WO2017197499A1 - Stabilized hemoglobin-avidin assembly and method of preparation - Google Patents

Abstract

The disclosure concerns a hemoglobin based oxygen carrier (HBOC) comprising avidin or streptavidin and a hemoglobin conjugate preferably comprising a conjugate of hemoglobin and biotin where the conjugate is associated with avidin. The HBOC is capable of binding oxygen and releasing same in circulation in animals or humans. The design of the HBOC is intended so that it would not be vasoactive in vivo. The disclosure also concerns a method of synthesis and a method of use, or the use, of the HBOC for providing oxygen transport in the circulatory system of a human or animal.

Description

STABILIZED HEMOGLOBIN-AVIDIN ASSEMBLY AND METHOD OF

PREPARATION

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

[0001 ] This application claims priority from U.S. provisional patent application 62/327, 196 filed on April 25, 2016. A sequence listing in electronic form is being filed concurrently. The content of the priority application and of the sequence listing are herewith incorporated herein by reference in their entirety.

TECHNOLOGICAL FIELD

[0002] The present disclosure relates to a hemoglobin-based oxygen carrier (HBOC) in which hemoglobin may be chemically linked to biotin to form an assembly upon addition to avidin, a method of preparing the HBOC, and the use of same in a method for increasing oxygen transport in vivo in an individual.

BACKGROUND

[0003] The potential use of modified hemoglobin as a substitute for red blood cells in transfusions is widely documented to fill a critical need in medical therapeutics. Circulating red blood cells serve to deliver oxygen to tissues. A significant decrease of red blood cells in circulation, typically from loss of blood in trauma or from disease, can result in serious and irreversible damage to organs due to the lack of available oxygen. While administration of plasma or saline can replace lost volume, oxygenation capacity must also be restored to reduce potential morbidity. Red blood cells present problems with respect to administration (typing), storage, timely availability, and are a source of potential infection. Thus, a product that would replace red blood cells to deliver oxygen to tissues in certain circumstances continues to be widely sought.

[0004] Hemoglobin is the oxygen-carrying component of the red cell. Unmodified hemoglobin is a tetrameric assembly of protein components consisting of two sets of paired subunits, each with a heme prosthetic group to which oxygen binds. Human adult hemoglobin is tetrameric having a molecular weight of approximately 64 kD. It is structurally comprised of two alpha and two beta subunits with the alpha/beta subunits forming pairs called dimers. Acellular hemoglobin cannot be used as a replacement for lost cellular material as it dissociates into its dimeric subunits. The dissociated form is not a useful oxygen carrier and also is the source of serious side effects. Thus, a chemical cross-link or its equivalent that prevents separation of the subunits is necessary for hemoglobin to be used in an acellular state. In that case it is expected that hemoglobin will deliver oxygen to cells if it is first oxygenated and if its affinity for oxygen is lower than that of the target cell. In addition, stabilized derivatives of hemoglobin have been previously tested clinically and it was found that in some cases the materials induced symptoms that are most likely related to induced hypertension. In those cases, the added hemoglobin derivatives appear to cause vasoconstriction that is likely to be a result of their scavenging of endothelial nitric oxide. This can occur if the modified tetramer extravasates through the endothelia where they scavenge endogenous nitric oxide, the signal required for relaxation of the muscles surrounding the blood vessel.

[0005] Hemoglobin (Hb)-based oxygen carriers (HBOCs) are also known as a component of a "blood substitute". Various potential HBOCs have been investigated over the last two decades with varying degrees of success.

[0006] For use in humans, blood-substitutes must contain no unstabilized hemoglobin. Early attempts to polymerize native hemoglobin by chemical reagents provided undefined mixtures that contained materials that remained vasoactive. Therefore, an HBOC suitable for human use requires a highly purified material that does not cause any side effects, vasoactivity being a central issue that was not widely recognized until clinical trials had been conducted.

[0007] For example, hemoglobin treated with a polymerizing agent was typically passed through a 100 kD filter to remove lower molecular weight hemoglobin derivatives from hemoglobin polymers.. However, those processes have not been capable of producing material that is free of species similar in size to the core tetramers and dimers..

[0008] It was anticipated that protein assemblies that are about twice the size of the hemoglobin tetramer would avoid extravasation and scavenging of nitric oxide. Thus, it was shown that an assembly consisting of two tetramers did not raise blood pressure in sensitive animals where even a small amount of a single tetramer produces a very significant blood pressure increase. In addition, measurements of the effects of the coupled hemoglobins, "bis- tetramers" were shown to be effective oxygen carriers that did not induce hypertension in the same animals. However, reports of production of an effective oxygen-carrying bis-tetramer derived from hemoglobin in a pure state note that the processes are inefficient and the final product requires extensive purification to avoid contamination by smaller assemblies that would be subject to extravasation, potentially inducing clinical complications.

[0009] To date, it is known that the administration of some potential HBOCs administered to normal and diseased animals results in (1 ) tissue oxygenation, (2) transient blood pressure elevation and (3) potential for heme loss and toxicity. Therefore, a need exists to improve the efficiency of the hemoglobin assembly and to improve the yield of the desired hemoglobin derivatives while avoiding contamination with smaller entities.

[0010] Thus, in some aspect, it would be highly desirable to be provided with an efficient process for producing HBOCs that would not cause vasoconstriction.

[001 1 ] Still, in some aspect, it would be highly desirable to be provided with HBOCs that would not extravasate through the epithelia, avoiding induced vasoconstriction.

[0012] It would also be highly desirable that the HBOCs have an affinity for oxygen that is useful in circulation.

BRIEF SUM MARY

[0013] The present disclosure concerns a novel approach to the formation of an HBOC such that it forms readily in a state that resists extravasation while providing a suitable oxygenation function.

