AU604719B2 - Small peptides which inhibit binding to t-4 receptors and act as immunogens - Google Patents

Description

i i A d) bv I t, -q Associate Director Declarant's Name David...

T. Mow., Ph.D.

E B. RICE CO PATENT ATTORNEYS This form is suitable for any type of Patent Application. No legalisation required.

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PCT

AU-AI-75408/87 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau is, INTERNATIONAL APPLICATION P I HD DE THEA T COOPERATION TREATY (PCT) (51) International Patent Classification 4 1) International Publication Number: WO 87/07613 C07K 5/10, 7/06, A61K 39/385 A1 A61K 39/42, 39/12, G01N 33/53 (43) International Publication Date: A61K 39/42,39/12, 17 December 1987 (17.12.87) (21) International Application Number: PCT/US87/01270 (74) Agents: STERN, Marvin, R. et al.; Holman Stern, 2401 Fifteenth Street, Washington, DC 20009 (22) International Filing Date: 27 May 1987 (27.05.87) (US).

(31) Priority Application Numbers: 869,919 (81) Designated States: AU, BJ (OAPI patent), CF (OAPI 878,586 patent), CG (OAPI patent), CM (OAPI patent), DK, 048,148 FI, GA (OAPI patent), HU, JP, KR, ML (OAPI patent), MR (OAPI patent), NO, SN (OAPI patent), (32) Priority Dates: 3 June 1986 (03.06.86) SU, TD (OAPI patent), TG (OAPI patent).

26 June 1986 (26.06.86) 11 May 1987 (11.05.87) Published (33) Priority Country: US With international search report.

(71) Applicant: THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, U.S. DEPART- MENT OF COMMERCE [US/US]; 5285 Port Royal A jQ. I 1 FEB B Road, Springfield, VA 22161 (US).

(72) Inventors: PERT, Candace, B. RUFF, Michael, R. AUSTRALIAN 8104 Custer Road, Bethesda, MD 20814 FAR- RAR, William, L. 10 Norwich Court, Gaithersburg, 11 JAN 1988 MD 20878 (US).

PATENT OFFICE (54) Title: SMALL PEPTIDES WHICH INHIBIT BINDING TO T-4 RECEPTORS AND ACT AS IMMUNOGENS (57) Abstract Short peptide of formula Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb where Ra represents an amino terminal residue Alaor D-Ala and Rb representes a carboxy terminal residue -Thr or -Thr amide or a derivative thereof with an additional Cysresidue at one or both of the amino and carboxy terminals, or a peptide of formula RI-R 2 -R3-R4-R5 where R I is an amino terminal residue Thr-, Ser-, Asn-, Leu-, Ile-, Arg- or Glu-, R 2 is Thr, Ser or Asp, R 3 is Thr, Ser, Asn, Arg, Gin, Lys or Trp, R 4 is Tyr and R 5 is a crboxy terminal amino group or a derivative thereof with a corresponding D- amino acid as the amino terminal residue and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys- residue at one or both of the amino and carboxy terminals, or a physiologically acceptable salt thereof. Such peptides bind to T4 receptors are useful in preventing viral infectivity by viruses which bind to the T4 receptors. These peptides are believed to act as competitive blocking agents.

This document contains the amendments made under Section 49 and is correct for printing.

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i' 9 i 'WO 87/07613 PCT/US87/01270 -1- 1 SMALL PEPTIDES WHICH INHIBIT BINDING TO 2 T-4 RECEPTORS AND ACT AS IMMUNOGENS 3 BRIEF DESCRIPTION OF THE INVENTION 4 This invention relates to synthetically produced short pe-ptide sequences which inhibit HTLV-III/LAV (here- 6 inafter referred to as HIV) binding to human cells by 7 blocking receptor sites on the cell surface, and thus 8 preventing viral infectivity of human T cell. The pep- 9 tides, while preventing infectivity, also induce antibody production against the envelope protein of the HIV virus.

11 Hence, these peptides also have use as vaccines to pre- 12 ven-t development of Acquired Immune Disease Syndrome 13 (AIDS). Monoclonal antibodies to the peptides could 14 also be used as diagnostic agents to identify the HIV virus. Hence, peptides and antibodies to the peptides 16 would have use in preparing test kits for identification 17 of HIV carriers or persons suffering from AIDS.

