NL8701254A - PROCESS FOR PREPARING CONJUGATES OF METALLOPROTEINS AND NEW DERIVATIVES THEREOF. - Google Patents

Description

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Process for preparing conjugates of metalloproteins and new derivatives thereof.

This invention relates to a convenient preparation of biologically active metalloprotein conjugates without loss of biological activity.

Such novel conjugates are useful for targeting the metalloprotein to a particular tissue or organ for therapeutic purposes to reduce the immunogenicity and antigenicity of that metalloprotein and / or increase their bioavailability and their therapeutic effect.

More particularly, the novel conjugates prepared by the method of the invention are conjugates of metalloproteins with purified human albumin and optionally with purified IgG immunoglobulin or suitable fragments thereof. In particular, conjugates are obtained "with antigen-binding fragments (Fab) of tissue-specific antibodies such as that against myosin. The corresponding metalloprotein-albumin-antimyosin conjugate is especially useful for targeting the metal-loprotein conjugate to myosin, a protein from the 20 heart muscle.

The metalloprotein conjugates of this invention have useful therapeutic effects, in particular, nullify the toxic effect of free oxygen radicals. Such useful properties are evidenced by a reduction in damage attributable to free oxygen radicals, such as arrhythmia, tissue necrosis and tissue damage from radiation and from poisoning with certain chemicals (paraquat) or with certain cytotoxic drugs (anthracyclines ).

The scope of this invention is not limited to certain metalloprotein conjugates, but particularly concerns the metalloproteins known as "peroxydis mutases". The preferred metals in this invention are copper and zinc. The structure of the corresponding apoproteins, which are exemplified by their monomeric polypeptide chains, is described in more detail below.

In scientific and patent literature, there is a very extensive description of metalloproteins relevant to this invention. See, for example, "Super-oxide Dismutase" by Larry W. Oberley, ed. (CRC Press Ine., Vol. I-II-III, 1982, 1985) and "WH Biological and Clinical Aspects of Superoxide and Superoxide Dismutase" from 10 WH Bannister. and JV Bannister, ed., 1980 (Elsevier

North Holland Inc.) and also U.S. Patent 4,340,675 (J.T. Johansen, July 20, 1982) and Canadian Patent 1,168,150 (from M.J. Poznansky, May 29, 1984).

In particular, copper-zinc dismutases have been shown to have dimers with a molecular weight of about 32,000, with per gram 2 grams of copper and 2 grams of zinc. The dimers are made up of two identical subunits that associate by non-covalent interactions and the subunits are known to be specific for the species and for the origin of the metalloportex.

It is also known that the biological effect is lost if the metals, especially copper, are removed from it. It is also known that in the presence of chelating agents at a low pH (3,5 3.5) total inactivation and removal of metals occurs, and moreover that even in the absence of chelating agents at low pH during dialysis partial loss of the biological activity (dismutase) occurs (JC Dunbar and JT Johansen in Carlsberg Res. Commun. 47 (1982) 163.

Not surprisingly, therefore, a significant loss of efficacy (30 to 40%) in the preparation of an albumin conjugate was noted by K. Wong et al.

(Agents and Actions, 1CI (1980) 231), likely attributable to dialysis purification and / or other operating conditions using a bovine metalloprotein (Cu-Zn peroxide dismutase). Other examples of biological inactivation of metalloproteins due to metal loss are also known to those skilled in the art (see e.g.

K.H. Beem et al. In Biochemistry JJ) (1977) 1931).

8701131

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The desirability of obtaining metalloprotein conjugates (with albumin, with antibodies or with other substances such as Ficoll, polyethylene glycol, etc.) has been reviewed by M.J. Poznansky and R.L. Juliano 5 in Pharmac. Reviews 36 (1984) 277-336.

In all previous cases, a suitable metalloprotein dimer was used to obtain the desired conjugate in the presence of a crosslinker and the appropriate carrier protein (albumin) and / or the appropriate antibody, followed by extensive purification to remove the unwanted proteins and / or reagents.

The invention provides a new method in which the monomeric or dimeric apoprotein (without metals) undergoes cross-linking in the presence of the carrier protein (albumin) and optionally the desired antigen-binding fragment (Fab) and as crosslinker glutaraldehyde, a carbodiimide (especially 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or EDCI), cis-aconitic anhydride, a mixed anhydride, cyanuric chloride and / or an imido-crosslinker, whereupon 20 * by the corresponding apoprotex albumin or optionally the mixed apoprotein albumin-antibody conjugate, usually under mild conditions at neutral pH and even at low temperature. According to the invention, the conjugate thus obtained, after quenching the crosslinking reaction with a suitable reagent (glycine in the case of glutaraldehyde), is purified by dialysis, by ultrafiltration by molecular sieves or by gel exclusion chromatography.