[0014] The present disclosure also concerns a method for producing HBOCs.

[0015] According to a first aspect, the present disclosure provides a hemoglobin based oxygen carrier (HBOC) comprising avidin and a hemoglobin conjugate comprising hemoglobin, said hemoglobin comprising two alpha subunits and two beta subunits, wherein said hemoglobin is bound to said avidin, wherein said HBOC is capable of binding 0₂ and releasing same in a similar manner as native hemoglobin in whole blood in vivo, and wherein said HBOC does not cause vasoconstriction when used in vivo. The hemoglobin conjugate may also further comprise biotin, in which case, the biotin is covalently linked to the hemoglobin, and avidin is affinity bound to the biotin and not to the hemoglobin. The HBOC may comprise more than one hemoglobin conjugate.

[0016] According to a second aspect, in the HBOC as described above, the hemoglobin conjugate comprises two hemoglobin molecules and four biotin molecules, each one of said four biotin molecules being linked to the beta subunit of said two hemoglobin molecules, and two of said four biotin molecules being affinity bound to one avidin molecule.

[0017] According to a third aspect, the present disclosure provides a method for preparing a hemoglobin based oxygen carrier (HBOC) as defined above, said method comprising the steps of: (i) biotinylating the hemoglobin to obtain the hemoglobin conjugate comprising biotin and hemoglobin, said hemoglobin comprising two alpha subunits and two beta subunits; and

(ii) contacting the hemoglobin conjugate with avidin under suitable conditions for self-assembling the biotin from the hemoglobin conjugate with said avidin resulting in the HBOC.

[0018] According to a fourth aspect, there is provided the use of the HBOC as described herein for increasing oxygen transport in vivo in an individual. There is also provided a method for increasing oxygen transport in vivo in an individual. The method comprises administering intravenously a hemoglobin based oxygen carrier (HBOC) comprising avidin and a hemoglobin conjugate, said hemoglobin conjugate comprising hemoglobin and biotin, said hemoglobin comprising two alpha subunits and two beta subunits, wherein said hemoglobin is covalently linked to said biotin, and said biotin is bound by affinity to said avidin, wherein said HBOC is capable of binding 0₂ and releasing in circulation said 0₂ in a similar manner as native hemoglobin in whole blood in vivo, and wherein said HBOC does not cause vasoconstriction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

[0020] Fig. 1 illustrates a diagram of the reaction for producing hemoglobin-biotin conjugates.

[0021 ] Fig. 2 illustrates a diagram of conjugation of biotinylated hemoglobin and avidin.

[0022] Fig. 3 illustrates a plot of the measurement of tail systolic blood pressure (SBP) in awake wild-type mice following tail-vein injection of modified PBS buffer containing N-acetyl cysteine, native hemoglobin or hemoglobin bis-tetramer. Protein solutions (3.5 g/dL) were administered at a dosage of 0.4 g/kg.

[0023] Fig. 4 illustrates a chart reporting the reverse-phase HPLC traces of Hb species after treatment with biotin-maleimide cross-linker. Peaks are as follows - Native: 20 min. (β- subunit modified), 30 min. (a-subunit). TrimesoyI: 30 min. (a-subunit), 50 min. (β-subunits cross-linked and modified). [0024] Fig. 5 illustrates a chart reporting the reverse-phase HPLC trace of fumaryl cross- linked Hb after treatment with biotin-maleimide cross-linker. Peaks are as follows - 50 min. (β-subunit modified), 80 min. (a-subunits cross-linked). Retention times have drifted due to solvent evaporation over time (solvents were mixed off-line).

[0025] Fig. 6 illustrates the mass spectrum of biotinylated β-subunit (from modification of native Hb). 15867.22 g/mol + C20H29N5O5S (451.54 g/mol) = 16318.76 g/mol

[0026] Fig. 7 illustrates the mass spectrum of biotinylated β-subunit (from modification of native Hb). 15867.22 g/mol + C20H29N5O5S (451.54 g/mol) = 16318.76 g/mol

[0027] Fig. 8 illustrates the mass spectrum of biotinylated trimesoyl cross-linked β- subunits. (2*(15867.22 g/mol-1.01 g/mol)) + C9O4H4 (176.13 g/mol) + (2XC20H29N5O5S (451.54 g/mol)) = 32811.63 g/mol

[0028] Fig. 9 illustrates a graph reporting the absorbance at 700 nm, from turbidity, resulting from the formation of one to one mixtures of native or cross-linked Hb and avidin in buffers of varying pH and ionic strength. Buffers: phosphate, pH 7.4 (l=24 mM); tris, pH 8.3 ( l=3 mM); tris, pH 9.0 (1=1 mM); borate, pH 9.2 (l=54 mM).

[0029] Fig. 10 illustrates a graph reporting the absorbance at 700 nm, associated with solution turbidity, of one to one mixtures of fumaryl cross-linked Hb and avidin in buffers of varying pH and ionic strength.

[0030] Fig. 1 1 illustrates a graph reporting the turbidity increase associated with increasing the fumaryl cross-linked Hb to avidin ratio. Buffer is tris, pH 9.0 (1=1 mM).

[0031 ] Fig. 12 illustrates a graph reporting the turbidity changes associated with adding inositol hexaphosphate (IHP) to fumaryl cross-linked Hb/avidin mixtures in HEPES, pH 7.2 ( l=2 mM).

