen pulse method as described in Gibson et al., Proc Natl Acad Sci USA, 1973, 70:1-4, the entire disclosure of which is herein incorporated by reference. The rate constant O2 and CO binding was determined by photolysis as described in Springer et al., 1978, supra, using the instrument described by Arcovito et al., J Biol Chem, 2001, 276:41073-79, the entire disclosure of which is herein incorporated by reference. Transfusion experiments were conducted as previously described (Bobofchak et al., 2003, supra). All procedures were approved by the relevant institutional animal care and use committees.
A cysteine residue was introduced into Hb Minotaur at position β9 without the substitution of the natural cysteines present at α112 and β93 as described in Bobofchak et al., 2003, supra. This mutant was polymerized to form Hb (Polytaur)n, which was a heterogeneous polymer with components having MW of 1,000 kDa or higher, as seen in size exclusion chromatography shown in FIG. 4. Oxygen-binding measurements indicated that Hb (Polytaur)n had oxygen-binding characteristics similar to Mb; i.e., high oxygen affinity (P50=about 2.0 torr) and loss of cooperativity (n=1). See Table 2, supra.
A mouse model was used to determine the effect of the Hb polymers Hb Polytaur, Hb (Polytaur)n and the chemically polymerized bovine Hb ZL-HbBv on arterial blood pressure following hypervolemic exchange as described in (Bobofchak et al., 2003, supra). The infarct volume (±SE) after 2 h focal cerebral ischemia was measured in control mice with no transfusion, and in mice transfused with either 5% albumin, 3% Hb Polytaur (P50=16.0 torr, n=1.7), 3% Hb (Polytaur)n (P50=2.0 torr, n=1.0) 0, or 6% ZL-HbBv (P50=4.0 torr, n=1.0). The results are presented in FIG. 5. The small increase in blood pressure observed in the three Hb groups is probably related to moderate hypervolemia after hypervolumetric exchange transfusion.
The effect of polymeric Hbs on transient cerebral ischemia was also investigated, as described in Bobofchak et al., 2003, supra and Nemoto et al., J Cereb Blood Flow Metab, 2003; Suppl 1; 23:48, the entire disclosure of which is herein incorporated by reference. FIG. 5 shows the reduction in infarct volume following middle cerebral artery occlusion (MCAO). An exchange transfusion was performed over a 20-min period starting at 10 min after MCAO. Infarct volume was reduced by 20% by 3% Hb Polytaur (P50=17.0, n=1.7); by 40% by 3% Hb (Polytaur)n (P50=−2.0; n=1) and by 40% by 6% ZL-HbBv (P50=−4.0; n=1). These results indicated that Hbs with a low P50 and no cooperativity preferentially unloaded O2 in ischemic tissue, even at low plasma concentrations. A rationale for this phenomenon is presented in FIG. 6, where the fractional saturation of Hb Polytaur and Hb (Polytaur)n with O2 is compared. These data indicated that at very low partial pressure of O2, (5.0-6.0 torr), as in hypoxic/ischemic tissues, Hb (Polytaur)n still released O2, whereas Hb Polytaur was nearly completely deoxygenated.
The normalized time course for oxygen dissociation from Mb O2 is shown in FIG. 8A. The normalized time course for the recombination of 0.27 mM O2 with 4.0 μM Mb or PolyMb following photolysis by a 5 ns laser flash, is shown in FIG. 8B. The observed time courses were fitted to a single exponential; the quality of the fit demonstrated that PolyMb behaved homogeneously with respect to O2 combination and dissociation, and very similarly to Mb. Thus, polymerization did not alter these properties. The calculated kinetic rate constants are listed in Table 3, together with the calculated affinity constants.
The photochemical method was used to determine the rate constant of CO combination at 1.0 mM CO and 4.0 μM Mb or PolyMb (Table 3). Also in this case, Mb and PolyMb behaved similarly; i.e., the time course of CO recombination conformed to a single exponential with the same rate constant for both proteins, thus confirming the conclusion derived from the experiments on MbO2.












TABLE 3






k′O2



k′CO


Protein
(×10 M−6s−1)
kO2s−1
K (×10 M−6)
P50(torr)
(×10 M−6s−1)







Mb
15.0
27.0
0.55
1.1
4.0


PolyMb
15.0
27.0
0.55
1.1
4.0









All documents referenced in this application are herein incorporated by reference in their entirety. A variety of modifications to the embodiments described above will be apparent to those skilled in the art from the disclosure provided herein. Thus, the invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof.