18 BACKGROUND OF THE INVENTION 19 The complete nucleotide sequence of the AIDS (HIV) virus has been reported by several investigators. (See 21 Lee Ratner, et al., Nature 313, p. 277, January 1985; 22 Muesing, et al., Nature 313, p. 450, February 1985; 23 and Wain-Habson, et al., Cell 40, pp. 9-17, January 24 1985.) The envelope gene has bon associated particularly with antigenicity and infectivity. However, the 26 envelope portion is also known to have regions which WO 87/07613 PCT/US87/0127G -2- 1 are highly divergent. The HIV virus envelope glycopro- 2 tein has been shown to affix covalently to the brain 3 membranes of humans, rats, and monkeys and to cells 4 of the immune system.

The realization that viruses may exert cell and 6 tissue tropism by attachment at highly specific sites 7 on cell membrane receptors has encouraged investigators 8 to seek agents which would bind at the viral receptor 9 sites of cell membranes and thus prevent binding of a specific virus to these cells. A demonstration of 11 specific receptor-mediated vaccinia virus infectivity 12 being blocked by synthetic peptides has been previously 13 demonstrated (Epstein, et al., Nature 318, pp. 663-667).

14 The HIV virus has been shown to bind to a surface molecule known as the CD4 or T4 region, which is present 16 on various cells susceptable to HIV infection, including 17 T lymphocytes and macrophages. (See Shaw, et al., Science 18 226, pp. 1165-1171 for a discussion of tropism of HTLV- 19 III.) In addition to symptoms arising from immunodefi- 21 ciency, patients with AIDS show neuropsychological de- 22 fects. The central nervous and immune systems share 23 a large number of specific cell-surface recognition 24 molecules, serv .ig as receptors for neuropeptide-mediated intercellular communication. The neuropeptides and 26 their receptors show profound evolutionary stability, 27 being highly conserved in largely unaltered form in 28 unicellular organisms as well as higher animals. Further- 29 more, the central nervous and immune systems show common DC4 (T4) cell-surface recognition molecules which serve 31 as receptors for the binding of HIV envelope glycoprotein 32 (gp 120). Since the same highly conserved neuropeptide 33 informational substances integrate immune and brain 34 function through receptors remarkably similar to those of HIV, we postulated a very similar amino acid sequence 36 between the HIV glycoprotein gp 120 and a short peptide 'WO 17/07613 PCT/US87/01270 3 1 previously identified in another context from the enve- 2 lope region of the Epstein Barr-Virus might indicate 3 the core peptide essential for viral receptor binding.

4 It was postulated that such a peptide would be useful in preventing infection of cells with the HIV by binding 6 with receptor cells and blocking the binding of HIV 7 gp 120, that suh peptides binding to the receptor sites 8 would give rise to production of antibodies directed 9 to the peptide sequence, and that these peptides might be used to provide immunological basis for prevention 11 of AIDS.

12 PURPOSE 13 It was the object of the present invention to 14 provide peptides that would act to alleviate symptoms of AIDS by preventing binding of HIV (AIDS virus) to 16 receptor sites of cells of brain membranes and the immune 17 system.

18 It was also an object of the present invention 19 to provide peptides for use as vaccines to be used to give rise to antibodies that would protect against deve- 21 lopment of AIDS in persons who might become exposed 22 to the HIV (AIDS virus).

23 It was a further object of the present invention 24 to provide diagnostic means of identifying presence of antibodies to HIV or HIV envelope protein.

26 DETAILED DESCRIPTION OF THE INVENTION 27 An octapeptide in the HIV envelope glycoprotein 28 (gp 120) was identified by computer-assisted analysis.

29 This peptide, termed "peptide T" because of the high threonine content, has been shown to inhibit binding 31 of gp 120 to the brain membranes. The peptide has the 32 sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Later analysis 33 disclosed a class of related pentapeptides having similar 34 binding properties.

1 I I 4 In a first aspect the present invention consists a peptide selected from the group consisting of the peptides having the formula I: Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb, peptides having the formula II:

R

1

-R

2

-R

3 -Tyr-R peptides having the formula I or II with an additional Cys residue at the amino terminus, peptides having the formula I or II with an additional Cys residue at the carboxy terminus, peptides having the formula I or II with an additional Cys residue at the amino terminus and at the carboxy terminus, peptides having the formula II, and physiologically 15 acceptable salts of any of the preceding peptides, wherein

S

Ra is an consisting of Rb is an consisting of

R

1 is an consisting of D-Ser, D-Asn,

R

2 is an consisting of 25 R 3 is an consisting of

R

5 is an consisting of amino acid residue selected from the group Ala and D-ala; amino acid residue selected from the group Thr, Thr-amide and Thr-Cys-amide; amino acid residue selected from the group Thr, Ser, Asn, Leu, Ile, Arg, Glu, D-Thr, D-Leu, D-Ile, D-Arg and D-Glu; amino acid residue selected from the group Thr, Ser and Asp; amino acid residue selected from the group Thr, Ser, Asn, Arg, Gln, Lys and Trp; and amino acid residue selected from the group Thr, Arg, Gly, Thr-amide, Arg-amide,

S

S 9 p 9 As P 'S 6 Gly-amide, D-Thr, D-Arg and D-Gly.