[0032] Figs. 13A to 13F are charts reporting the size-exclusion HPLC traces of Hb-avidin conjugates with Hb bis-tetramer as a reference, Hb αβ-dimer (32 kDa, 40 min. ); Hb cross- linked tetramer (64 kDa, 36 min. ); Avidin (67 kDa, 35 min. ); Hb bis-tetramer (128 kDa, 32 min. ), Avidin + 1 < Hb dimer (99 kDa, 30 min. ); Avidin + 2* Hb dimers (131 kDa, 29 min. ); Aggregation > 131 kDa (< 28 min. ), where Fig. 13A reports the absorbance of excess biotinylated native Hb + avidin, Fig. 13B reports absorbance of Hb bis-tetramer, Fig. 13C reports the absorbance of excess fumaryl cross-linked biotinylated HB + avidin, Fig. 13D reports the absorbance of excess trimesoyl cross-linked biotinylated Hb + avidin, Fig. 13E reports the absorbance of biotinylated native Hb + excess avidin, and Fig. 13F reports the absorbance of fumaryl cross-linked biotinylated Hb + excess avidin.

[0033] Fig. 14 illustrates a chart reporting the absorption spectrum of (non-cross-linked) Hb-avidin conjugate with excess biotinylated Hb.

[0034] Fig. 15 illustrates a chart reporting the absorbance changes accompanying addition of HABA to avidin (4 μΜ avidin). Avidin was in the reference beam such that the resulting spectra above are absorptions due to the dye alone. Total [HABA] = 6 (curve 1 ), 13 (curve 2), 22 (curve 3), 37 (curve 4) or 55 μ Μ (curve 5).

[0035] Fig 16 illustrates a chart reporting the absorbance changes accompanying addition of HABA to the Hb-avidin conjugate (1 μ Μ avidin). The reference spectrum of the conjugate is provided as SI. The segment at 420 nm can be ignored because the spectrometer is at the limit of detection. Total [HABA] = 5 (curve 1 ), 1 1 (curve 2), 19 (curve 3), 33 (curve 4) or 50 μΜ (curve 5).

[0036] Fig. 17 illustrates a chart reporting the binding curves for the Hb-avidin conjugate (·) and avidin titrated with HABA (x). The curves are generated from an equation for hyperbolic saturation binding.

[0037] Fig. 18 is a photograph of a native PAGE analysis of the Hb-avidin conjugates (4% stacking gel, 6% separating gel, 1 hour, 200 V). Lane 1 : Native Hb-avidin conjugate (-195 kDa) with excess Hb; Lane 2: Native Hb-avidin conjugate (-131 kDa) with excess avidin; Lane 3: Fumaryl cross-linked Hb-avidin conjugate (-195 kDa) with excess Hb; Lane 4: Fumaryl cross-linked Hb-avidin conjugate (-131 kDa) with excess avidin; Lane 5: Trimesoyl cross-linked Hb-avidin conjugate (-195 kDa) with excess Hb.

[0038] Fig. 19 is a photograph of a native PAGE analysis of the Hb-avidin conjugates with a high percentage separating gel. Lane 2 is empty because avidin runs in the opposite direction (pi = 10.5).

[0039] Fig. 20 is a photograph of a native PAGE analysis of the Hb-avidin conjugates. The anode and cathode are reversed here.

[0040] Figs. 21 A and 21 B illustrates plots of spectral changes associated with heating native Hb (21 A) and native biotinylated Hb (21 B) at 60 °C for 10 min.

[0041 ] Fig. 22 illustrates a graph reporting the oxygen binding curve of the Hb-avidin conjugate ( ... ) compared to the curve for native Hb (— ). DETAILED DESCRIPTION

[0042] As used herein, the term "avidin" is used interchangeably for avidin and/or streptavidin, as both avidin and streptavidin have affinity for biotin.

[0043] As used herein, the term "biotin" is meant to include biotin, biotin derivatives prepared and commercially used with a linker and/or adapter to facilitate the attachment of biotin to a protein of interest. For example, biotin is being referred to herein when biotin- maleimide is used to attach biotin to the subunit of the hemoglobin. Such Biotin-maleimide, although a biotin derivative, as it is composed of biotin and a functional linker, it is nevertheless referred to herein as biotin. Those biotin, biotin derivatives, with linkers or adapters are known in the art.

[0044] As used herein, the expression "capable of binding oxygen and releasing same in a similar manner as in whole blood" is meant to refer to the property of the HBOC of the present disclosure that such HBOC would indeed have oxygen carrier capabilities. In fact, the HBOC described herein should be capable of binding oxygen in a high partial pressure of oxygen and to release it at the lower partial pressure of oxygen.

[0045] The present disclosure concerns a new hemoglobin-based oxygen carrier (HBOC) for use as a component of a blood substitute that is designed to avoid vasoactivity in use. To this end, the inventors hypothesized that chemical modifications that increase the overall size of human hemoglobin would prevent vasoconstriction associated with tetramer extravasation and scavenging of nitric oxide (NO). An efficient route to creating a larger species that avoids extravasation is by selective formation of cysteine-linked biotin conjugates of hemoglobin that undergo self-assembly with avidin. The triple protein hemoglobin-avidin-hemoglobin (HbAvHb) so-produced, binds and releases oxygen with moderate affinity and cooperativity, much like native hemoglobin within circulating red blood cells. The inventors then evaluated the HBOC oxygenation potential, the heme stability and circulatory clearance, to evaluate its utility as blood substitutes. It is anticipated that these materials may serve as red blood cell substitutes in transfusion, for increasing oxygen transport in vivo in an individual, and as carriers for pharmaceuticals in circulation.

