DK173667B1 - Small peptides that inhibit binding to T-4 receptors and act as immunogens, pharmaceutical product comprising at least 1 of the peptides, and test equipment for antibody detection - Google Patents

Description

in DK 173667 B1

The present invention relates to synthetically produced short peptide sequences that inhibit HTLV-III / LAV (hereinafter referred to as HIV) binding to human cells by blocking receptor sites on the cell surface, thereby preventing viral infection of human T cells. The peptides that prevent soreness also induce antibody production against the capsule protein of the HIV virus. Therefore, these peptides also have use as vaccines to prevent the development of Acquired Immune Deficiency Syndrome (AIDS). Monoclonal antibodies to the peptides could also be used as diagnostic agents to identify the HIV virus. The present invention further relates to a pharmaceutical product comprising at least 1 of the peptides of the invention as well as test equipment for detecting antibodies to antigen which bind to the T4 receptor.

15 The complete nucleotide sequence of AIDS (HIV) virus has been reported by several investigators. (See Lee Ratner et al., Nature 313, page 277, January 1985; Muesing et al. Nature 313, page 450, February 1985, and Wain-Habson et al., Cell 40, pages 9-17, January 1985.) The capsule gene has in particular been associated with antigenicity and infectiousness. However, the capsule protein is also known to have highly divergent regions. The HIV virus capsule glycoprotein has been shown to be covalently attached to human, rat and monkey brain membranes and to cells of the immune system.

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The recognition that viruses can exert cell and tissue tropism by attachment to highly specific sites on cell membrane receptors has animated researchers to seek agents that would bind to the viral receptor sites of cell membranes, thereby preventing binding of a specific virus to these cells. One demonstration that specific receptor-mediated vaccinia virus infectivity is blocked by synthetic peptides has been previously done (Epstein et al., Nature 318, pages 663-667).

The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region found on various cells susceptible to HIV infection, including T lymphocytes and macrophages. (See Shaw et al., Science 226, pages 1165-1171 for a discussion of the tropism of HTLV-III}.

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In addition to immunodeficiency symptoms, patients with AIOS exhibit neuropsychological defects. The central nervous system and the immune system generally have a large number of specific cell surface recognition molecules that serve as receptors for neuropeptide mediated intercellular communication. Neuropeptides and their receptors exhibit profound evolutionary stability, being largely preserved in largely unmodified form 1 single-celled organisms and in higher animals. Furthermore, the central nervous system and immune system exhibit dropped DC4 (T4) cell surface recognition molecules that serve as receptors for the binding of HIV capsule glycoprotein (gp 120). As the same 15 highly conserved neuropeptide information agents integrate immune and brain function through receptors similar to those of HIV, the inventors hypothesized that a very similar amino acid sequence between the HIV glycoprotein gp 120 and a short peptide previously identified in another co. -20 menhang from the capsule region of the Epstein Barr virus, could indicate the core peptide that is essential for viruses! receptor binding. It was postulated that such a peptide would be useful in preventing infection of cells by HIV by binding to receptor cells and blocking the binding of HIV gp 120 that such peptide binding to the receptor sites would give rise to production of antibodies directed against the peptide sequence. and that these peptides could be used to provide immunological basis for the prevention of AIDS.

It is an object of the invention to provide peptides that may act to alleviate the symptoms of AIDS by preventing the binding of HIV (AIDS virus) to receptor sites of cells of brain membranes and the immune system.

It is also an object of the invention to provide a pharmaceutical product containing as active ingredient at least one peptide of the invention for use as vaccines for use in providing antibodies that would protect against the development of AIDS in persons who could be exposed to HIV (AIDS virus).

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It is a further object of the invention to provide test equipment to identify the presence of antibodies to HIV or HIV capsule protein.

The invention is further illustrated in the drawings, in which Figure 1A shows a cross-linking of, 25I-gp 120 to home membranes and T cells (a) only 125I-gp 120, (b) monkey, (c) rat, 5 (d) human brain and (e) human T cells.

