Synthetic Amino Acid Copolymer Research Articles

New insights into the mechanism of action of copolymer 1 in experimental allergic encephalomyelitis and multiple sclerosis.

Copolymer 1 is a synthetic amino acid copolymer, effective in suppression of experimental allergic encephalomyelitis (EAE) induced in a variety of species. Copolymer 1 can suppress both acute and chronic relapsing EAE induced by either whole brain homogenate or the purified encephalitogens myelin basic protein (MBP) and proteolipid protein (PLP). Thus, the suppressive effect of copolymer 1 in EAE is a general phenomenon and is not restricted to a certain species, the disease type, or the encephalitogen used for EAE induction. The suppressive activity of copolymer 1 is, however, limited to EAE, and copolymer 1 has no nonspecific immunological activity. On the other hand, a marked degree of immunological cross-reactivity in both the cellular and humoral immune responses was demonstrated between MBP and copolymer 1. This cross-reactivity may be the underlying mechanism for the specific suppressive effect of copolymer 1 in EAE. In vivo and in vitro studies using both murine and human cell cultures suggest that the mechanism for copolymer 1 activity in EAE and multiple sclerosis involves, as an initial step, the binding of copolymer 1 to the major histocompatibility complex class II molecules on antigen-presenting cells. Following this step, two pathways may be activated: (1) induction of antigen-specific suppressor T cells by determinants shared between MBP and copolymer 1, or (2) competition with MBP and other myelin-associated antigens, PLP and myelin oligodendrocyte glycoprotein, for the activation of effector T cells. These two mechanisms can act either separately or in concert to interfere in the autoimmune processes that lead to the neurological damage in EAE and multiple sclerosis.

Copolymer 1 inhibits chronic relapsing experimental allergic encephalomyelitis induced by proteolipid protein (PLP) peptides in mice and interferes with PLP-specific T cell responses

Copolymer 1 (Cop 1) is a synthetic amino acid copolymer effective in suppression of experimental allergic encephalomyelitis (EAE) and developed as a candidate drug for multiple sclerosis (MS). In the present study, we induced chronic relapsing (CR)-EAE in (SJL/J X BALB/c) F 1 mice by either whole spinal cord homogenate or two synthetic peptides of proteolipid protein (PLP), p139–151 and p178–191. When Cop 1 was added to the encephalitogenic inoculum, mice were almost completely resistant to disease induction. T cell lines to p139–151 and p178–191 were specific to these peptides. Their antigen-specific responses were inhibited by Cop I in a dose-dependent manner, while their polyclonal response to the superaniigen staphylococcal enterotoxin A (SEA) was not affected by Cop 1. Using biotinylated PLP derivatives, we demonstrated that the two PLP peptides bound to I-A' molecules, and that their binding was completely inhibited by unlabelled Cop 1. Furthermore, Cop 1 could displace the PLP peptides from the MHC binding site. These results support the potential of Cop 1 as a broad-spectrum drug for MS.

Synthetic Copolymer I and Myelin Basic Protein Do Not Require Processing Prior to Binding to Class II Major Histocompatibility Complex Molecules on Living Antigen-Presenting Cells

In the present study we attempted to examine whether copolymer 1 (Cop 1), a synthetic basic random copolymer of amino acids (a candidate drug for multiple sclerosis (MS)), and myelin basic protein (MBP) undergo processing prior to their binding to MHC class II molecules on antigen-presenting cells (APC). The direct binding of biotinylated Cop 1 and MBP to living APC was monitored by flow cytometry using phycoerythrin (PE)-streptavidin. The time course for either Cop 1 or MBP binding was similar at 37°C and on ice. Both Cop 1 and MBP bound to glutaraldehydefixed APC. Furthermore, these biotinylated antigens bound also in the presence of protease inhibitors and lysosomotropic agents, suggesting that proteolysis is not required prior to their interaction with the MHC determinants. Finally, short fragments of Cop 1 molecule did not bind to most of the APC, suggesting that the polymeric nature of Cop 1 is important for its efficient and promiscuous binding.

Direct binding of myelin basic protein and synthetic copolymer 1 to class II major histocompatibility complex molecules on living antigen-presenting cells--specificity and promiscuity.

Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and is being tested as a candidate drug for multiple sclerosis. It has been previously demonstrated that Cop 1 is immunologically cross-reactive with the autoantigen myelin basic protein (BP) and competitively inhibits the response to BP of T-cell lines and clones of different major histocompatibility complex (MHC) restrictions, of both mouse and human origin. In the present study we demonstrated the direct binding of Cop 1, using its biotinylated derivative, to MHC molecules on living antigen-presenting cells. Binding of biotinylated BP and peptide p84-102 (an immunodominant epitope of BP) was also demonstrated. Cop 1 and BP bound in a promiscuous manner to different types of antigen-presenting cells of various H-2 and HLA haplotypes. The specificity of the binding was confirmed by its inhibition with either the relevant anti-MHC class II antibodies or unlabeled analogs. Cop 1 exhibited the most extensive and fast binding to antigen-presenting cells. In addition, Cop 1 inhibited the binding of biotinylated derivatives of BP and of p84-102 to the MHC class II molecules and even displaced these antigens when already bound. Thus, these results suggest that Cop 1 indeed competes with BP for MHC binding and, thereby, inhibits T-cell responses to BP. The binding of Cop 1 to different DR alleles, probably because of its multiple MHC binding motifs, may indicate its potential as a broad-spectrum drug for multiple sclerosis.

Polymeric drugs as immunomodulatory vaccines against multiple sclerosis

AbstractCOP‐1, a synthetic basic random amino acid copolymer, is effective in suppression of experimental allergic encephalomyelitis in guinea pigs, rabbits, mice, rhesus monkeys and baboons, and of the exacerbating‐remitting type of multiple sclerosis in humans. COP‐1 is cross‐reactive at the monoclonal antibody level with the basic protein of the myelin sheath of the brain (BP), and the suppressive effect may be related to this immunological cross‐reaction. Indeed, competition for the major histocompatibility complex (MHC) class II antigen, between BP and COP‐1, may be one explanation for its beneficial effect in the disease. This polymeric drug, COP‐1, may, therefore, be considered also an immunomodulatory vaccine, and may serve as a prototype to vaccines against autoimmune diseases.

Physical and biological properties of a new synthetic amino acid copolymer used as wound dressing.

A new synthetic amino acid copolymer has been evaluated as wound covering. It is permeable to water vapor in the region of 4.1 kg/m2/24 h, it does not allow microbial proliferation after in vitro inoculation, it is impermeable to bacteria, and is stable and flexible. In vivo experiments were designed to provide qualitative and quantitative evaluation on its possible use as a skin substitute in full-thickness skin excision in the guinea pig. Two excisions, approximately 12-14 cm2 were performed on each side of the spine, leaving the panniculus carnosus. One site was treated with the membrane, the other with gauze. Each animal served as its own control. Photographs with a fixed focal-length camera were taken in identical conditions for all wounds immediately and 7, 14, and 21 days after excision. They were analyzed by planimetry. Histological studies were performed at 7, 14, and 21 days. The rate of healing between 0 and 21 days of the wounds treated with the copolymer membrane was significantly accelerated in comparison with wounds covered with a dry dressing (p less than 0.05). This increased epithelialization rate was confirmed by histology, which also suggested a reduction of the inflammatory response of the wound. In vivo biodegradation studies were also performed by subcutaneous implantation in the rat followed at 15, 30, 60, and 90 days by histology and physicochemical analyses. The results demonstrate that the membrane is not biodegradable.

Synthetic copolymer 1 inhibits human T-cell lines specific for myelin basic protein.

Copolymer 1 (Cop 1) is a synthetic basic random copolymer of amino acids that has been shown to be effective in suppression of experimental allergic encephalomyelitis and has been proposed as a candidate drug for multiple sclerosis. Cop 1 is immunologically cross reactive with myelin basic protein (BP) and was shown to inhibit murine BP-specific T-cell lines of various H-2 restrictions. In the present study these findings were extended to include human T-cell lines. Cop 1 competitively inhibited the proliferative responses and interleukin 2 secretion of six BP-specific T-cell lines and 13 clones with several DR restrictions and epitope specificities. Conversely, BP inhibited--albeit to a lesser extent--the response of all the Cop 1-specific T-cell lines and clones, irrespective of their DR restrictions. Another random copolymer of tyrosine, glutamic acid, and alanine, denoted TGA, had no effect on these lines. Neither Cop 1 nor BP inhibited the response of lines and clones specific for purified protein derivative. Cop 1 and BP exerted their cross-inhibitory effects only in the presence of antigen-presenting cells. These results suggest that Cop 1 can compete with BP for the binding to human major histocompatibility complex molecules. In view of recent studies implicating BP reactivity in multiple sclerosis, these findings suggest a possible mechanism for the beneficial effect of Cop 1 in this disease.