## Claims

1. An isolated hemoglobin comprising a first and a second polypeptide, each having the amino acid sequence of the normal human hemoglobin alpha chain, and a third and fourth polypeptide, each having the sequence of normal bovine hemoglobin beta chain, wherein at least one polypeptide comprises at least one mutation which introduces a polymerization site.

2. The isolated hemoglobin of claim 1, wherein the at least one mutation which introduces a polymerization site is the substitution of the alanine at position 9 for cysteine on at least one of the polypeptides having the sequence of normal bovine hemoglobin beta chain.

3. The isolated hemoglobin of claim 2, wherein the at least one mutation further comprises substitution of the naturally ocurring cysteine residues with an amino acid residue which is not cysteine.

4. The isolated hemoglobin of claim 1, wherein the naturally ocurring cysteines are substituted with alanines.

5. The isolated hemoglobin of claim 1, wherein at least one of the polypeptides having the amino acid sequence of the normal human hemoglobin alpha chain comprises a mutation of residue 104 from cysteine to serine, and wherein at least one of the polypeptides having the amino acid sequence of the normal bovine hemoglobin beta chain comprises a mutation of residue 9 from alanine to cysteine and a mutation of residue 93 from cysteine to alanine.

6. The isolated hemoglobin of claim 1, wherein at least one of the polypeptides having the amino acid sequence of the normal bovine hemoglobin beta chain comprises a mutation of residue 9 from alanine to cysteine.

7. A polymer comprising the isolated hemoglobin of claim 1.

8. A polymer comprising the isolated hemoglobin of claim 5.

9. A polymer comprising the isolated hemoglobin of claim 6.

10. The polymer of claim 9, wherein the polymer has a molecular weight at least 1000 kilodaltons.

11. A nucleic acid comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of the normal human hemoglobin alpha chain modified with a mutation of residue 104 from cysteine to serine.

12. A nucleic acid comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of the normal bovine hemoglobin beta chain modified with a mutation of residue 9 from alanine to cysteine and a mutation of residue 93 from cysteine to alanine.

13. A nucleic acid comprising a nucleotide sequence which encodes a polypeptide having the amino acid sequence of the normal bovine hemoglobin beta chain modified with a mutation of residue 9 from alanine to cysteine.

14. A polypeptide encoded by the nucleic acid of claim 11.

15. A polypeptide encoded by the nucleic acid of claim 12.

16. A polypeptide encoded by the nucleic acid of claim 13.

17. A blood substitute comprising the isolated hemoglobin of claim 1, or a polymer thereof.

18. A blood substitute comprising the isolated hemoglobin of claim 5, or a polymer thereof.

19. A blood substitute comprising the isolated hemoglobin of claim 6, or a polymer thereof.

20. The isolated hemoglobin of claim 1, wherein at least one of the alpha chain polypeptides or at least one of the beta chain polypeptides further comprises a modification to the heme binding pocket.

21. The isolated hemoglobin of claim 20, wherein the modification to the heme pocket comprises a mutation that alters the oxygen affinity of the hemoglobin.

22. The isolated hemoglobin of claim 21, wherein the oxygen affinity is higher than the oxygen affinity of whole blood.

23. An isolated hybrid hemoglobin comprising at least one human hemoglobin subunit polypeptide and at least one non-human mammalian hemoglobin subunit polypeptide.

24. The isolated hybrid hemoglobin of claim 23, comprising two human hemoglobin alpha subunit polypeptides, and two non-human mammalian hemoglobin subunit polypeptides.

25. A method of producing the isolated hemoglobin of claim 1, comprising modifying at least one subunit polypeptide to introduce a polymerization site.

26. A method of supplementing the oxygen-carrying capacity of a subject's blood, comprising administering to the patient an effective amount of the blood substitute comprising an isolated hemoglobin or a polymer thereof, wherein the isolated hemoglobin comprises a first and a second polypeptide, each having the amino acid sequence of the normal human hemoglobin alpha chain, and a second and third polypeptide, each having the sequence of normal bovine hemoglobin beta chain, and wherein at least one polypeptide comprises at least one mutation which introduces a polymerization site.