[0046] The inventors tested the stability of the HBOC described herein, and its oxygen transport capacity, i.e. its capacity to bind and release oxygen like native hemoglobin in red blood cells, that is to bind oxygen in a high partial pressure of oxygen in the lungs and to release it at the lower partial pressure of oxygen like in peripheral tissues. [0047] In practice, as can be seen in Figs. 1 and 2, the present invention make use of the natural binding affinity of biotin for avidin to obtain the HBOC ad described herein. Accordingly, the hemoglobin based oxygen carrier (HBOC) comprises avidin and a hemoglobin conjugate that comprises hemoglobin and biotin. The hemoglobin comprises the two alpha subunits and the two beta subunits, each beta subunit of the hemoglobin is covalently linked to one biotin, and said biotin is bound by affinity to said avidin. The resulting HBOC as described herein is capable of binding 0₂ and releasing same in a similar manner as native hemoglobin in whole blood in vivo, and does not cause vasoconstriction when used in vivo. For example, as one avidin can bind up to four biotins, upon self- assembly of the HBOC, it will be understood that the HBOC may comprise more than one hemoglobin conjugate. However, likely due to spatial limitations, it was found that avidin will not bind more than two hemoglobin conjugates. Biotin can be bound in many ways to hemoglobin using known techniques in the art, or following the manufacturer's instruction. To achieve the binding, the inventors attached the biotin to the thiol moiety of an available cysteine residue on the surface of the beta subunit of the hemoglobin. Preferably, the biotinylation of hemoglobin is carried out using a bifunctional biotin reagent under an atmosphere of carbon monoxide. There are reagents that are commercially available with such bifunctional linkers. The cysteine residue used for the binding is the one preferably located at a position corresponding to amino acid residue 93 of SEQ ID NO: 1. Again, there are many variants of hemoglobin known in the art and very well characterized. Hence, the cysteine should not be limited to the one at position 93, but is one that preferably correspond to position 93 upon a simple alignment with SEQ ID NO: 1. As seen in Fig. 2, the biotinylated hemoglobin and the avidin when brought into contact self-assemble into a bis-tetramer stabilized hemoglobin, the HBOC as referred herein. Preferably, the biotinylated hemoglobin is stirred with the avidin in a neutral buffered solution, under an atmosphere of carbon monoxide.

[0048] In an effort to also further stabilize the HBOC, the inventors also introduced cross-linking the subunits of the hemoglobin to prevent dissociation of the subunits, which would cause extravasation and thus vasoconstriction, in addition to causing kidney toxicity. As mentioned above, each subunit of hemoglobin is well-characterized with known sequences. Either the beta subunits, the alpha subunits or both can be cross-linked. For example where the cross-link is between the beta subunits, both beta subunits were modified via epsilon-amino groups of lysine or via the alpha amino group of an N-terminal residue. Preferably, the lysine residue is the one located at a position corresponding to amino acid residue 82 or 144 of SEQ ID NO: 1. When the beta subunits are linked together via the alpha amino group of the normal N-terminal valine, it is that which is designated as amino acid residue 1 of SEQ ID NO: 1. When the two alpha subunits are linked together, the subunits are preferably cross-linked via the epsilon amino groups of lysine residues, and more preferably the amino group f the lysine residue located at a position corresponding to amino acid residue 99 of SEQ ID NO:2. Various techniques are known for the cross-linking of the alpha subunits or of the beta subunits.

[0049] To test for vasoactivity, two classes of hemoglobin bis-tetramers were prepared in parallel using different chemical approaches. These two classes of hemoglobin bis- tetramers are also cross-linked hemoglobins formed via "click" chemistry resulting in the formation of triazole groups in the inter-protein linkage. In essence, these two classes of hemoglobin bis-tetramers comprise two hemoglobin molecules, each containing two alpha and two beta subunits cross-linked via triazole groups, which are smaller in size than biotin and avidin. These two classes of hemoglobin bis-tetramers have shown not to elicit unfavorable hemodynamic responses that are observed in its (mono)tetrameric counterpart. These results, combined with the observation that bis-tetramers retain full NO-binding ability, suggest that the enlarged size of hemoglobin bis-tetramers is responsible for the lack of vasoactivity. They are stable in circulation and do not convert into vasoactive tetramers. These two classes of hemoglobin bis-tetramers only differ from those described herein by the absence of biotin/avidin in these two classes. Since the hemoglobin bis-tetramer described herein can only be larger in size due to the presence of biotin/avidin, it is also understood that the hemoglobin bis-tetramer, when prepared as described herein with biotin and avidin, will also not extravasate, which is to be avoided due to accompanying scavenging of nitric oxide that is needed to avoid hypertension.

[0050] An indication of vasoconstriction from a lack of endothelial nitric oxide is the increase of blood pressure (hypertension). Tests of the effects of defined hemoglobin bis- tetramers have been previously evaluated to test the general approach. The systolic blood pressures (SBP) of test animals were collected using a tail cuff apparatus (CODA noninvasive blood pressure system, Kent Scientific). The mice (wild-type or db/db) were restrained in a tube holder (Kent Scientific) then the desired solution (either buffer, native Hb or Hb bis-tetramer) was introduced by tail vein injection. The protein solutions (3.5 g/dL) were transfused at a dosage of 0.4 g/kg (e.g. 300 μΙ_ in a 25 g mouse). This is a 15% top-load infusion in wild-type mice if we assume a mouse blood volume of 80 μί/g (e.g. 2 ml_ in a 25 g mouse). Db/db mice were also administered 0.4 g of protein/kg. However, the protein solutions were more concentrated (7 g/dL) to maintain the same percent top-load (e.g. 300it in a 50 g mouse). [0051 ] For the assembly of the HBOC as described herein, avidin can be used in a ratio avidin/biotin of at least 1 :2. However, it is preferred to use a ration of at least 1 : 1 , more preferably at least 2: 1 , and most preferably 4: 1 to make sure that there do not remain any free biotinylated hemoglobin that can cause in use a vasoconstriction. In such case, the method would not necessitate a further step of purification to remove any free hemoglobin or free biotinylated hemoglobin. As can be seen in Fig. 3, the use of the hemoglobin bis- tetramers causes a similar reaction as native Hb.