In a second aspect the present invention consists a peptide selected from the group consisting of the peptides having the formula II:

RI-R

2

-R

3 -Tyr-R peptides having the formula II with an additional Cys residue at the amino terminus, -4a peptides having the formula II with an additional Cys residue at the carboxy terminus, peptides having the formula II with an additional Cys residue at the amino terminus and at the carboxy terminus, and physiologically acceptable salts of any of the preceding peptides, wherein

R

1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, D-Thr, D-Ser and D-Asn; R2 is an amino acid residue selected from the group consisting of Thr, Ser and Asp;

R

3 is an amino acid residue selected from the group consisting of Thr, Ser, Asn and Arg; and

R

5 is an amino acid residue selected from the group 1 consisting of Thr, Arg, Gly, Thr-amide, Arg-amide and 15 Gly-amide.

In a third aspect the present invention consists a peptide selected from the group consisting of the peptides having the formula III:

SR

1

-R

2

.R

3

-R

4 2G wherein

R

1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Glu, Arg, Ile and Leu;

*R

2 is an amino acid residue selected from the group conp-ting of Thr, Ser, Asp; 25 .3 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Arg Gln, Lys, and Trp; and 4 R 4 is Tyr or a derivative of Tyr.

In a fourth aspect the present invention consists a peptide selected from the group consisting of 30 Ala-Ser-Thr-Thr-Thr-Asn-Thr, Thr-Thr-Asn-Tyr-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.

While the preferred amino acids at R 5 have been 'RA4i TT ~J i i 4b designated, it is known the amino acid at this position may vary widely. In fact, it is possible to terminate the peptide with R 4 (Tyrosine) as the carboxy terminal amino acid wherein R 5 is absent. Such peptides retain the binding properties of the group taught herein. Serine and threonine appear to be interchangeable for purposes of biological properties taught herein. The active compounds of the invention may exist as physiologically acceptable salts of the peptides.

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

Most preferred peptides, as well as peptide T above, are the following octapeptides of formula 9 8 *8S4 4**O 9 98 9 988* 88* 9*I 9.

999 9: 0e 9. 9 98 9 9 99*@ 9 9 S Ij/

(N*

i a -r IWO 87/07613 PCT/US87/01270 1 D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr 2 and D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-amide 3 and the following pentapeptides of formula 4 Thr-Asp-Asn-Tyr-Thr Thr-Thr-Ser-Tyr-Thr 6 Thr-Thr-Asn-Tyr-Thr 7 and their analogues with D-Thr as the amino terminal 8 residue and/or an amide derivate at the carboxy terminal.

9 The compounds of the invention may be beneficially modified by the methods known to enhance passage of 11 molecules across the blood-brain barrier. Acetylation 12 has proven to be especially useful for enhancing binding 13 activity of t*'e peptide. The terminal amino and carboxy 14 sites are pazticularly preferred sites for modification.

The peptides of this may also be modified in a 16 constraining conformation to provide improved stability 17 and oral availability.

18 The following abbreviations are used hereinafter: 19 Amino Acid Three Letter Code One Letter Code arginine arg R 21 asparagine asn N 22 aspartic acid asp D 23 cysteine cys C 24 glycine gly G serine ser S 26 threonine thr T 27 tyrosine tyr Y 28 Unless otherwise indicated the amino acids are, of course, 29 in the natural form of L-stereoisomers.