The apoprotein conjugate thus purified has a molecular weight which is a function of the amount of crosslinker used and the duration of the conjugation reaction.

After determining the average molecular weight, the purified conjugate is restored with an appropriate metal solution, first copper and then zinc in the case of Cu-Zn-metalloprotexne, at carefully controlled pH, as prescribed by Beem et al. (In J Biol Chem 249 (1974) 7298 and in Biochemistry 16 (1977) 1931). The recovered metalloprotein conjugate is biologically active and ready for therapeutic use.

40 A surprising and unforeseen aspect of this δ 7 ή · ί ·) λ V / ν '; vi ·; - 4 - The invention is the possibility to use apoproteins as starting materials, since it is well known that metals, especially zinc, have a stabilizing effect on the active site of the protein (see HM Steinman in Chapter 2 of part 1 5 of "Superoxide Dismutase", loc. cit.), and that restoration of the biological activity in the apoprotein conjugate complex is possible.

Another advantageous aspect of this invention is that apoproteins are directly available by recombinant biotechnology and can be used as starting materials without extensive purification.

Another advantage of this invention is the ability to restore the biological activity of the final product with metals other than zinc, for example, with cobalt or with radioactive Zn, which are necessary to study the pharmacokinetic properties of the conjugate (see SN Giri and HP Misra in Med. Biology 62 (1984) 285).

Examples 20 I) Of the peptide of the formula R-Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-Val-

Gln-Gly-Ser-Ile-Asn-Phe-Glu-Gln-Lys-Glu-Ser-Asp-Gly-Pro-

Val-Lys-Val-Trp-Gly-Ser-Ile-Lys-Gly-Leu-Thr-Glu-Gly-Leu- 25 His-Gly-Phe-His-Val-His-Gln-Phe-Gly-Asn-Asp -Thr-Ala-Gly-: Cys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Leu-Ser-Arq-Lys-

His-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val-Gly-Asp-Leu-

Gly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Val-Val- 30 Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp -His-Cys-Ile-

Ile-Gly-Arg-Thr-Leu-Val-Val-His-Glu-Lys-Ala-Asp-Asp-Leu-

Gly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-Ala-

Gly-Ser-Ar g-Leu-Ala-Cys -Gly-Val-Ile-Gly-I le-Ala-GIn-OH 55 where R * = H (obtained from E.coli cross-linked by recombinant technology) or where R = Ac (obtained from human erythrocytes by dialysis against 1 x 10 3 M EDTA at pH 3.8 and purified over a Sephadex G-25 column), (there is a disulfide bond between the two underlined Cys) ^ ^ m§ sanine with 500 mg of purified (degreased) human albumin 8 / U \ co vi - 5 - taken up in 10 ml of 0.02 M sodium phosphate buffer (pH = 7.4), followed by 100 µl of 25% glutaraldehyde in distilled water.

The reaction was allowed to proceed at 4 ° C for 4 hours and then quenched by adding 120 mg of glycine, after which the mixture was dialyzed against multiple changes of 0.13 M NaCl / 0.16 M glycine followed by gel filtration over a column Bio-gel A with an exclusion limit of 500,000 Dalton. The product was eluted with 0.02 M sodium phosphate buffer (pH 7.4) containing 0.13 M NaCl; it was frozen in carbonic acid / ethanol and freeze dried.

According to gel filtration analysis, the apparent average molecular weight of the apoprotein-albumin conjugate was 300,000 (range 100,000-500,000). The ratio of dimer to albumin was about 1:10.

In order to restore the metal content, two equivalents of Cu 2+ and Zn were added per 2+ moles of apoprotexalbumin conjugate and left to stand for 2 hours at 4 ° C. The pH was adjusted to 7.8 by adding enough 0.1 M KOH 20.2 + 2+ containing two equivalents of Cu and Zn per mole of conjugate.

The solution was passed through a column of Sephadex G-25 which had first been equilibrated with phosphate buffered saline, pH 7.8.

The metalloprotein albumin thus obtained was lyophilized and tested for SOD activity, which was 900 units per mg of dry matter.