[0052] The HBOC as described herein may then be used in a composition for use in a method for increasing oxygen transport. Such composition comprises the hemoglobin based oxygen carrier (HBOC) as defined herein together with a suitable excipient or carrier.

[0053] The method for increasing oxygen transport in vivo in an individual preferably comprises the step of administering intravenously a hemoglobin based oxygen carrier (HBOC) as defined herein.

[0054] The present invention may be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Example 1 - Biotinylation of Hb

Protein sources

[0055] Purified human hemoglobin A (Hb) was from Oxygenix Co. Ltd. Avidin from egg white was obtained from BioShop. Biotin-maleimide (N-Biotinoyl-N'-(6- maleimidohexanoyl)hydrazide) was purchased from Sigma-Aldrich. Fumaryl cross-linked Hb (a99-fumaryl-a99, β2) was prepared according to Snyder et al. (Snyder, S. R. et al. , PNAS, 84, 7280-7284, 1987). Trimesoyl cross-linked Hb (a2, 82-trimesoyl- 82) was prepared by published methods from tris(3,5-dibromo-salicyl)trimesate (Kluger, R. et al. , Biochemistry 31 , 7551 -7559, 1992).

Biotinylation of Hb

[0056] Native carbon monoxide bound Hb (and subsequently fumaryl/trimesoyl cross- linked Hb) was treated with 20 eq. of the biotin-maleimide reagent (Fig. 1 ).

[0057] To native Hb (0.075 μηιοΙ, 0.075 mM in 1 ml_ 50 mM phosphate, pH 6.5), in the carbon monoxide bound state was added the biotin-maleimide cross-linker (20 eq. , 24 μΙ_ of a 62.5 mM stock solution in DMSO). The Hb containing solution was stirred at room temperature for 1 h in a crimp sealed vial flushed with CO(g). Excess reagent was removed from the protein solution by four cycles of centrifugation (14000 * g, 5 min. ) through a filter (30 kDa cut-off) followed by dilution to the original volume with 50 mM phosphate, pH 6.5. The biotinylated Hb species were analyzed by reverse-phase HPLC using a 330 A C-4 Vydac column (4.6 mm * 250 mm) with a solvent gradient from 20 to 60% acetonitrile:water with 0.1 % trifluoroacetic acid (see Figs. 4 and 5). The eluent was monitored at 220 nm. Observed drifts in retention times may be attributed to solvent evaporation over time (solvents were mixed off-line) and variations in column equilibration time, temperature and degassing. The composition of the protein associated with the peaks was investigated by isolation of the fractions and analysis using electrospray ionization (ESI) high resolution mass spectrometry (AI MS Lab, Department of Chemistry, University of Toronto)(see Figs. 6-8). Biotinylated fumaryl/trimesoyl cross-linked Hb were prepared by the same method.

[0058] Conducting protein modification under an atmosphere of carbon monoxide minimizes the probability of methemoglobin formation. Two biotin molecules were incorporated per Hb tetramer, as determined by mass analysis (Figs. 6-8) with the β-subunit cys-93 residues as the expected sites of modification. Protein was manipulated in the stable carbon monoxide bound state to minimize the probability of methemoglobin formation.

Example 2 - Self-assembly of Hb-biotin-avidin-biotin-Hb

Hb-avidin conjugation

[0059] To a solution of avidin (0.019 μητιοΙ, 86 μΙ_ of 0.22 mM in 50 mM phosphate buffer, pH 6.5) was added the solution of modified Hb (biotinylated Hb) (3 eq. , 0.057 μηιοΙ, 710 μΙ_ of 0.08 mM solution in 50 mM phosphate, pH 6.5). The resulting solution was stirred at room temperature for 1 h in a crimp-sealed vial flushed with carbon monoxide. Final products were stored under an atmosphere of carbon monoxide at 4 °C. The resulting conjugates were analyzed by size-exclusion HPLC using a Superdex™ G-200 HR size-exclusion column (10 mm x 300 mm) and a tris-HCI (37.5 mM, pH 7.4) elution buffer containing magnesium chloride (0.5 M). The eluent was monitored at 280 nm. Conjugates of fumaryl/trimesoyl cross- linked Hb and avidin were prepared by the same method. Conjugates with 1 : 1 Hb:avidin were made by combining 1 eq. of biotinylated Hb to approximately 5 eq. of avidin. Final products contained less than 1 % percent methemoglobin.

[0060] In Figs. 9-12, absorbance at 700 nm was acquired for Hb species/avidin mixtures and Hb/lysozyme mixtures (5 μΜ of each protein in 1 mL of the specified buffer) using a Cintra 40 UV-vis spectrometer. The Hb species were in the CO-bound state. Independent studies with added inositol hexaphosphate (IHP) (2 eq. , 2.58 μί of a 4 mM solution in H₂0) were conducted in the same manner. In a separate experiment, a 1 : 1 mixture of native Hb and avidin was fixated using 13 eq. of glutaraldehyde (1 1 μΙ_ of a freshly prepared 0.05 M solution in water) for every 1.0 eq. of Hb (0.2 mM in 210 μΙ_ of 10 mM HEPES buffer, pH 8.0). The reaction was terminated by the addition of 100 μΙ_ of 1 M Tris, pH 8.0 then concentrated through a 30 kDa cut-off filter.

