SE468716B - Novel peptide and immunologically active compounds - Google Patents

Abstract

The patent describes a peptide of the formula NH2-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr- -Ser-Arg-Arg-Ser-Val-Ala-Ser-X2-COY in which X1 and X2 each represents a possible amino acid coupling unit, and Y represents -OH or -NH2, and an artificially prepared compound in free or carrier- associated form having the ability to bind to glycoconjugates, especially immunoglobulins, which compound is selected from the group which consists of the said peptide and functional analogues and functional derivatives thereof.

Description

In which X 1 and X 2 each represent an optional amino acid linking moiety, and Y represents -OH or -NH. Examples of suitable amino acid linkers are -Lys- and -Cys-. These coupling devices facilitate coupling of a carrier, such as bovine serum albumin, to the peptide.

The peptide of the invention has been synthesized in accordance with per se solid state techniques.

As is well known in the art, a peptide is synthesized in either acid form (COOH) or amide form (CONH2) depending on available resin-bound starting amino acid.

In another aspect of the invention there is provided an artificially prepared compound in free or carrier-associated form capable of binding to glyco-conjugates, which compound is selected from the group consisting of the peptide of the formula NH 2 -X 1 -Gln-Thr -Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr- -Ser-Arg-Arg-Ser-Val-Ala-Ser-X2-COY 1 and X2 each represent an option in which X is an amino acid linker, and Y represents -OH or -NH 2; and functional analogues and functional derivatives thereof.

As the term "artificially produced compound" is used in this specification and appended claims, the term is intended to include compounds which have been produced in an artificial manner, i.e., which have been obtained by human effort and not by natural causes which are independent of human involvement. Although the compound, specifically the peptide, of the invention has been chemically synthesized, the compound can be prepared using any other technique, for example, degradation, cloning, etc., and it is intended that the term "art. "J ..... \ 0 3 officially manufactured" shall cover products manufactured by any such technology.

When the compound of the invention is in carrier-associated form, it may be associated with any carrier to which it may be coupled by physical / chemical interaction, such as covalent bonding, ionic bonding, hydrogen bonding or hydrophobic bonding.

Examples of such carriers are mineral carriers, for example glass, aluminum hydroxide, calcium phosphate, etc., plastic surfaces, for example microplates, spheres, etc., lipids, liposomes, carbohydrates, amino acids, peptides, proteins, membranes or fractions thereof and whole cells or fractions. hence.

The term "glyco-conjugate", as used in the present specification and claims, is intended to include glycoproteins, glycolipids, peptidoglycans and proteoglycans, whether in free or carrier-associated form (both in vivo and in vitro).

Binding mechanism has not yet been established, but it is firmly believed that the compound of the invention binds to the glycol moiety of the glyco-conjugates defined above, although there may also be other sites on the glyco-conjugates which may be of equal importance in the interaction between said compound. and the glyco-conjugates. It is further believed that it is the binding conformation of the peptide of the invention in aqueous solution (pH ranging from 1 to 13) which is responsible for this specific binding to the glyco-conjugates.

In view of the above, the compound of the invention may be selected from a variety of different compounds as long as they have a similar ability to bind to glyco-conjugates (for example glycoproteins) as the peptide of the invention.

Thus, the term "functional analogs" of the peptide of the invention is intended to cover, inter alia, shorter or longer monomers or polymers of the peptide (for example functional fractions or fragments, or polymers). of fractions or fragments of the peptide) with or without exchange of one or more amino acid residues for other amino acid residues, as long as the analogs have a similar ability to bind to glyco-conjugates (especially glycoproteins, for example immunoglobulins) as the peptide of the invention .

Furthermore, the term "functional derivatives" of the peptide of the invention is intended to cover compounds which have a similar ability to bind to glyco-conjugates (especially glycoproteins, for example immunoglobulins), as the peptide of the invention. Examples of such compounds are compounds which have substantially the same structure as the peptide of the present invention, but which has one or more amino acid residues exchanged for other chemical groups, i.e. organic as well as inorganic molecules or elements.