V 30 A comparison of amino acid sequences of 12 penta- 31 pepties is presented in Table 1. Although historically 32 our initial computer search revealed peptide T (contained 33 in the ARV isolate) to be the relevant moiety, as addi- 34 tional viral sequences became available, it became clear that the relevant, bioactive sequence, might be a shorter 36 pentapeptide comprising, nominally, peptide T I_ WO 87/07613 PCT/US87/01270 6 1 or the sequence TTNYT. In the isolates, we compared 2 (Table substantial homologies were discerned only 3 in this, shorter, region. The majority of changes are 4 the interconversions of serine and threonine two closely related amino acids. The tyrosine of posi- 6 tion 7 of peptide T is an invariant feature of all these 7 constructs indicating that it may be obligatory for 8 bioactivity. Substitutions occurring at position 9 include T, G, R or S. Position 4 and 6 were first restricted (with one exception) to S, T and N, all amino 11 acids containing uncharged polar groups with closely 12 similar steric properties. An assessment of general 13 sequence concordance among 5 various AIDS viral isolates 14 10) reveals that the region around and including the pt=ptide T sequence is a highly variable area. Such 16 variability may indicate specialization through strong 17 selective diversification of the function(s) which may 18 be defined at this locus. Like the opiate peptides, 19 these peptide T analogs seem to exist in multiple forms, reminiscent of met and leu enkephalin. These pentapep- 21 tide sequences represented in these various AIDS virus 22 isolates are biologically active and capable of interac- 23 ting as agonists of the CD4 receptor previously known 24 largely as a surface "marker" of T helper cells.

I I I; i WO 87/07613 PCT/US87/01270 7 1 Table 1. Comparison of ENV Sequence from Multiple 2 AIDS Virus Isolates 3 Isolate Sequence Reference 4 peptide T ASTTTNYT Pert, et al.

PNAS (in press) 6 1 ARV (195-199) TTNYT Willey, et al.

7 LAV TTSYT PNAS 83, p. 5038, 1986 8 Z3 SSTYR 9 NY5 NTSYT BIO(HTLV-III) TTSYT Starcich, et al.

11 WMJ-1 SSTYR Cell 45, p. 637, 1986 12 HAT-3 NTSYG 13 Sequential 14 isolates STNYR WMJ-1 SSTYR Hahn, et al.

16 WMJ-2 SSRYR Science 232, p. 1548, 17 WMJ-3 SSTYR 1986 18

TTSYS

19 lNumbers refer to relative positions of amino acids within the ARV env sequence 21 The seven amino acid peptide CYS-THR-THR-ASN-TYR- 22 THR-CYS is also active. Addition of cysteines to a 23 core does not adversely affect activity.

24 The pepties were custom synethesized by Peninsula Laboratories under a confidentiality agreement between 26 the inventors and the manufacturer. The Merrifield 27 method of solid phase peptide synethesis was used.

28 (See U.S. Patent No.3,531,258 which is incorporated 29 herein by reference.) The synethesized peptides are V 30 especially preferred. While peptide T and the pentapep- 31 tide which is a portion thereof could be ioslated from 32 the virus, the peptides prepared in accord with Merri- 33 field are free of viral and cellular debris. Hence, 34 untoward reactions to contaminants does not occur when the synthesized peptides are used.

WO 87/07613 PCT/US87/01270 8 1 The peptides of the invention may be produced 2 by conventional methods of peptide synthesis. Both 3 solid phase and liquid phase methods may be used. We 4 have found the solid phase of Merrifield to be particularly convenient. In this process, the peptide is synthe- 6 sized in a stepwise manner while the carboxy end of 7 the chain is covalently attached to the insoluble support.

8 During the intermediate synthetic stages, the peptide 9 remains in the solid phase and therefore can be conveniently manipulated. The solid support is a chloro- 11 methylated styrene-divinylbenzene copolymer.

12 An N-protected form of the carboxy terminal amino 13 acid, a t-bu.toxycarbonyl protected (Boc-) amino 14 acid, is reacted with the chloromethyl residue of the chloromethylated styrene divinlybenzene copolymer resin 16 to produce a protected amino acyl derivative of the 17 resin, where the amino acid is coupled to the resin 18 a benzyl ester. This is deprotected and reacted with 19 a protected form of the next required amino acid thus producing a protected dipeptide attached to the resin.

21 The amino acid will generally be used in activated form, 22 by use of a carbodiimide or active ester. This 23 sequence is repeated and the peptide chain grows one 24 residue at a time by condensation at the amino end with the required N-protected amino acids until the required 26 peptide has been assembled on the resin. The peptide- 27 resin is then treated with anydrous hydrofluoric acid 28 to cleave the ester linking the assembled peptide to 29 the resin, in order to liberate the required peptide.

Side chain functional groups of amino acids which must 31 be blocked during the synthetic procedure, using conven- 32 tional methods, may also be simultaneously removed.

33 Synthesis of a peptide with an amide group on its carboxy 34 terminal can be carried out in conventional manner, using a 4-methylbenzhydrylamine resin.