11) The peptide of the formula H-Val-Gln-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-Ser-30 Gly-Val-Val-Lys-Phe-Glu- Gln-Ala-Ser-Glu-Ser-Glu-Pro-Thr- l * ir-Val-Ser-Tyr-Glu-lle-Ala-Gly-Asn-Ser-Pro-Asn-Ala-Glu-

Arg-Gly-? He-His-Ile-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-Gly-; Cys-Val-Ser-Ala- <31y-Prc) -His-Phe-Asn- Pro-Phe-Lys-Lvs-Thr-! 35 His-Gly-Ala-Pro-Thr-Asp- <3lu-Val-Arg-His-Val- <3ly-Asp-Met-

Gly-Asn-Val-Lys-üir-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-Sar-

Phe-Lys-Asp-Ser-Leu-Ile-Lys-Leu-Ile-Gly-Pro-Thr-Ser-Val-

Val-Gly-Arg-Ser-Val-Val-Ile-His-Ala-Gly-Gln-Asp-Asp-Leu- 6 7 0 1 2 3 4 40 '- 6 -

Gly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-Ala-

Gly-Pro-Arg-Pro-Ala-Cys-Gly-Val-Ile-Gly-Leu-Thr-Asn-QH (in which there was a disulfide bond between the two underlined Cys), obtained by the method of US 5 U.S. Patent 4,340,675 with subsequent dialysis against 1 x 10 M EDTA at pH = 3.8, as in Example I, was cross-linked with glutaraldehyde in the same manner as in Example I in the presence of t purified human albumin.

The apoprotein albumin protein was similarly purified 2+ and the product, as in Example I, with Cu and 2+

It was incubated to give a biologically active metallo-protein-albumin conjugate at about 800 units per mg of dry matter.

III) A metal-free peptide according to Example 15 I and Example II was used as starting material. A mixture of 2mg peptide, 50mg human albumin and 2mg anti-myosin antibodies from which the Fc portion with pepsin had been removed (see L. Hudson and FC Hay in "Practical Immunology" (Oxford, Blackwell Scientific Publications, 1976) 20 and 50 µl 25% glutaraldehyde was stirred gently at 4 ° C for 4 hours after which 300 mg of glycine was added to stop the reaction After overnight dialysis at 4 ° C against 1% NaCl and 1% glycine, unreacted peptide and small polymers were removed by ultrafiltration over Amicon XM 300. Then metal was reintroduced in accordance with Example I to give a biologically active metalloprotein-albumin-antimyosin complex with 500 dismutase units per mg of dry matter.

It will be apparent to those skilled in the art that the foregoing description is illustrative only and that a number of changes and alterations can be made without departing from the spirit and scope of this invention. For example, various other types of antibodies than that of Example III can be used, as can other cross-linkers, and conditions other than those described can also be used.

1.1 *; <* ί V 'Λ 4 O / 3 1, ^ t

Claims (2)