Since it is believed that it is the binding conformation of the peptide of the invention in aqueous solution which is responsible for the binding of the peptide to the defined glyco-conjugates, it follows that the functional analogs and functional derivatives (of the peptide of the invention) should comprise at least a conformation substantially corresponding to the binding conformation of said peptide in aqueous solution. The word "includes" is used here to indicate that something is included, but that this something is not necessarily the only thing included.

In a preferred embodiment of the invention, there is provided an artificially prepared compound in free or carrier-associated form capable of binding to immunoglobulins {which compound is selected from the group consisting of the peptide of formula 15.

Ch CO \\] __ .xf) \ NH2-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr- -Ser-Arg-Arg-Ser-Val-Ala-Ser-X2 -COY in which X 1 and X 2 each represent an optional amino acid linking moiety, and Y represents -OH or -NH 2; and functional analogues and functional derivatives thereof.

The peptide and compound of the invention have highly interesting properties which make them particularly useful in the field of immunology.

The compound of the invention binds to glyco-conjugates, especially glycoproteins, for example immunoglobulins, and can thus be used in enzyme-associated form in ELISA (enzyme-linked immunosorbent assay) instead of conjugates conventionally used in such assays.

The compound of the invention can be used as a general sorbent for glyco-conjugates, for example immunoglobulins or other immunologically active substances, for example B2 microglobulin. As a sorbent, it can be added to or utilized for extracorporeal body fluids (in vivo) when plasmapheresis is used to free the body fluid from immune complexes. Other examples of the compound's utility as a sorbent in plasmapheresis are in the treatment of such diseases as allergies, leukemias and AIDS.

As a sorbent, it can further be used (in vitro) in the purification of poly- and monoclonal antibodies or other immunologically active substances.

Furthermore, the compound of the invention in carrier-associated form has a wide applicability (in vitro) in the field of immunochemistry. The carrier may specifically be a marker (label), such as gold particles, colloidal gold, enzymes (for example alkaline phosphatase or horseradish peroxidase), avidin, biotin or any radioactive isotope, (for example iodine).

-F>.

QX CO 10 15 20 25 30 35 35 The compound of the invention binds to immunologically active cells and consequently it can be used in carrier-associated form for targeting (in vivo).

Examples of carriers in this respect are various pharmaceuticals, cytostatic agents and hormones. One application of target control that is of particular interest is target control of artificial and synthetic vaccines as well as subunit vaccines for immune-competent cells.

The compound of the invention may further be used to block receptors on cell membranes, a property which renders said compound useful for immunosuppression in patients receiving grafts, such as kidney, liver or bone marrow.

A further interesting property of the compound of the invention in free form is that it acts as a lymphocyte activating factor (LAF) and induces mitosis in cells, and thus the compound possesses a strong adjuvant activity.

SYNTHESIS OF THE PEPTIDE OF THE INVENTION Solid phase synthesis (Merrifield) of the peptide of the invention has been carried out in a conventional manner, by coupling the amino acids to each other, forming peptide bonds, starting with a solid phase (resin) to which the C-terminal of the first amino acid is cup. - laden, whereupon the C-terminus of the following amino acid is coupled to the N-terminus of the first amino acid, etc. Finally, the constructed peptide is released from the solid phase.

Specifically, the following peptide was synthesized: NH 2 -Lys-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr- -Ser-Arg-Arg-Ser-Val-Ala-Ser-COOH Synthesis of the peptide fragments was performed with using a Biosearch Sam Two (Biosearch, Inc. California USA) peptide synthesis machine, using version 2.38 of Biosearch's peptide synthesis automation software for t-BOC 10 amino acids. Standard coupling constants (default) were used for 1 g of starting material (degree of substitution = 0.33 meq / g).

The resin used to synthesize the peptide was a standard Merrifield resin. The suitably protected amino acids were: Boc-Ala-OH (Boc-L-Alanine) Boc-Asn-OH (Boc-L-Asparagine) Boc-Lys (CL-Z) -OH (Boc-eo-Chloro-Benzyloxycarbonyl -L-Lysine) Boc-Ser (Bzl) -OH (Boc-O-Benzyl-L-Serine) Boc-Tyr (Cl2-Bzl) -OH (Boc-O-2,6-Dichlorobenzyl-L-Tyrosine ) Boc-Arg (Tos) -OH (α-Boc-N-Tosyl-L-Arginine) Boc-Gln-OH (Boc-L-Glutamine) Boc-Pro-OH (Boc-L-Proline) Boo-Thr ( Bz1) -OH (Boc-O-Benzyl-L-Threonine) Boc-Val-OH (Boc-L-Valine) For details regarding amino acid concentration, solvent volumes, reaction times, etc., the manufacturer's (Biosearch) recommendations were followed.