36 The compounds of the invention were found to effec- 37 tively block receptor sites of cells and to prevent

-I

WO 87/07613 PCT/US87/01270 9 1 cell infectivity with HIV (AIDS virus) in monkey, rat 2 and human brain membranes and cells of the immune system.

3 As an aspect of the invention, therefore, we pro- 4 vide a pharmaceutical composition comprising a peptide compound of the invention in association with a pharma- 6 ceutically acceptable carrier or excipient, adapted 7 for use in human or veterinary medicine. Such composi- 8 tions may be presented for use in conventional manner 9 in admixture with one or more physiologically accpetable carriers of excipients. The compositions may optionally 11 further contain one or more other therapeutic agents 12 which may, if desired, be a different antiviral agent.

13 Thus, the peptides according to the invention 14 may be formulated for oral, buccal, parenteral, topical or rectal administration.

16 In particular, the peptides according to the inven- 17 tion may be formulated for injection or infusion and 18 may be presented in unit dose form in ampoules or in 19 multidose containers with an added preservative. The compositions may take such forms as suspensions, solu- 21 tions, or emulsions in oily or aqueous vehicles, and 22 may contain formulatory agents such as suspending, stab- 23 ilizing and/or dispersing agents. Alternatively, the 24 active ingredient may be in powder form for constitution with a suitable vehicle, sterile, pyrogen-free 26 water, before use.

27 The pharmaceutical compositions according to the 28 invention may also contain other active ingredients 29 such as antimicrobial agents or preservatives.

The compositions may contain from 0.001-99% of 31 the active material.

32 The invention further provides a process for pre- 33 paring a pharmaceutical composition which comprises 34 bringing a peptide of the invention into association with a pharmaceutically acceptable excipient or carrier.

m n lA acceptable salts of any of the preceding peptides, wherein Ra is an amino acid residue selected from the group consisting of Ala and D-ala; /2 I WO 87/07613 PCT/US87/01270.

10 1 For administration by injection or infusion, the 2 daily dosage as employed for treatment of an adult human 3 of approximately 70 kg body weight will range from 0'.2 4 mg to 10 mg, preferably 0.5 to 5 mg, which may be administered in 1 to 4 doses, for example, depending on the 6 route of administration and the condition of the patient.

7 It was postulated that the affinity constants 8 are similar to those of morphine. On the basis of this 9 affinity, dosage of .33-.0003 mg/kg per day was suggested.

This has proven to be effective. A blood concentration 11 10"6 to 10 1 1 molar blood concentration is suggested.

12 In monkeys, 3 mg/kg per day achieves a serum concentra- 13 tion of 150 x 10- 9 M. This concentration is 15 times 14 greater than necessary to achieve a concentration of 10- 8 M. Primates generally require 10 times the dose 16 used in humans.

17 A further aspect of this invention relates to 18 vaccine preparations containing a peptide according 19 to the invention, to provide protection against infection by AIDS virus. The vaccine will contain an effective 21 immunogenic amount of peptide, 1 pg to 20 mg/kg 22 of host, optionally conjugated to a protein such as 23 human serum albumin, in a suitable vehicle, sterile 24 water, saline or buffered saline. Adjuvants may be be employed, such as aluminum hydroxide gel. Administra- 26 tion may be by injection, intramuscularly inter- 27 peritoneally, subcutaneously, or intravenously. Admini- 28 stration may take place once or at a plurality of times, 29 at 1-4 week intervals.

Antigenic sequences from crab as well as proteins 31 from other invertebrates can also be added to the pep- 32 tides of the invention to promote antigenicity.

33 A yet further aspect of this invention relates 34 to test kits for the detection of the AIDS virus and antibodies to the AIDS virus containing a peptide accord- 36 ing to the invention as source of antigen, or a monoi 'WO 87/07613 PCT/US87/01270 11 1 clonal antibody elicited by a peptide according to the 2 invention. For example, a peptire according to the 3 invention may be used in a test kit to detect AIDS infec- 4 tion and to diagnose AIDS and pre-AIDS conditions by using it as the test reagent in an enzyme-linked immuno- 6 sorbent assay (ELISA) or an enzyme immunodot assay.

7 Such test kits may include an insoluble porcus surface 8 or solid substrate to which the antigenic peptide or 9 monoclonal antibody has been preabsorbed or covalently bound, such surface or substrate preferably in the form 11 of microtiter plates or wells; test sera; heteroantisera 12 which specifically bind to and saturate the antigen 13 or antibody absorbed to the surface or support; various 14 diluents and buffers; labelled conjugates for the detection of specifically bound antibodies and other signal- 16 generating reagents such as enzyme substrates, cofactors 17 and chromogens.