[0061 ] A spontaneous association of oppositely charged globular proteins has been observed for other combinations. Solutions of Hb (pi = 6.9) and of avidin (pi = 10.5) are transparent at 700 nm. So, an increase in absorption at that wavelength indicates turbidity of the solution following a self-assembly process. For comparison, the combination of avidin with native Hb does not result in turbidity, while mixtures of cross-linked Hb with avidin produce notable changes in the character of the solutions (Fig. 9). The inventors determined the magnitude of the effect in buffers of various ionic strength and pH (Fig. 10). In general, the aggregation was most apparent in buffers of low ionic strength, such as <10mM. The effect is enhanced with a larger ratio of Hb to avidin (Fig. 1 1 ) and by the addition of inositol hexaphosphate (IHP), which associates with the polycationic site on Hb that binds 2,3-DPG (see (Fig. 12).

[0062] The inventors also observed that a similar association between biotinylated Hb and avidin is effective in forming bis-tetramers of Hb associated with avidin as a specific assembly, nominally: Hb-biotin-avidin-biotin-Hb (designated as HbAvHb - Fig. 2).

[0063] In their initial approach, the inventors sought to modify avidin with the biotin- maleimide reagent and then introduce Hb. However, this did not produce any of the desired higher molecular weight species. However, the alternative approach, incubating biotinylated Hb with avidin, gave the desired result (Fig. 2).

[0064] Avidin's biotin-binding sites are pairs located on opposing faces of the 67 kDa protein. Thus, the inventors expect a maximum of two neighboring Hb tetramers per avidin. Excess biotinylated Hb in combination with avidin achieves saturation of the binding pockets. A single high molecular weight product was obtained, as deduced from size-exclusion HPLC (Fig. 13A). The product peak elutes much earlier than the -128 kDa Hb bis-tetramer reference material (Fig. 13B), which elutes at 32 min. , suggesting a product of larger diameter. This is as expected for a conjugate with Hb:avidin 2: 1 (-195 kDa) as shown in Fig. 2. The peak at 40 min. is consistent with the 32 kDa αβ-dimer, derived from the biotinylated species that is not interacting with avidin.

[0065] When biotinylated Hb is combined with a large excess of avidin, an assembly with Hb:avidin in a 1 : 1 ratio (-131 kDa) results (Fig. 13E). Scavenging of dissociating biotinylated dimers by avidin during their passage through the column can account for the absence of the 32 kDa peaks. These fragments were observed in native gel electrophoresis (Fig. 18, Lane 2), confirming that the tetramer is intact in the assembly. An alternative explanation is that the direct interaction of the oppositely charged proteins stabilizes the tetramer against salt- induced dissociation. However, this is unlikely, considering the importance of the cys-93 modification.

[0066] Conjugates from fumaryl (Snyder, S. R. et al. , PNAS 84, 7280-7284, 1987) and trimesoyl (Kluger, R. et al. , J Am. Chem. Soc. 114, 9275-9279, 1992) cross-linked derivatives (Figs. 13C and 13D) were also prepared. Aggregation, related to the propensity for these species to interact and/or weak avidin-biotin associations, produced an irregular tail to the major peak at 29 min. Unfolding of the protein is less likely because the defect is a non-issue for the non-cross-linked derivative. Furthermore, the shoulder is of a significant mass as it begins to elute with the void volume. It remains present if PBS buffer is the eluent. The effect is heavily exaggerated for the unsaturated fumaryl cross-linked conjugate (Fig. 13F), which has its biotin pockets completely accessible to approach. Ignoring the irregular segment, HPLC analysis reveals that the elution time of the cross-linked conjugate is the same as that of the non-cross-linked one, providing further support of the synthesis of the molecule as illustrated in Fig. 2.

Occupancy assay of (non-cross-linked) Hb-avidin conjugate with HABA dye

[0067] To independent solutions of native avidin and the Hb-avidin conjugate (4 μ Μ or 1 μ Μ of avidin, respectively in 1.0 ml_ 50 mM phosphate buffer, pH 6.5) was added 4'- hydroxyazobenzene-2-carboxylic acid (HABA) (1 -20 μΙ_ of a 5 mM solution). The reference cell contained either avidin or the hemoglobin-avidin conjugate at the same concentration. The absorption spectrum from 200-700 nm was acquired using a UV-vis spectrometer. An increase in absorbance at 500 nm (ε = 34.5 M-1 ) is associated with bound HABA. Binding curves were derived from the changes in absorbance. The same assay was performed on non-conjugated biotinylated Hb as a control.

[0068] It is unlikely that the Hb tetramer within the conjugate is cross-linked through avidin by both biotin moieties on each tetramer interacting with a biotin-binding pocket because of conflicting geometrical requirements. An assay with 4-hydroxyazobenzene-2- carboxylic acid (HABA), which binds weakly to avidin's biotin binding sites, reveals the number of unoccupied sites. A binding event is accompanied by an increase in absorption at 500 nm. The spectral changes associated with HABA binding to native avidin only (4 μΜ avidin) are presented in Fig. 15. The assay performed on the Hb-avidin conjugate (1 μΜ avidin) is shown in Fig. 16. [0069] A binding curve for the association of HABA with the conjugate was prepared and compared against that for avidin (Fig. 17). Saturation of avidin with HABA occurs with approximately four equivalents of the dye bound (KD ~6μ Μ).21 , 22 Saturation of the conjugate occurs with approximately 1.1 equivalents of the dye bound, with an equal dissociation constant. The inventors were expecting a complete two equivalents to bind, as per the proposed architecture, but do not know the reason for the deviation. The inventors can speculate that access to the binding site depends on hemoglobin's docking mode, where spatial crowding varies as a function of the statistically significant orientations. The important conclusion is that avidin's binding sites are only partially saturated. Two Hb tetramers are then linked to avidin by likely two rather than four biotin-avidin associations to give HbAvHb, which is consistent with the initial description of the conjugate made herein.