Specifically, the following chemicals are used as listed below in the synthesis, cleavage and purification of the synthetic peptide. C-1 '/ C 31 C) \ SUBSTANCE Acetylimidazole Dimethylformamide Diisopropylethylamine Hydroxybenzyltriazole Methylene chloride Ninhydrin Potassium hydroxide t-Boc amino acids Hydrogen fluoride Resorcinol Dimethylsulfide Hydroxybenzyltriazole Ethyl ether pla fl Double distilled from 1 g / l ninhydrin, then 5 g / l KOH. The first 10% distillate was discarded. p.a.

HPLC p.a. pIaI p.a. (synthesis) sequanal (HPLC) HPLC p.a.

SUPPLIER Aldrich Fluka Aldrich Aldrich Fisons (UK) Aldrich Bachem AGA Aldrich Aldrich Rathburn Merck Fluka The finished, resin-bound peptide was removed from the synthesis chamber and washed with methanol, and dried to constant weight under vacuum.

Digestion of the Peptide from the Resin Carrier Aliquots (0.5 g) of the washed and dried resin-bound peptide were transferred to a cylindrical (PTFE) polytetrafluoroethylene chamber containing 1 g of resorcinol, 6.5 ml of dimethyl sulfide and a PTFE coated magnet. The chamber was cooled to -80 ° C in a dry ice-ethanol bath. Hydrofluoric acid (HF) (final volume 10 ml) was then released into the chamber, which was then kept at 0 ° C for 120 minutes on a magnetic stirrer. The acid was then removed using a water aspirator. The chamber was again cooled to -80 ° C and 10 ml of HF was admitted into the chamber, which was placed over a magnetic stirrer at 0 ° C for 45 minutes. Then the acid was removed again using a water aspirator and the remaining mixture was transferred to a sintered glass filter. The resin / peptide mixture was then washed with 100 ml of 10% by volume acetic acid and then with 50 ml of diethyl ether. The acetic acid wash was also washed with 50 ml of diethyl ether in a separatory funnel, and the ether phase was removed from the aqueous phase.

The combined ether washes and acetic acid wash were then evaporated and lyophilized, respectively, and the peptide product was recovered. The peptide was recovered only from the aqueous phase.

The crude product was stored in glass tubes in a desiccator at room temperature.

Purification of the synthetic peptide The various components of the crude synthesis product were dissolved by applying them to a high-pressure chromatography column with "reverse-phase" C-18 silica, using acetonitrile / H 2 O (0.1 vol% trifluoroacetic acid ( TFA)) as the mobile phase. 20 pg-100 mg of the crude peptide was dissolved in up to 1 ml of H 2 O (0.1% TFA), centrifuged and injected into a 1 ml loop of a Rheodyne (California, USA) HPLC injector. The solution was pumped through a pre-packed l0x500 mm Oligosil "reverse-phase" C-18 column (Scandinavian Genetec, Sweden) using a 2152 gradient controller and two 2150 HPLC pumps (LKB, Sweden). The gradient was linear from 0 to 100% acetonitrile after 60 min.

The flow rate was 2 ml / min. The elution of the synthesis products was monitored at 206-238 nm using a variable wavelength LKB 2131 absorption monitor. Fractions were collected by hand and purified again if necessary by lyophilization of the relevant fractions, redissolution and repetition of the chromatographic procedure. The final products were lyophilized and stored in glass tubes at -20 ° C.

PREPARATION OF THE PEPTIAN ANTIGEN FOR IMMUNO ANALYSIS The peptide synthesized above was coupled to a carrier, ie bovine serum albumin (BSA) in the following manner to form a peptide antigen (coating antigen), which was used in ELISA. 1 mg of peptide and 3.6 mg of BSA were dissolved in 2.0 ml of phosphate buffered saline (PBS), pH 7.4.