18 The peptide according to the invention may be 19 used as an immunogen to elicit monoclonal antibodies which specifically bind to the relevant portion of the 21 envelope sequence of the AIDS virus, using conventional 22 techniques; such monoclonal antibodies form a further 23 feature of the invention.

24 EXPERIMENTAL METHODS AND DATA Radiolabelling of gp 120, Preparation of Brain 26 Membranes, Binding and Crosslinking of gp 120 to Receptor, 27 and Immunoprecipitation of T4 Antigen. HTLV-IIIb of 28 HIV was propaged in H9 cells, and the gp 1)0 was isolated 29 by immunoaffinity chromatography and preparative NaDodSO 4 /PAGE. Purified gp 120 was labelled with 31 1251 by the chloramine-T method.

32 Fresh human, monkey and rat hippocamus were quickly 33 homogenized (Polytron, Brinkmann Instruments) in 100 34 vol of ice-cold 50 mM Hepes (pH The membranes collected by centrifugation (15,000 x g) were washed a 3 purified T cells (ref. 16; gift of Larry Wahl) were 4 preincubated for 15-30 min in phosphate-buffered saline (PBS). Membranes derived from 20 mg (initial wet weight) 6 of brain (lOO pg of protein) were incubated with 28,000 7 cpm of 1 2 5I-gp 120 for 1 hr at 37 0 C in .200 1 (final 8 volumne) of 50 mM Hepes containing 0.1% bovine serum i J^ WO 87/07613 PCT/U S87/01279 12 1 i9 albumin and the peptidasoriginal buffer ve inhibitors bacitracin (0.005%),used fresh 10 aprotinin leupeptin and chymostatin 11 Incubations were rapidly vacuum-filtered 12 stored counted to determine the receptor-bound material 3 urified T cells rfcipitation. 16; gift of Larryecipitates were 14 preicubated by incubation (overnight 15-30 min in phospat 4Ce-buffered saline (PBS). Membranes derived f rom 20 mg (initial wet weight)dase 16 I-iodof brainated brain membranes or intact T cells with28,000 17 indicated mAbs at 10 jig per reaction. A solid-phase 18 immunoabsorbant (immunobeads, Bio-Rad) was used to pre- 7 c ipitate immune complexes prior to their resolution 208 v olumne) of 50 mM Hpesontrol incubations containe serum 9 albumin and the peptida subclase inhibitors bacitracin (0.005%)trol b (OT8).

122 Chemical Neuoranatomy and Computer-Assisted Densi- 23 tomet0.001r. Incubryostat-cut 25- m sections were rapidly vacuumfresh-filtered 24 humand counted to determine the brain wereeptor-bounted and dried onto gelatin-coipitated slides, and receptors were visualized 26 as dprepared by incubation (overnight at 4 or without Triton X-100/PBS solubilized, lactoperoxidase/glucose oxidase/ 2716 I-iodinated brain membranes or intact T cells with 2817 inducted overnigmAbs a at10 Cg per reaction. A solid-phase 18 immunoabsorbant igens, and visualized with used to pre-lled 19 cipitate immune complexes prior to their resolution 0 goatby NaDodSnti-muse a ntibody. Incubations of slide-mounted 21 primary mAb or a subclass control mAb (OKT8).

22 Chemical Neuoranatomy and Computer-Assisted Densi- 23 tomssue try se ctions to label the antigen-receptor with 24 human, monkey and rat brain were thaw-mounted and dried onto gelatincoawere conduted in -ml slides, and receptors were visualizeth 26 as described Incubations, with or without anti- 27 bodies (10 g/ml) against T4, T4A, T8, and TIl, were i 28 conducted overnight at 0° C in RPMI medium, cross-linked 29 onto their antigens, and visualized with I-labelled goat anti-mouse antibody. Incubations of slide-mounted 31 tissue sections to label the antigen-receptor with 32 125I-gp 120 were conducted in 5-ml slide carriers with f 33 lM) or without unlabelled gp 120 or mAb OKT4A 34 pg/Ml) (Ortho Diagnostics).

Separation of T-Lymphocyte Subsets. Subsets of 36 T cells were obtained by treatment of Percoll densityi n _I 1. UII.mI-iCuIi <3a I.nzecrlv.LLy. noweve.-i L. 26 envelope portion is also known to have regions which WO 87/07613 PCT/US87/01270 1327 1 purified peripheral blood T cells with specific mono- 2 clonal antibodies (T4 or T8) at 10 jg/ml. The treated 3 cells were then panned (21) on a plastic Petri dish 4 that was coated with goat [F(ab') 2 J anti-mouse immunoglobulin (Sero Lab, Eastbury, MA) for 30 min at 40 C.