Method for obtaining 2 biologically active metalloprotein-albumin conjugates or metalloprotexalbumin-antibody conjugates in high yield and without loss of biological activity, characterized in that a metal-free apoprotexne in the presence of human albumin and optionally of crosslinking an antibody fragment with a crosslinker, purifying the metal-free conjugates and restoring their biological activity by adding the desired metals to those conjugates. Method according to claim 1, characterized in that the metal-free apoprotex is selected from the peptides in the following amino acid sequence: R-Ala-Hir-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp -Gly-pro-Val-15 Gln-Gly-Ser-Ile-Asn-Phe-Glu-Gln-Lys-Glu-Ser-Asp-Gly-Pro- Val-Lys-Val-JTrp-Gly-Ser-Ile- Lys-Gly-Leu-Thr-Glu - Gly-Leu-His-Gly-Phe-His-Val-His-Gln-Phe-Gly-Asn-Asp-Thr-Ala-Gly-Cys-llir-Ser-Ala -Gly-Pro-Hi s -? He-Asn-Pro-Leu-Ser-Arg-Ly s-Gly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Val- Fall- 20 Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp-His-Cys-Ile-Ile-Gly-Arg-Thr-Leu-Val-Val-iiis-Glu-Lys-Ala -Asp-Asp-Leu-Gly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-Ala-Gly-Ser-Arg-Leu-Ala-Cys-Gly-Val -Ile-Gly-Ile-Ala-Gln-CH where there is RH or acetyl and there is a disulfide bond between the two underlined Cys. Method according to claim 1, characterized in that the metal-free apoprotexne has the following amino acid sequence: H-Val-Gln-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val- Ser-Gly-Val-val-Lys-Phe-Glu-Gln-Ala-Ser-Glu-Ser-Glu-pro-Thr-Thr-Val-Ser-iyr-Glu-Ile-Ala-Gly-Asn-Ser- Pro-Asn-Ala-Glu-Arg-Gly-Phe-Hi sI le-His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-Glv- 3 7 3 1 25 4 30 "-8- Cys- Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-Thr- His-Gly-Ala-Pro-Thr-Asp-Glu-Val-Arg-ilis-Val-Gly- Asp-Met- Gly-Asn-Val-Lvs-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-Ser- Phe-Lys-Asp-Ser-Leu-Ile-Lys-Leu-Ile- Gly-Pro-Thr-Ser-Val- 5 Val-Gly-Arg-Sar-Val-Val-Ile-iiis-Ala-Gly-Gln-Asp-Asp-Leu- Gly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr -Gly-Asn-Ala-Gly-Pro-Arg-Pro-Ala-Cys-Gly-Val-Ile-Gly-Leu-Thr-Asn-CH with a disulfide bond between the two underlined Cys. Method according to claim 1, characterized in that the optional antibody fragment with anti-no binding capacity (Fab) is a fragment of antibodies against myosin. 5. Process according to claim 1, characterized in that the crosslinker used is glutaraldehyde, a carbodiimide, cis-acetic anhydride, cyanuric acid chloride and / or an imido ester. Process according to claim 5, characterized in that the crosslinker is glutaraldehyde. Process according to any one of the preceding claims, characterized in that the metals are Cu2 + and Zn2 +. Novel, biologically active metalloprotein-albumin conjugate or metalloprotein-albumin-antibody conjugate, prepared by a process according to claim 1, the monomer of which has the structure of: R-Ala-Thr-Lys-Ala-Val- Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-Val-Gln-Gly-Ser-Ile-Asn-Phe-Glu-GIn-Lys-Glu-Ser-Asp-Gly-Pro-Val-Lys- Val-Trp-Gly-Ser-Ile-Lys-Gly-Leu-Thr-Glu-Gly-Leu-His-Gly-Phe-His-Val-His-Gln-Phe-Gly-Asn-Asp-Thr-Ala- Gly- 30 Cys-Thr-Ser-Ala-Gly-Pro-Eis-Phe-Asn-Pro-Leu-Ser-Arq-Lvs- His-Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val -Gly-Asp-Leu- • Gly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp-Val-Val-Ile-Glu-Asp-Ser-Val-Ile-Ser- Leu-Ser-Gly-Asp-Eis-Cys-Ile- & 7 λ π * - I 'i 4? Ϊ́ -3- Ile-Gly-Arg-Thr-Leu-Val-Val-His-Glu-Lys-Ma -Asp-Asp-Leu- Gly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-Ala- Gly-Ser-Arg-Leu-Ala-Cys-Gly-Val -I le-Gly-I le-Ala-Gln-OH where there is RH or acetyl and between the two underlined 5 Cys is a disulfide bond, with Cu2 + and Zn2 + as metals and with a human albumin to metalloprotein ratio between 20: 1 and 1: 1, and optionally with an antigen binding fragment which is a fragment of antibodies to myosin. A novel, biologically active metalloprotein-metalloprotein-albumin-antibody conjugate prepared by a method according to claim 1, the monomer of which has a structure according to: H-Val-Gln-Ala-Val-Ala-Val-Leu-Lys- Gly-Asp-Ala-Gly-Val-Ser- Gly-Val-Val-Lys-Phe-Glu-Gln-Ala-Ser-Glu-Ser-Glu-Pro-Thr- Thr-Val-Ser-Tyr-Glu-Ile-Ala-Gly-Asn-Ser-Pro-Asn-Ala-Glu- Arg-Gly-Phe-His-Ile-His-Glu-Phe-Gly-Asp-Ala -ltir-Asn-Gly-Cys — Va 1 —Ser — Ala-Gly — Pro-uis — Phe — Asn-Pro-Phe — Lys — Lys-Thr— His-Gly-Ala-Pro-Thr-Asp-Glu -Val-Arg-Kis-Val-Gly-Asp-Met-Gly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys-Gly-Ser- 20 Phe-Lys-Asp-Ser-Leu-Ile-Lys-Leu-Ile-Gly-Pro-Thr-Ser-Val-Val-Gly-Arg-Ser-Val-Val-Ile-His-Ala-Gly-Gln -Asp-Asp-Leu- Gly-Lys-Gly-Asp-Tnr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-Ala-Gly-Pro-Arg-Pro-Ala-Cvs -Gly-Val -I le-Gl v-Leu-Thr-Asn-OH in which between the two underlined Cys is a disulfide-2 + 2+ bond, with Cu and Zn as metals and with a ratio of human albumin to metalloprotein between 20 : 1 and 1: 1, and optionally with an antigen binding fragment which is a fragment of antibodies to myosin. 10. New copper-zinc-protein-albumin and copper-zinc-protein-albumin-antibody conjugates, the protein of which has a structure according to: - · J: · * f * / / V J i. v i ;! > ·· -10- R-Ala-Thr-Lys-Ala-Val-Cys-val-Leu-Lys-Gly-Asp-Gly-Pro-Val- Gln-Gly-Ser-lle-Asn-Phe-Glu- Gln-Lys-Glu-Ser-Asp-Gly-Pro- Val-Lys-Val-Trp-Gly-Ser-Ile-Lys-Gly-Leu-Thr-Glu-Gly-Leu- Eis-Gly-Phe-His- Val-His-Gln-Phe-Gly-Asn-Asp-Thr-Ala-Gly-5 Cys-Thr-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Lsu-Ser-Arg-Lys- His -Gly-Gly-Pro-Lys-Asp-Glu-Glu-Arg-His-Val-Gly-Asp-Leu-Gly-Asn-Val-Thr-Ala-Asp-Lys-Asp-Gly-Val-Ala-Asp -Val-Val-Ile-Glu-Asp-Ser-Val-Ile-Sar-Leu-Ser-GXy-Asp-His-Cys-Ile-Ile-Gly-Arg-Thr-Leu-Val-Val-His-Glu -Lys-Ala-Asp-Asp-Leu-10 Gly-Lys-Gly-Gly-Asn-Glu-Glu-Ser-Thr-Lys-Thr-Gly-Asn-Ala- Gly-Ser-Arg-Leu-Ala- Cjrs-Gly-Val-Ile-Gly-Ile-Ala-Gln-OH where there is RH or acetyl and between the two underlined Cys is a disulfide bond, the optional antibody of which is an anti-myosin antibody and with a ratio between human albumin and metalloprotein between 20: 1 and 1: 1. 11. New copper-zinc-protein-albumin and copper-zinc-protein-albumin-antibody conjugates, the protein of which has a structure according to: 2. H-Val-Gln-Ala-Val-Ala-Val-Leu-Lys-Gly-Asp-Ala-Gly-Val-Ser-Gly-Val-Vad-Lys-Phe-Glu-Gln-Ala-Ser- Glu-Ser-Glu-Pro-Thr-Thr-Val-Ser -Tyr-Glu-Ile-Ala-Gly-Asn-Ser-Pr o-Asn-Ala-Glu- ^ g ^^ y-Phe-His-Ile -His-Glu-Phe-Gly-Asp-Ala-Thr-Asn-Gly-: Q ^ s-Val-Ser-Ala-Gly-Pro-His-Phe-Asn-Pro-Phe-Lys-Lys-Thr- 25 His-Gly-Ala-PrcXThr-Asp-Glu-Val-Arg-His-Val-Gly-Asp ^ not- Gly-Asn-Val-Lys-Thr-Asp-Glu-Asn-Gly-Val-Ala-Lys -Gly-Ser-Phe-Lys-Asp-Ser-Leu-Ile-Lys-Leu-Ile-Gly-Pr o-Thr-Ser-Val-Val-Gly-Arg-Ser-Val-Val-Ile-His- Ala-Gly-Gln-Asp-Asp-Leu-Gly-Lys-Gly-Asp-Thr-Glu-Glu-Ser-Leu-Lys-Thr-Gly-Asn-Ala-30 Gly-Pro-Arg-Pro-Ala -Gys-Gly-Val-Ile-Gly-Leu-Thr-Asn-Ol where between the two underlined Cys is a disulfide, the optional antibody of which has an anti-p 7 Ai / A u / v 5 LJ Hr! l - is body against myosin, and with a ratio of human albumin and metalloprotein between 20: 1 and 1: 1. -o-o-o-o- Λ ί 'I "·. Z; * j" · / -} "* * 4, v, - - j v;