To this solution was added 30 microliters of a 2.5% (w / v) aqueous glutardialdehyde solution.

The reaction mixture is incubated at room temperature (20-25 ° C) for 1 hour, stopped by adding 0.5 ml of a 5 M aqueous ethanolamine solution and then dialyzed against one liter of PBS at + 4 ° C for 4 hours, exchanging the dialysis fluid after 30 min and after 2 h.

The contents of the dialysis bag are then gel filtered through a column (2.5x60 cm) containing SephacrylC> S-300 gel (Pharmacia, Uppsala, Sweden) which is equilibrated with PBS. 3.0 ml fractions were collected.

The fractions containing coupled material were pooled and the pooled material was used in ELISA as a coating antigen and added directly, without any prior dilution, to the microplate.

ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA) The following is a general description of ELISA.

The conjugate and the dilution of the conjugate, as well as the time that elapsed before the microplates were read, can vary between the different analyzes. The differences are given according to the general description. f> 10 15 20 25 30 35 -F C) \ CÛ \ ~ 1 ... x Cs * 11 êL-ëëëïí-ëêêßfššllëlëël-YÄL-Iêê EQUIPMENT: Microtiter plates (Dynatech, mod. no. l29B).

Antigen for coating microplates (eg peptides and proteins).

Coating buffer: Phosphate buffered saline (PBS).

Incubation buffer: PBS + 0.05 vol% Tween 20 Aqueous washing liquid: 0.9% NaCl + 0.05% Tween 20 Serum (eg human and animal reference and test sera) Conjugate (eg alkaline phosphatase-conjugated porcine anti- human IgG antibodies (Orion Diagnostica) and goat anti-rabbit Ig antibodies (Sigma).

Enzyme substrate: p-nitrophenylphosphate tablets (Sigma), 1 mg / ml substrate buffer (see below) Substrate buffer: 1M aqueous diethanolamine, pH 9.8, + 0.5 mM MgCl 2 + 0.02% NaN 3.

Pipettes and test tubes DESIGN: l.

Coating of microplates.

Microplates were incubated with 0.1 ml of antigen in PBS at room temperature (20-25 ° C) overnight.

Incubation with bovine serum albumin (BSA) and serum.

The plates were washed four times and then incubated with 0.1 ml of 1% (w / v) BSA in PBS at 37 ° C for 1 hour. After washing, 0.1 ml of serum diluted appropriately in incubation buffer was added to the wells and plate (plates). ) was incubated at room temperature for 1 hour.

Incubation with conjugate.

After washing, 0.1 ml of conjugate, which was suitably diluted in incubation buffer, was added to the wells and the plate (s) were incubated at room temperature for 2 hours.

CT-CZ * - 5 10 15 20 25 30 \ J (j \ 12 After washing, 0.1 ml of the enzyme substrate solution was added to the wells. The plates were kept at room temperature and the absorbance at 405 nm was read after the time given below.

SPECIFIC DESCRIPTION OF ELISA ENZYME-LINKED IMMUNOSORBENT ASSAYS: Enzyme-linked immunosorbent assay (ELISA) for measuring the reaction between the synthetic peptide and antibodies in rabbit or human sera, or the reaction between the synthetic peptide and the peptide alone.

The peptide used as the coating antigen in the ELISA was first treated as described under "Preparation of Peptide Antigen for Immunoassay".

The serum samples were from two different rabbits (rabbit 1 and rabbit 2) and from a human (human 1) and from a pool of human sera from different individuals (human 2). All sera were used in a 1/500 dilution. The conjugate used was an alkaline phosphatase-goat anti-rabbit Ig conjugate (Sigma) diluted 1 / S00 or an alkaline phosphatase-pig anti-human IgG conjugate (Orion Diagnostica) diluted 1 / 100.

The plates were read at the times specified below in an automated ELISA reader (Titertek, Multiscan).