Figures 1B and 1C show immunoprecipitation of 125 I-labeled retrieval membranes and human T cells, respectively ((f, i) no primary antibody control, (g, j) OKT4 Mab, (h, k) OKT8 Mab.

Figure 2A shows a displacement of specific 125 I-gp 120 binding to healthy rat hippocampal palm membranes. Each determination was performed in triplicate. The results of an experiment performed three times with similar results are shown.

Figure 2B shows specific binding that can be displaced by 0.1 µg / ml OKT4 and 4A was between 27 and 85% of the total binding, which was 2,201 ± 74 cpm in the experiment shown.

Figure 2C shows that viral infectivity is blocked by peptide T and its synthetic analogs.

15 Each determination was performed twice. The results represent a single experiment that was repeated three times with similar results.

FIG. 3 shows the ability of various octapeptide T derivatives to block viral infectiousness.

An octapeptide in the HIV capsule glycoprotein (gp 120) was identified by computer-assisted analysis. This peptide called peptide "T" due to its high threonine content has been shown to inhibit binding of gp120 to the home membranes. The peptide has the sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Later analysis revealed a class of related pentapeptides that have similar binding properties.

In a first embodiment of the invention, a peptide which inhibits binding to T4 receptors and acts as immunogen is characterized in that it has the formula: R'-R2-R3-R4-R5-R6-R7-Tyr -R8-R9 (I) where, R1 is an amino terminal residue Cys or absent.

R2 is Ala or D-Ala or an amino terminal residue Ala or D-Ala or absent, R3 is Ser or absent, R4 is Thr or an amino terminal residue Cys or absent, R5 is Thr, Ser, Asn, Leu, Ile, Arg, GIu or amino terminal Thr, Ser, Asn, Leu, Ile, Arg, GIu or a corresponding amino terminal D-amino acid, R6 is Thr, Ser or Asp, R7 is Thr, Ser, Arg, Asn, Gin, Lys or Trp, R8 is Thr, Arg, Gly, Ser or a corresponding carboxy-terminal amino acid and / or a corresponding carboxy-terminal amide derivative or absent, R 9 is a carboxy-terminal residue Cys or absent.

or a physiologically acceptable salt thereof.

B1 carboxy-terminal amino acid in which R8 is not present. Such peptides retain the binding properties of the group described in the application. Serine and threonine appear to be interchangeable with respect to the biological properties described herein. The active compounds of the invention may exist as physiologically acceptable salts of the peptides.

This class of peptides has been shown to bind to the T4 viral receptors.

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The most preferred peptides, like peptide T above, are the following octapeptides of the formula; D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr and D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-amide and the following pentapeptides of the formula:

Thr-Asp-Asn-Tyr-Thr 20 Thr-Thr-Ser-Tyr-Thr

Thr-Thr-Asn-Tyr-Thr and their analogs down D-Thr as the amino-terminal residue and / or an amide derivative of the carboxy terminal.

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The compounds of the invention can be successfully modified by the methods known to increase the passage of molecules across the blood-brain barrier. Acetylation has been found to be particularly useful in enhancing the binding activity of the peptide. The terminal amino and carboxy sites are particularly preferred sites for modification.

The peptides can also be modified in a forcing conformation to provide improved stability and oral accessibility.

The following abbreviations are used in the following: 35 6 DK 173667 B1

Amino acid 3 letter code 1 letter code arginine arg r

asparagine asn N

5 aspartic acid asp D

cysteine cys C

g 1 yc i n g 1 y G

serine sees S.

threonine thr T

10 tyrosine bull γ

Unless otherwise stated, the amino acids are, of course, the natural form of L stereoisomers.

A comparison of amino acid sequences of 12 pentapeptides is shown in Table 1. Although historically the original computer study revealed that peptide T (containing the 1 ARV isolate) was the relevant molecule, it became clear that as additional viral sequences became available, relevant bioactive sequence could be a shorter pentapeptide comprising nominal peptide T [4-8] or the sequence TTNYT, in the isolates compared (Table 1), only major homologs were distinguished in this shorter range. The majority of the changes are the interconversions of serine (S) and threonine (T), two closely related amino acids. The tyrosine 1 position 7 1 peptide T is an invariant feature of all these constructs, demonstrating that it may be mandatory for bioactivity. Substitutions occurring in position 5 include T, G, R or S. Positions 4 and 6 were first restricted (with one exception) to S, T and N, all of which are amino acids containing uncharged polar groups having very similar steric properties. An assessment of general sequence condensation among 5 different AIDS viral isolates (9, 10) reveals that the region around and including the peptide T sequence is a highly variable region.