Synthetic approaches to vaccines for infectious and autoimmune diseases

The development is outlined of some synthetic vaccines against infectious diseases, in particular cholera, shigella and influenza. In the last case, use of the synthetic adjuvant MDP in combination with a haemagglutinin peptide has led to a synthetic vaccine with built-in adjuvanticity. The production of vaccines both by chemical synthesis and genetic engineering is described. The successful use of the synthetic amino acid copolymer COP-1 as an immunomodulatory vaccine to suppress the onset of allergic encephalomyelitis in experimental animals has led to clinical trials with patients suffering from exacerbating remitting multiple sclerosis. T-cell vaccination is an alternative approach to immunization against autoimmune diseases.

Cross-reactions and specificities of monoclonal antibodies against myelin basic protein and against the synthetic copolymer 1.

Antibody cross-reactivity is here demonstrated between basic protein (BP), the encephalitogenic molecule of myelin, and copolymer 1 (Cop 1), the synthetic amino acid copolymer, which has a suppressive effect on experimental allergic encephalomyelitis and is effective in reducing the number of relapses in exacerbating-remitting multiple sclerosis. This cross-reactivity is conclusively established using mouse monoclonal antibodies (mAbs). About a third of anti-rat BP mAbs and most of anti-mouse BP mAbs cross-reacted with Cop 1. This cross-reactivity could be demonstrated with anti-BP mAbs of different specificities. In addition, several anti-Cop 1 hybridomas cross-reacted with BP. This cross-reactivity was verified in several assay systems, including competitive inhibition experiments. Moreover, some anti-BP mAbs and anti-Cop 1 mAbs reacted in a heteroclitic manner and favored the cross-reactive antigen over the immunogen. In contrast to the mAbs, no cross-reactivity could be demonstrated with the antisera of immunized mice. This observation may reflect the different B-cell populations expressed in the mAb response as compared to the polyclonal response. Thus, the use of mAbs has uncovered specificities that are not evident in antisera and has revealed pronounced cross-reactivity between BP and Cop 1 at the B-cell level. These results further establish the immunological interrelationships between Cop 1 and BP, demonstrated earlier at the T-cell level.

Effect of a synthetic polypeptide (Cop 1) on patients with multiple sclerosis and with acute disseminated encephalomyelitis: Preliminary report

Three patients with acute disseminated encephalomyelitis (ADE) and 4 patients in the terminal stages of multiple sclerosis (MS) were subjected to treatment with Cop 1, a synthetic copolymer of amino acids, which had previously been shown to have a beneficial effect in the treatment of experimental allergic encephalomyelitis (EAE). Under the treatment, the ADE patients recovered completely within 3 weeks, but 1 of 2 control cases treated with steroids showed complete recovery as well. The MS patients did not show any significant change in their motor function; however, 2 of them showed some improvement in vision and speech capacity. It is too early to conclude whether this improvement is related to the treatment. No side effect was observed in any of the patients treated with Cop. 1.

Studies on the administration of glucagon and insulin antibodies to rats.

Glucagon antisera were raised in rabbits immunised with glucagon linked to a synthetic amino-acid copolymer. Antibodies concentrated from these sera by ammonium sulphate precipitation or specifically purified using an immunoadsorbent of glucagon covalently linked to cellulose, were injected into anaesthetised rats which had been starved for 5 hours. The antibodies disappeared slowly from the serum. Neither the concentrated nor the purified antibodies, however, produced a consistent effect on the levels of blood glucose. In contrast both anti-insulin serum and insulin antibodies purified by a similar technique as that used for the purification of glucagon antibodies, caused a transitory hyperglycemia approximately proportional to the amount of antibody injected. Insulin antibodies were rapidly neutralised in the serum. Possible explanations for the failure of glucagon antibodies to induce hypoglycemia are discussed.

Protection against Experimental Allergic Encephalomyelitis

EXPERIMENTAL allergic encephalomyelitis (EAE) is an acute neurological autoimmune disease induced in laboratory animals such as guinea-pigs, rabbits, rats and monkeys by administration in complete Freund's adjuvant of a basic encephalitogenic protein (BE) isolated from the brain or spinal cord of several animal species1. We have recently shown2 that the disease may be suppressed by intravenous injection of a synthetic amino-acid copolymer (denoted Cop 1). This copolymer is composed of alanine, glutamic acid, lysine and tyrosine, in a residue molar ratio of 6.0 : 1.9 : 4.7 : 1.0, and has an average molecular weight of 23,000. This basic polymer is not encephalitogenic2 and is not a general non-specific immunosuppressive agent (our unpublished results). Whereas it efficiently suppresses the disease when administered after the basic encephalitogen, it cannot prevent the disease when injected intravenously in saline before the basic brain protein2.