The results were as follows: 10 15 20 25 30 35 ms O \ co ~ a _ .. à QN 13 Serum Conjugate Plates of- Absorbance read at at 405 nm Rabbit 1 goat anti-rabbit 3 min 1.21 Rabbit 2 goat anti rabbit 3 min 1.05 Human l porcine anti-human 20 min 1.11 Human 2 porcine anti-human 20 min 1.26 no goat anti-rabbit 5 min 1.23 no porcine anti-human 13 min 0.84 As the above results showed positive reactions in all cases, these data support that the synthesized peptide reacts with both rabbit and human sera - in the latter case both with serum from an individual (human 1) and with a pooling of human sera (human 2) and / or with in these cases used alkaline phosphatase-goat anti-rabbit Ig conjugate and alkaline phosphatase-pig anti-human IgG conjugate respectively.

The fact that the synthesized peptide reacted with and bound to the respective conjugates was further demonstrated by the ability of the conjugates alone to bind to the peptide. That this binding was not caused by the interaction between the peptide and alkaline phosphatase in these conjugates was confirmed in a similar experiment where enzyme alone was used and no binding was observed.

SPECIFIC BINDING OF THE SYNTHESIZED PBP TIME TO HUMAN IgG The ability of the synthesized peptide to specifically bind to human IgG was further demonstrated in the following experiments.

The synthesized peptide (0.5 mg) was applied to a Sepharosec) 4B (Pharmacia, Uppsala, Sweden) column. The CNBr-activated Sepharose "4B gel (10 ml) had been previously reacted with an excess (800 mg) of human IgG overnight at room temperature for to immobilize IgG by covalent binding 150 mg of human IgG was found to be covalently bound to the gel matrix.- Approximately 85% of the synthesized peptide was retained i.e. bound to this column at pH 7.2 in PBS at 4 ° C and at 25 ° C. fraction of the synthesized peptide that did not bind to the column probably comprises contaminants, i.e. peptides with other sequences, It is well known that such contaminants occur when peptides are synthesized according to Merrifield.

In a control experiment using only Sepharoseqp 4B gel (10 ml) in the column, the CNBr-activated sites were blocked with ethanolamine (2 ml, 1M ethanolamine + 10 ml PBS) at room temperature overnight.

Then, the synthesized peptide (0.5 mg) was applied to the column and the column was purged with PBS.

All of the synthesized peptide was recovered from the eluate.

Consequently, the peptide did not bind to Sepharose 4B as such but to the human IgG bound to Sepharoseqg 4B gel.

By using such a column (ie human IgG-Sepharose segp 4B) it is possible to fractionate the active peptide (s) from the inactive peptide (s).

The active peptide fraction could be eluted from the column by adding 1% by volume of aqueous acetic acid. The peptide (s) thus eluted could again be shown to bind to the same column after readjustment of pH 7.2.

Taken together, these experiments further support that the synthesized peptide actually bound to human IgG in the ELISA experiments described above. fw 10 15 20 25 30 35 4-68 716 15 ANALYSIS REGARDING LYMPHOCYTE OR THYMOCYTE PROLIFERING ACTIVITY: LAF ANALYSIS In this experiment, the synthesized peptide of the invention is shown to exhibit a lymphocyte or thymocyte proliferating activity which is the one exhibited by a well-known mitogen, ie phythemagglutinin. êuësëaaäss_9së_u§§u§§nin9_§9 @ _an! ëa§§_i_§aëly§§: në Medium: 90 ml RPMI medium, Flow Laboratories 10 ml Fetal calf serum, Sigma, USA 2 ml Aqueous L-glutamine solution 14.6 g / liter, Sigma, USA 1 ml Sodium pyruvate (100 mM), Gibco Limited, UK 1 ml Penicillin-Streptomycin (10,000 units / ml), Gibco Limited, UK Mitogen: Phytohaemagglutinin, PHA HAI6 2 mg / 5 ml H20, Wellcome Diagnostics (Methyl, 1 ', 2'-3H) -Thymidine: 4.44 Bq / nmol, 120 Ci / mmol, Amersham International plc, UK Gas: air including 4% CO 2, Base gas B level. OTC-20, AGA, Sweden Centrifuge: IEC clinical centrifuge, 603 B Climate cabinet: WEDCO Incorporated, EZ-l7MM Incubation plates: NUNCLON Delta, InterMed, Denmark Cell harvester: Dynatech, Automash 2000 Scintillation counter: Beckman LS-lOillationPhase liquid , LKB products AB, Sweden êsëk§i! §¿N9_s! -Âë: §ë§ën§§§ All measures were performed under sterile conditions.