Native PAGE of Hb-avidin conjugates

[0070] 2-Dimensional Tris-HCI polyacrylamide gels contained 6% or 12% separating gel (pH 8.8) and 4% stacking gel (pH 8.8). The buffered solution containing the sample was adjusted to pH 6.8 and running buffer was adjusted to pH 8.3. Gels run with reverse polarity are noted specifically. Finished gels were stained with Coomassie Brilliant Blue. A comprehensive procedure for native polyacrylamide gel electrophoresis (PAGE) was followed.

[0071 ] This analysis is consistent with the proposed formulation of the conjugate. A 12% gel (Fig. 19) provides sufficient separation of native Hb (64 kDa) from the reference bis- tetramer (-128 kDa) and demonstrates the reverse mobility of avidin (pi = 10.5). However, the progress of the conjugates through the 12% gel is hindered, probably because the pore size is too small to allow passage of the nominally 180 A diameter of the constructs. A lower concentration (6%) separating gel was employed to exaggerate the separation between the saturated and unsaturated conjugates (Fig. 18). The fully saturated conjugates (lanes 1 , 3 and 5), with excess biotinylated Hb present in the mixture, bear overall more negative surface charge. Therefore, they can progress through the gel despite their larger size. Lanes 2 and 4 contain the unsaturated conjugates with Hb:avidin likely to be present as 1 : 1. With near neutral isoelectric points, their progression is severely retarded despite their smaller size. Thus, they remain at the origin.

[0072] A reverse polarity gel (run with cathode and anode switched) served as a control (Fig. 20). Bands from avidin are visible from that gel, while those from the saturated conjugates that run in the opposite direction are not. Example 3 - Thermal stability of biotinylated (non-cross-linked) Hb

[0073] The UV-vis spectrometer Peltier-controlled cell was maintained at 60.0 °C and protein solutions in the carbon monoxide bound state ( 10 μ Μ in 1 mL of 0.01 M phosphate buffer, pH 6.5) were cooked for 10 min. The absorbance spectrum (500 to 700 nm) was acquired at 1 min intervals.

[0074] Modification of the solvent-accessible thiols of cysteine residues of the β-subunits affects the heat-stability of the tetramer. The carbon monoxide bound protein provides a well- defined substrate to evaluate the perturbation. Subjecting the biotinylated tetramer to higher temperatures reveals that the modification decreases the protein's intrinsic heat-stability. Native Hb maintains its structural stability upon heating at 60 °C for ten minutes (Fig. 21 A). In contrast, biotinylated Hb denatures under the same conditions and a turbid solution is produced as a result the protein's unfolding (Fig. 21 B). Caccia et al.(Caccia, D. et al. , Bioconjugate Chemistry 20, 1356-1366, 2009) described the same fate for PEG-modified Hb, noting enhanced tetramer dissociation as a result of cysteine modification. As can be seen, the stability of the hemoglobin bis-tetramers as described herein is comparable to the one of native Hb.

Example 4 - Oxygen-binding properties of (non-cross-linked) Hb-avidin conjugate

[0075] The material prepared using an excess of avidin (1 : 1 Hb:avidin conjugate) was utilized for oxygenation studies. The oxygen pressure at half-saturation (P50) and Hill's coefficient of cooperativity at half-saturation (n50) were determined using a Hemox Analyzer with the sample maintained at 27 °C. Hb samples (5 mL, 7 μΜ), prepared in phosphate buffer (0.01 M, pH 7.4), were oxygenated prior to analysis by stirring under a stream of oxygen with photo-irradiation for 1.5 h at 0 °C. Samples were then transferred to a cell connected to the Hemox Analyzer for acquisition of the oxygen desaturation curve. The conversion to the deoxy state was achieved by flushing the cell with nitrogen. The data for native Hb were fit to the Adair equation using computation of an optimal non-linear least squares fit.

[0076] The unsaturated conjugate with a 1 : 1 ratio of Hb:avidin (prepared using an excess of avidin) was analyzed for its oxygenation properties. The heterogeneous oxygen-binding curve yields an affinity (P50) of 3.4 torr and a Hill coefficient (n50) = 1.7 (Fig. 22). High affinity and reduced cooperativity has been observed for another cysteine-modified Hb derivative, PEGylated-Hb, a material that has been used in various applications. Nonetheless, a degree of cooperativity and reasonable oxygen affinity are retained. [0077] While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A hemoglobin based oxygen carrier (HBOC) comprising an assembly comprising avidin and one or more hemoglobin conjugates, each conjugate comprising hemoglobin having two alpha subunits and two beta subunits, wherein said hemoglobin binds with avidin, wherein said assembly is capable of binding oxygen and releasing same in a similar manner as in whole blood.

2. The HBOC of claim 1 wherein each hemoglobin conjugate comprises hemoglobin and biotin, wherein said hemoglobin is covalently linked to said biotin, and said hemoglobin conjugate is bound to said avidin.

3. The HBOC of claim 1 or 2, wherein the assembly comprises two hemoglobin conjugates, each one of said two hemoglobin conjugates comprising two biotins attached chemically to the hemoglobin.

4. The HBOC of claim 1 or 2, wherein, for each one of said two hemoglobin conjugates, each of the hemoglobin conjugates is bound to avidin.

5. The HBOC of claim 1 , 2, 3 or 4, wherein the biotin is attached via a chemical crosslink to the beta subunits of the hemoglobin.