35 Such variability may indicate specialization through strong selective spread of the function or functions that may be defined at this location. Like the opiate peptides, these peptide T analogs appear to exist in multiple forms, similar to meth and leu enkephalin. Oisse pentapeptide sequences represented in these various AIDS virus isolates are biologically active and capable of acting as agonists of the CD4 receptor, previously known largely as a pea surface marker of T helminth cells.

Table 1.

Comparison of ENV Sequence from Multiple AIDS Virus Isolates 10 Isolate Sequence Reference Peptide T ASTTTNYT C.B. Pert et al.

PNAS (in print) 15 1ARV (195-199) TTNYT R.L. Willey et al.

LOW TO PNAS 83, page 5038, 1986

Z3 CONTROL

NY5 NTSYT

BIO (HTLV-III) TTSYT B.R. Starcich et al.

WHJ-1 SSTYR Cell 45, page 637, 1986

HAT-3 REVIEW

sequential

25 isolates STNYR

WHJ-1 SSTYR B.L. Hahn et al.

WMJ-2 SSRYR Science 232, page 1548, 1986

WMJ-3 CONTROL

30 TTSYS

1 Number refers to relative positions of amino acids within the ARV env sequence (9).

The 7 amino acid peptide CYS-THR-THR-ASN-TYR-THR-CYS is also active. Adding cysteines to a nucleus does not adversely affect activity.

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The peptides were synthesized on request by Peninsula Laboratories under a confidentiality agreement between the inventors and the manufacturers. The Merrifield method for solid phase peptide synthesis was used. (See U.S. Patent No. 3,531,258, to which reference is made.) The synthesized peptides are particularly preferred. Peptide T and the pentapeptide, which are part thereof, could be isolated by the virus, but the peptides made according to Merrifield are free of viral and cellular residues. Thus, adverse reactions to contaminants do not occur when the synthesized peptides are used.

The peptides of the invention can be prepared by conventional methods of peptide synthesis. Both solid phase and liquid phase methods can be used. It has been found that according to 15 Merrifield solid phase is very convenient. In this process, the peptide is synthesized in a stepwise manner while the carboxy end of the cation is covalently linked to the insoluble bar. During the intermediate synthetic steps, the peptide remains in the solid phase and can therefore be conveniently manipulated. The solid bars are a chloromethylated styrene-divinylbenzene copolymer.

An N-protected form of the carboxy-terminal amino acid, e.g. a t-butoxycarbonyl protected (Boc) amino acid is reacted with the chloromethyl residue of the chloromethylated styrene-divinylbenzene copolymer to produce a protected aminoacyl derivative of the resin, wherein the amino acid is coupled to the resin as benzyl ester. This is released for protection and is reacted with a protected form of the closest desired amino acid to produce a protected dipeptide attached to the resin. The amino acid will generally be used in activated form, for example, using a carbodiimide or active ester. This sequence is repeated and the peptide cad grows one residue at a time by condensing the amine end with the desired N-protected amino acids until the desired peptide has been collected on the resin. The peptide resin is then treated with anhydrous hydrofluoric acid to cleave the ester which binds the total peptide to the resin to release the desired peptide. Functional side chain groups of amino acids, which may be blocked during synthesis using conventional methods, may also be removed simultaneously. Synthesis of a peptide with an amide group on the carboxy terminal can be carried out in the usual manner using a 4-methylbenzhydrylamine resin.

5

The compounds of the invention were found to effectively block receptor sites of cells and to prevent cell infectivity with HIV (AIDS virus) in monkey, rat and human brain membranes and immune system cells.