The spleen was removed from an NMRI mouse and placed in 5 ml of medium at room temperature. Thereafter, the spleen was homogenized through a 100 mesh wire mesh. The homogenate was allowed to settle for 2 minutes and the suspension was decanted from the sediment, after which the cells were centrifuged for 8 minutes at 1220 rpm. The supernatant was discarded and the cells were washed again by suspension in 5 ml of medium and centrifugation for 8 minutes at 1220 rpm. The supernatant was discarded and the cells were suspended in 5 ml of medium. The cells were diluted 1/100 and counted in a Bürker chamber and then diluted to a final concentration of 5x10 6 cells / ml. äèš: ënelx§ The outer rows of wells in the incubation plates were not used for samples but were filled with 200 μl of medium to create a stable temperature zone. "Negative" controls were placed in 12 wells / plate and consisted of 100 μl of medium and 100 μl of cell suspension. 100 μl samples were then placed in new wells and a 1: 2 series dilution was made in the remaining wells of the plate.

At least triplicate of the samples were made. As a reference (control), the mitogen phytemagglutinin (PHA) was used.

To separate wells were added in triplicate 2.5 pg, 5 pg and 7 pg PHA in medium and 1 pg and 10 pg of the synthesized peptide of the invention in medium.

The plate was placed in a desiccator which had distilled water on the bottom, after which the air in the desiccator was exchanged for the above-mentioned gas in 2 minutes. The lid was placed on the desiccator, then the desiccator was placed in the climate cabinet and incubated at 37 ° C, 90% humidity for 48 hours. After the incubation, 25 μl of 3H-thymidine was added to each well (0.010 pCi / well) and the plate was incubated for another 16 h the climate cabinet.

Then the wells were harvested quantitatively by means of the cell harvester and the test samples were collected on filter paper. To ensure complete harvesting of the cells, the wells were washed 3 times with 0.9% (w / v) aqueous sodium chloride solution. The cells thus harvested on the filter paper were punched out and placed in scintillation jars. 6 ml of scintillation fluid was added to each jar. The jars were shaken and allowed to stand for 15 minutes before measuring the radioactivity in the scintillation counter for 5 minutes / jar.

BÉELU Additives cpm (range of triplicate) No mitogen 600- 870 PHA 2.5 pg 6700- 8100 PHA 5 pg 9800-10400 PHA 7 pg 7000- 8100 The peptide of the invention: 1 pg 3000- 4200 10 pg 7000- 8500 The above the results clearly show that the synthesized peptide of the invention exhibits a thymocyte proliferating activity similar to that exhibited by the well-known mitogen PHA.

Claims (3)

1. (IX CO se 10 15 20 25 30 ('i. ~' \ 18 CLAIMS 1. Peptide of the formula H-X1-Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser - -Arg-Arg-Ser-Val-Ala-Ser-X2-Y 1 and X 2 each represent an optional coupling-facilitating amino acid residue, -OH or -NH. Artificial compound in free or carrier associate in which X and Y represent a radical capable of binding to glyco-conjugates, characterized in that it is selected from the group consisting of the peptide of the formula H-X1- Gln-Thr-Arg-Ala-Asn-Pro-Asn-Pro-Tyr-Thr-Ser- -Arg-Arg-Ser-Val-Ala-Ser-X2-Y 1 and X2 each represent an option in which X coupling-facilitating amino acid residue , and Y represents -OH or -NH 2; and functional analogs and functional derivatives thereof comprising at least one conformation corresponding substantially to the binding conformation of said peptide in aqueous solution. Artificial compound in free or carrier-associated form capable of binding to immunoglobulins, characterized in that it is selected from the group consisting of the peptide of the formula H-X1-Gln-Thr-Arg-A1a- Asn-Pro-Asn-Pro-Tyr-Thr-Ser- -Arg-Arg-Ser-ValfAla-Ser-X2-Y in which X1 and X2 each represent an optional coupling-facilitating amino acid residue, and Y represents -OH or -NH2 ; Q and functional analogs and functional derivatives thereof comprising at least one conformation which substantially corresponds to the binding conformation of said peptide in aqueous solution.