6 The nonadherent cells were then removed, washed and 7 analyzed for reactivity by flow cytometry. The separated 8 T4 and T8 cell populations have 5% contamination of 9 other T cell subsets. Cells were then cultured with phytohemagglutinin (1 t g/ml) for 72 hr and exposed to 11 HIV as described below. Infected cells were phenotypi- 12 cally characterized when cytotoxicity assays were per- 13 formed.

14 Virus Infection. The HTLV-III virus used for infection was isolated from an interleukin 2 (IL-2)- 16 dependent cultured T-cell line established from fresh 17 AIDS patient material and passaged in:o HuT 78, a per- 18 missive IL-2-independent cell line.

19 DESCRIPTION OF THE DRAWINGS 125 Figure 1A shows a cross-linking of 2I-gp 120 21 to brain, membranes and T cells 25 I-gp 120 only; 22 monkey; rat; human brain; and human 23 T cells.

24 Figures 1B and 1C show immunoprecipitation of 125I-labelled monkey brain membranes and human T cells, 26 respectively; no primary antibody control; (g,j) 27 OKT4 Mab; OKT8 Mab.

28 Figure 2A shows a displacement of specific 29 125 I-gp 120 binding to fresh rat hippocampal membranes.

Each determination was performed in triplicate; the 31 results of one experiment, which was performed three 32 times-with similar results, is shown. Specific binding 33 displaceable by 10 ug/ml of OKT4 and 4A ranged between 34 27 and 85% of total binding, which was 2,201 1 74 cpm in the experiment shown.

II 36 between the HIV glycoprotein gp 120 and a short peptide 87/07613 PCT/US87/01270 WO 87/07613 14 1 Figure 2B shows that viral infectivity is blocked 2 by peptide T and its synthetic analogs. Each determin- 3 ation was performed in duplicate. Results represent 4 a single experiment which was repeated three times with similar results.

6 Example 1. A single radiolabelled cross-linking 7 product of about 180 Kd is obtained after specific bind- 8 ing of 125 I-gp 120 to membranes from either squirrel 9 monkey, rat or human brain membranes which are indistinguishable from that of human T cells (Fig. 1A). This 11 result indicates that gp 120 can be coupled to an approxi- 125 12 mately 60 Kd protein; unreacted I-gp 120 runs adjacent 13 to the no membrane control (lane a).

14 Immunoprecipitation of radioiodinated human brain membranes with OKT4 and OKT8 (10 ig/ml) (Fig. 1B) shows 16 that brain membranes contain a T4 antigen of about 17 Kd, indistinguishable from that identified on human T 18 lymphocytes (Fig. 1C); by contrast, OKT8 immunoprecipi- 19 tates a low (about 30 Kd) molecular weight protein from T lymphocytes (Fig. 1C) which is absent in brain mem- 21 branes (Fig. 1B) indicating that brain T4 is not derived 22 from resident lymphocytes. Similar results are observed 23 with monkey and rat (not shown) hippocampal membranes.

24 These results show that the T4 antigen serves as the viral receptor and is a highly consesrved 60 Kd molecule 26 shared by the immune and central nervous systems.

27 The realization that Epstein-Barr and HTLV-III/LAV 28 share an almost identical octapeptide sequence caused 29 the synthesis and study of "peptide Figure 2 demonstrates the high (0.1 nM range) affinity and saturability 31 (Fig. 2A) of 125 1-gp 120 binding to freshly prepared 32 rat brain membranes. Specificity (Fig. 2B) is demon- 33 strated by blockade with OKT4 and OKT4A, but not OKT3 34 (0.1 yg/ml). Peptide T and two of its synthetic analogs (but not the irrelevant octapeptide substance P 1-8]) i WO 87/07613 PCT/US87/01270 15 1 significantly inhibited 12 5 I-gp 120 binding in the 0.1 2 nM range (Fig. 2C). Substitution of a D-threonine-amide 3 in position 8 resulted in at least a 100-fold loss of 4 receptor binding activity. The classical (D-Ala] substitution for [L-Ala] results in a consistenly 6 more potent, presumably more peptidase-resistant, analog 7 than peptide T; amidation of the C terminal threonine 8 also consistently produces somewhat greater potency 9 (Figure 3).