10

Therefore, one aspect of the invention is a pharmaceutical product comprising at least one peptide compound of the invention in association with a pharmaceutically acceptable carrier or adjuvant suitable for use in human or veterinary medicine.

Such agents may be available for use in the usual manner in admixture with one or more physiologically acceptable excipients or excipients. The agents may optionally further contain one or more other therapeutic agents which, if desired, may be another antiviral agent.

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Thus, the peptides of the invention can be formulated for oral, buccal, parenteral, topical or rectal administration.

In particular, the peptides of the invention may be compounded for injection or infusion and may be in unit dosage form in ampoules or in multi-dose containers with preservative added. The agents may take such forms as suspensions, solutions or emulsions in oily or aqueous carriers, and may contain adjuvants such as suspending agents, stabilizers and / or dispersing agents. Alternatively, the active ingredient may be in powder form to be formulated with a suitable bar, for example sterile pyrogen-free water, prior to use.

Pharmaceutical agents according to the invention may also contain other active ingredients such as antimicrobial or preservatives.

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The agents may contain from 0.001 to 99% of the active material.

The invention further relates to a process for the preparation of a pharmaceutical agent comprising bringing a peptide according to the invention into association with a pharmaceutically acceptable excipient or carrier.

For administration by injection or infusion, the daily dose used to treat an adult human 10 with a body weight of about 70 kg will range from 0.2 mg to 10 mg, preferably 0.5 to 5 mg, which can be administered in For example, 1 to 4 doses, depending on the route of administration and the patient's condition.

15 It was postulated that the affinity constants are similar to those of morphine. On the basis of this affinity, doses of 0.33-0,0003 mg / kg per day were suggested. This has proven to be effective. A blood concentration of 10 "to 10" H molar blood concentration is suggested. In monkeys, 3 mg / kg per day gives a serum 20 concentration of 150 x 10 9 m. This concentration is 15 times greater than necessary to achieve a concentration of 10 ~ 8 M. Primates require 1 dose 10 times the dose. used for humans.

Another aspect of the invention relates to vaccine compositions containing a peptide of the invention to provide protection against infection by AIDS virus. The vaccine will contain an effective immunogenic amount of peptide, e.g. 1 µg to 20 mg / kg each, optionally conjugated to a protein such as human serum albumin, in a suitable bars, e.g. sterile water, saline or saline with buffer. Additives may be used, such as aluminum hydroxide gel. Administration can last by injection, e.g. intramuscular, interperitoneal, subcutaneous or intravenous. Administration can be done once or several times, for example 35 at 1 to 4 week intervals.

Antigenic sequences from crabs and proteins from other vertebrates can also be added to the peptides of the invention to promote antigenicity.

11 DK 173667 B1

Yet another aspect of the invention relates to test equipment for the detection of AIDS viruses and antibodies to AIDS virus containing a peptide of the invention as a source of antigen, or a nonclonal antibody triggered by a peptide of the invention. For example, an S peptide of the invention can be used in a test equipment to detect AIDS infection and to diagnose AIDS and pre-AIDS conditions by using it as a test reagent in an enzyme-linked immunosorbent assay (ELISA) or an enzynin immunodot assay. Such test equipment may include an insoluble porous surface or solid substrate to which the antigenic peptide or monoclonal antibody has been previously absorbed or covalently bonded to which surface or substrate is preferably in the form of nicrotite plates or wells; sample sera, heteroantisers specifically bound to and nights the antigen or antibody absorbed on the surface or carrier, various diluents and buffers, labeled conjugates for the detection of specifically bound antibodies, and other signal-developing reagents such as enzyme substrates, cofactors and chromogens.

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Pept according to the invention can be used as an immunogen to trigger monoclonal antibodies that specifically bind to the relevant part of the capsule sequence of the AIDS virus using conventional techniques. Such monoclonal antibodies form another part of the invention.

Experimental methods and data

Radiolabelling of gp120, preparation of brain membranes, 3D binding and cross-linking of gp120 to receptor and immunoprecipitation of T4 antigen. HTLV-IIIb of HIV was propagated in H9 cells and gp120 was isolated by immunoaffinity chromatography and preparative NaDodSO4 PAGE. Purified gp 120 was labeled with * 25I by the chloramine-T method.