6. The HBOC of claim 2, wherein the biotin is attached to the thiol moiety of a cysteine residue of the beta subunit of the hemoglobin.

7. The HBOC of claim 6, wherein said cysteine residue is located at a position corresponding to amino acid residue 93 of SEQ I D NO: 1.

8. The HBOC of any one of claims 1 -7, wherein the two beta subunits of each hemoglobin are cross-linked together.

9. The HBOC of claim 8, wherein amino groups of lysine residues of the beta subunits of each hemoglobin are cross-linked together.

10. The HBOC of claim 8, wherein the N-terminal residues of the beta subunits of each hemoglobin are cross-linked via their alpha amino groups to amino groups of lysine residues.

1 1 The HBOC of claim 9, wherein said lysine residues are located at a position corresponding to amino acid residue 82 or 144 of SEQ ID NO: 1.

12. The HBOC of claim 10, wherein the N-terminal residues is a valine residues located at a position corresponding to amino acid residue 1 of SEQ ID NO: 1.

13. The HBOC of any one of claims, 1 -1 1 wherein the two alpha subunits of each hemoglobin are cross-linked together

14. The HBOC of claim 12, wherein lysine residues of the alpha subunits of each hemoglobin are cross-linked together.

15. The HBOC of claim 13, wherein said lysine residues are located at a position corresponding to amino acid residue 99 of SEQ I D NO:2.

16. A method for preparing a hemoglobin based oxygen carrier (HBOC) as defined in claim 1 , said method comprising the step of contacting a hemoglobin conjugate with avidin under suitable conditions for assembling the hemoglobin conjugate with said avidin resulting in the HBOC, said hemoglobin conjugate comprising hemoglobin with a chemical linkage between like subunits.

17. A method for preparing a hemoglobin based oxygen carrier (HBOC) as defined in claim 2 or 3, said method comprising the steps of:

(i) biotinylating the hemoglobin to obtain the hemoglobin conjugate comprising biotin and hemoglobin; and

(ii) contacting the hemoglobin conjugate with avidin under suitable conditions for assembling the hemoglobin conjugate with said avidin resulting in the HBOC.

18. The method of claim 17, wherein at step i) the biotin is attached to the thiol moiety of a cysteine residue of the beta subunit of the hemoglobin under an atmosphere of carbon monoxide.

19. The method of claim 18, wherein said cysteine residue is located at a position corresponding to amino acid residue 93 of SEQ I D NO: 1.

20. The method of claim 17, wherein at step ii) the hemoglobin conjugate is in a neutral buffered solution, stirred with the avidin under an atmosphere of carbon monoxide.

21. The method of any one of claims 17-20, wherein the avidin is used in a ratio avidin/biotin of at least 1 :2, preferably at least 1 : 1 , more preferably at least 2: 1 , and most preferably 4: 1.

22. The method of any one of claims 17-21 , wherein the two beta subunits of each hemoglobin are cross-linked together.

23. The method of claim 22, wherein lysine residues of the two beta subunits of each hemoglobin are cross-linked together.

24. The method of claim 23, wherein said lysine residues are located at a position corresponding to amino acid residue 82 or 144 of SEQ ID NO: 1.

25. The method of any one of claims 17-24, wherein the two alpha subunits of each hemoglobin are cross-linked together.

26. The method of claim 25, wherein lysine residues of the two alpha subunits of each hemoglobin are cross-linked together.

27. The method of claim 26, wherein said lysine residues are located at a position corresponding to amino acid residue 99 of SEQ I D NO:2.

28. A composition for use in a method for increasing oxygen transport, said composition comprising the hemoglobin based oxygen carrier (HBOC) as defined in any one of claims 1 -14 and a suitable excipient or carrier.

29. A method for providing oxygen transport in vivo in an individual, said method comprising administering intravenously a hemoglobin based oxygen carrier (HBOC) comprising an assembly comprising avidin and one or more hemoglobin conjugates, each conjugate comprising hemoglobin having two alpha subunits and two beta subunits, wherein said hemoglobin binds with avidin, wherein said assembly is capable of binding oxygen and releasing same in a similar manner as in whole blood.

30. The method of claim 29, wherein each hemoglobin conjugate comprises hemoglobin and biotin, wherein said hemoglobin is covalently linked to said biotin and said hemoglobin conjugate is bound to said avidin.

31. The method of claim 30, wherein the assembly comprises two hemoglobin conjugates, each one of said two hemoglobin conjugates comprising two biotin cross- linked to the hemoglobin.

32. The method of claim 30 or 31 , wherein the biotin is attached to the thiol moiety of a cysteine residue of the beta subunit of the hemoglobin.

33. The method of claim 32, wherein said cysteine residue is located at a position corresponding to amino acid residue 93 of SEQ I D NO: 1.

34. The method of any one of claims 30-33, wherein the two beta subunits of each hemoglobin are cross-linked together.

35. The method of claim 34, wherein lysine residues of the beta subunits of each hemoglobin are cross-linked together.

36. The method of claim 34, wherein valine residues of the beta subunits of each hemoglobin are cross-linked together.

37. The method of claim 35, wherein said lysine residues of the beta subunits of each hemoglobin are located at a position corresponding to amino acid residue 82 of SEQ ID NO: 1.

38. The method of claim 36, wherein said valine residues are located at a position corresponding to amino acid residue 1 of SEQ ID NO: 1.

39. The method of any one of claims 30-38, wherein the two alpha subunits of each hemoglobin are cross-linked together.

40. The method of claim 39, wherein lysine residues of the alpha subunits of each hemoglobin are cross-linked together.

41. The method of claim 40, wherein said lysine residues are located at a position corresponding to amino acid residue 99 of SEQ ID NO:2 .