Fresh human, monkey and rat hippocamus were rapidly homogenized (Polytron, Brinkmann Instruments) in 100 volumes of ice-cold 50 mM Hepes (pH 7.4). The membranes collected by single trifugation (15,000 x g) were washed in the original buffer volume and used fresh or stored at -70 ° C. Prior to use, brain membranes and highly purified T cells (ref. 16, gift from Larry Wahl) were preincubated for 15-30 minutes in saline 5 with phosphate buffer (PBS). Membranes from 20 mg (onset) brain («199 µg protein) were incubated with 2B,000 cpm 125 I-gp 120 for 1 hour at 37 ° C in 200 liters (final volume) 50 mM Hepes containing 0.1% bovine serum albumin and the peptidase inhibitors bacitracin (0.005%), aprotinin (0.005%), leupeptin 10 (0.001%) and chymostatin (0.001%). The incubations were rapidly vacuum-filtered and counted to determine the receptor-bound material.

Immunoudfsldning. Immunoprecipitations were prepared by incubation (overnight at 4 ° C) of 0.5% Triton X-100 / P8S solubilized, 1actoperoxidase / glucose oxidase / I-iodinated brain membranes or intact T cells with the mAbs shown in an amount of 10 µg per reaction. A solid phase immunoabsorbent (immunoreader, Bio-Rad) was used to precipitate immune complexes before their digestion with NaDodSO4 / PAGE. Control incubations contained no primar mAb or a subclass control mAb (OCT8).

Chemical neuro-anatomy and computer-aided densitometry. The cryo-state 25 µm sections of freshly frozen human, monkey, and rat brain were thaw mounted and dried on gelatin-coated glass and receptors were made visible as described (18). Incubations down or without antibodies (10 µg / ml) against T4, T4A, T8 and Til were carried out overnight at 0 ° C in RPMI medium, cross-linked on their antigens and made visible with 125I-labeled 30 goat anti-ause antibody. Incubations of pre-assembled glass sections to label the antigen receptor with l25l-gp 120 were performed in 5 ml of pre-prepared glass barriers with (lpM) or without unlabeled gp 120 or mAb OKTA4 (10 pg / ml) (Ortho Diagnostics).

35 Separation of T lymphocyte under study. T cells were obtained by treating Percoll weight-filled peripheral blood T cells with specific monoclonal antibodies (T4 or T8) at a concentration of 10 pg / ml. The treated cells were then spread (21) onto a plactic petri dish which was coated with goat [F (ab *> 2l anti-mouse Innunoglobulin (Sero Lab, Eastbury, HA) for 30 minutes at 4 ° C). non-adherent cells were then removed, washed, and analyzed for flow cytometry responsiveness. The separated T4 and T8 cell populations 5 have <5% contamination of other T cells examined. Cells were then cultured with phytohemagglutinin (1 pg / ml) for 72 h. Exposed to HIV as described below Infected cells were chemically characterized when cytotoxicity assays were performed.

Viral infection. The HTLV-III virus used for infection was isolated from an interleukin 2 (IL-2) dependent cultured T cell line from fresh AIDS patient material and passed into HuT 78, an optional IL-2 independent cell line.

Example 1

A single radiolabelled crosslinking product of approximately 180 Kd is obtained after specific binding of 12SI-gp120 to membranes from either squirrel, rat or human brain membranes indistinguishable from that of human T cells (Fig. 1A). This result shows that gp 120 can be coupled to a protein of about 60 Kd.

20 Unreacted 12Sl-gp 120 runs next to the control without membrane (track a).

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Immunoprecipitation of radioiodinated human brain membranes with OKT4 and OKT8 (10 µg / ml) (Fig. 1B) shows that brain membranes contain a T4 antigen of approximately 60 Kd, indistinguishable from that identified on human T lymphocytes (Figs.

1C). In contrast, OKT8 immunocompresses a low molecular weight (about 30 Kd) protein from T lymphocytes (Fig. 1C), which is absent in brain membranes (Fig. 1B), indicating that brain T4 is not derived from inherent lymphocytes. Similar results are observed with monkey and rat (not shown) hippocampal membranes. These results show that the T4 antigen serves as the viral receptor and is a highly conserved 60 Kd molecule, which is downstream of the immune and central nervous systems.

The recognition that Epstein-Barr and HTLV-1II / LAV fall into a roughly identical octapeptide sequence caused the synthesis and study of "peptide T". Figure 2 shows the high (0.1 nM range) affinity and satiety (Figure 2A) of 125 I-gp 120 binding to freshly made rat brain membranes. Specificity (Fig. 2B) is demonstrated by blocking with OCT4 and OCT4A but not OCT3 (0.1 pg / ml). Peptide T and two of its synthetic analogs (but not the irrelevant octapeptic substance P [1 * -8]) significantly inhibited 125 µgp 120 binding in the 0.1 nM range (Fig. 2C). Substitution of a 0-threoninamide at position 8 resulted in at least a 100-fold loss of receptor binding activity. The classical substitution of [0-Ala] instead of [L-Ala] results in a consistently more potent, apparently more peptide-resistant analogue than peptide T. Amidation of the C-terminal threonine also consistently provides somewhat greater potency (Figure 3 ).

15 DK 173667 B1 May the synthetic peptides be tested for their ability to block viruses! -infection of human T cells, experimenters were blind to binding measurement results. At 10 7, the three peptides active in the binding assay are capable of reducing detectable amounts of reverse transcriptase activity nearly 9 times. Similarly, the less active binding-displacing [D-Thre] peptide T exhibited much reduced blocking of viral infection, requiring concentrations 100 times greater to achieve significant inhibition. Thus, it is not only the order of strengths of the four peptides (D- [Ala] * - peptide T-amide> D- [Ala] i-peptide T> peptide T> D-1Threlg-pep-time T-amide) but also their absolute concentrations for inhibition of receptor binding and viral contagion that are closely linked (Figure 3).

15

Example 2

An approximately 60 Kd protein, similar to and possibly identical to human T cell T4 antigen, was present in 1 apparently 20 molecular fora on membranes made of human brain. Furthermore, the radiolabelled HIV capsule glycoprotein (* 25I-gp 120) can be covalently cross-linked to a molecule found in three mammalian brains whose size and immunoprecipitation properties were indistinguishable from the T4 antigen. Using a method for visualizing antibody-bound receptors on brain slices, the neuroanatomical distribution pattern of brain T4, which is closest to cortical neuropil and analogously organized in all three mammalian brains, was presented. Radiolabeled HIV viral capsule glycoprotein was also bound in an identical pattern on adjacent brain sections, which again suggests that T4 was the HIV receptor.

Example 3 Chemical neuroanatomy, computer-aided densitometry. Cryo-state cut 25 µm sections of freshly frozen human, monkey and rat brain were thaw mounted and dried on gel coated slides and receptors made visible as described by 16 DK 173667 B1

Herkenhaa and Pert, J. Neuro-sci., 2, pp. 1129-1149 (1982). Incubations with or without antibodies (10 µg / ml) against T4, T4A, T8 and Til were performed overnight at 0 ° C in RPMI, cross-linked to their antigens and made visible with 125 I-goat anti-5 mouse antibody. Tissue incubations mounted on specimen vials to label the antigen / receptor with 125 I-gp 120 were performed in 5 ml specimen vials with (10 µM) or without unlabeled gp 120 or Hab OCT4A (10 pg / ml) (Ortho Diagnostics) as described above for membranes.

10

Computer-aided conversion of autoradlographic film transparency to quantitative color images was performed. Co-exposure of standards with known increases in monkey brain radioactivity developed a linear curve (4> 0.99) 15 of log O.D. versus cpn, of which the relative concentration of radioactivity can well be extrapolated. Cell staining of brain sections with thlonin was performed by classical methods and visualization of receptors overlying stained tissue.

Example 4

Attempts have been made to determine the distribution of T4 antigen on a series of frontal or crown squirrel monkey cross sections. These experiments show that there are detectable amounts of T4 monoclonal antibody that bind to cytoarchitecturally important regions of the brain stem (for example, substantia nigra), but the striking pattern of cortical enrichment is evident at any level of the neuroaxis. 0KT8, a T-lymphocyte-directed monoclonal antibody from the same subclass as 0KT4, 3Q, shows no observed pattern. Generally, the more superficial layers within the cerebral cortex contain the closest concentrations of the T4 antigen. The frontal and perilimbic cortex overlying the amydala are particularly receptive throughout the deep layers. The hippocampal formation has the densest concentration of receptors in the monkey, rat and human brain. Dark error microscopy of squirrel dipped in photographic emulsion revealed that the band of closest receptor labeling is located within the molecular layers of the tate gyrus and the actual hippocampus (containing very few neurons). Thus, receptors appear to be properly distributed over the neuropil (the neuronal cravings of dendrites and axons) or may be localized to a specific subset of unstained astroglial cells.

Evidence has been found for the specificity of the chemical neuroanatomy and results showing that T4 and the viral capsule recognition molecule are indistinguishable. Coronal 10 sections of rat brain revealed a very similar cortex / hippo-campus-rich pattern of receptor distribution, whether used for visualization 0KT4 or l2!> I_gp 120. Furthermore, this pattern did not occur when incubation occurred in the absence of unmarked gp 120 (1 μΜ), OKT4A (10 µg / ml) or OKT4 15 (10 µg / ml). Other mouse Mab directed against other human T cell surface antigens including OKT8 and OKT11 showed no detectable pattern on rat brain when visualized with 125 I goat anti-mouse IgG secondary antibody, as well as no reproducible detectable antigen / receptor with sec-20 dart antibody alone.

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Claims (9)

18 DK 173667 B1 1. Peptide which inhibits binding to T4 receptors and acts as an immunogen, characterized in that it has the formula 2. Peptide selected from ala-ser-thr-thr-thr-asn-tyr-thr, thr-thr-asn-tyr-thr, 5-ser-ser-thr-tyr-thr, thr-thr-asn-tyr-thr thr, ser-ser-thr-tyr-arg, asn-thr-ser-tyr-thr, thr-thr-ser-tyr-thr, asn-thr-ser-tyr-gly, ser-thr-asn-tyr -arg, ser-ser-arg-tyr-arg, thr-thr-ser-tyr-ser, and cys-thr-thr-asn-tyr-thr-cys. Pharmaceutical product containing as active ingredient at least one peptide according to claim 1 in a pharmaceutical carrier. Pharmaceutical product containing as active ingredient at least one peptide according to claim 2 in a pharmaceutical carrier. A pharmaceutical product comprising a peptide according to claim 1 conjugated to a protein. R1-R2-R3-R4-R5-R6-R7-Tyr-R8-R9 (I) where, R is an amino terminal residue Cys or absent. R2 is Ala or D-Ala or an amino terminal residue Ala or D-Ala or absent, R3 is Ser or absent, R 4 is Thr or an amino terminal residue Cys or absent, R 5 is Thr, Ser, Asn, Leu, Ile, Arg, Glu or amino terminal Thr, Ser, Asn, Leu, Ile, Arg, Glu or a corresponding amino terminal D amino acid, R 6 is Thr, Ser or Asp, R 7 is Thr, Ser, Arg, Asn, Gln, Lys or Trp, R8 is Thr, Arg, Gly, Ser or a corresponding carboxy-terminal amino acid and / or a corresponding carboxy-terminal amide derivative or absent, R9 is a carboxy-terminal residue Cys or absent. 19 DK 173667 B1 or a physiologically acceptable salt thereof. Pharmaceutical product according to claim 5, characterized in that the protein is human serum albumin. Test equipment for detecting antibodies to antigen that bind to the T4 receptor, characterized in that it contains an antigenic peptide according to claim 1 bound to a porous surface or a solid substrate. DK DK 173667 B1 Equipment according to claim 7, characterized in that the antigenic peptide is bound to indentations in a microtiter plate. Pharmaceutical product containing as active ingredient at least two or more peptides according to claim 1. 5