When the synthetic peptides were tested for their 11 ability to block viral infection of human T cells, experi- 12 mentors were blind to binding assay results. At 10 7 13 the three peptides active in the binding assay are able 14 to reduce detectable levels of reverse transcriptase activity by almost 9-fold. The less active binding 16 displacer [D-Threl -peptide T similarly showed greatly 17 reduced blockade of viral infection, requiring concen- 18 trations 100-fold higher to achieve significant inhibi- 19 tion. Thus, not only the rank order of potencies of the four peptides [Ala] 1 -peptide T-amide D- Ala]- 21 peptide T peptide T D-[Thre] 8 -peptide T-amide), but 22 also their absolute concentrations in inhibiting receptor 23 binding and viral infectivity are closely correlated 24 (Figure 3).

Example 2. An approximate 60 Kd protein, which 26 is similar if not identical to human T cells T4 antigen, 27 was present in apparently conserved molecular form on 28 membranes prepared from human brain; furthermore, the 29 radiolabelled HIV envelope glycoprotein 125 I-gp 120) can be covalently cross-linked to a molecule present 31 in three mammalian brains whose size and immunoprecipi- 32 tation properties were indistinguishable from the T4 33 antigen. Using a method for visualizing antibody-bound 34 receptors on brain slices, the neuoanatomical distribution pattern of brain T4, which is densest over cortical 36 neuropil and analogously organized in all three mammalian 37 brains, was presented. Also, radiolabelled HIV viral

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or :ru~rr~lu~-xl~~-~Ylua IWO 8707613 PCT/US8701270 -16- 1 envelope glycoprotein bound in an identical pattern 2 on adjacent brain sections, once again suggesting that 3 T4 was the HIV receptor.

4 Example 3. Chemical Neuroanatomy, Computer-Assisted Densitometry. Cryostat-cut 25 micron sections 6 of fresh-frozen human, monkey, and rat brain were thaw- 7 mounted and dried onto gel-coated slides and receptors 8 visualized as described by Herkenham and Pert, J. Neuro- 9 sci., 2, pp. 1129-1149 (1982). Incubations, with or without antibodies (10 pg/ml) against T4, T4A, T8 and li T1l, were conducted overnight at 00 C in RPMI, cross- 12 linked onto their antigens and visualized with 1I-goat 13 anti-mouse antibody. Incubations of slide-mounted tissue 14 sections in order to label the antigen/receptor with 125 I-gp 120 were conducted in 5 ml slide carriers with 16 (10- 6 M) or without unlabelled gp 120 or Mab OKT4A 17 1 g/ml) (Ortho Diagnostics) as described above for mem- 18 b-anes.

19 Computer-assisted transformation of autoradiographic film opacity into quantative color images was perfor- 21 med. Co-exposure of standards known increments of radio- 22 activity with the monkey brain sections generated a 23 linear plot (4 .99) of log O.D. versus cpm from which 24 the relative concentration of radioactivity can be meaningfully extrapolated. Cell staining of brain sections 26 with thionine was performed by classical methods and 27 visualization of receptors overlying stained tissue.

28 Example 4. Experiments have been conducted to 29 determine the distribution of T4 antigen on a rostral to caudal series or coronal sections of squirrel monkey 31 brain. These experiments show that there are detectable 32 levels of T4 monoclonal antibody binding to cytoarchitec- 33 tionally meaningful areas of the brain stem the 34 substantia nigra), but the striking pattern of cortical enrichment is apparent at every level of the neuoraxis.

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WO 87/07613 PCT/US87/01270 17 1 OKT8, a T-lymphocyte directed monoclonal antibody from 2 the same subclass as OKT4, exhibits no observable pat- 3 tern. Generally, the more superficial layers within 4 the cerebral cortex contain the densest concentrations of the T4 antigen; the frontal and perilimbic cortex 6 overlying the amydala are particularly receptor-rich 7 throughout the deep layers. The hippocampal formation 8 has the desest concentration of receptors in the monkey, 9 rat and human brain. Dark field microscopy of squirrel monkey sections dipped in photographic emulsion revealed 11 that the band of densest receptor labelling is located 12 within the molecular layers of the dentate gyrus and 13 hippocampus proper (which contain very few neurons).

14 Thus, receptors appear to be rightly distributed over the neuropil (the neuronal extensions of dendrites and 16 axons) or may be localized to a specific subset of un- 17 stained astroglial cells.

18 Evidence of the specificiLy of the chemical neuro- 19 anatomy and results showing that T4 and the viral envelope recognition molecule are indistinguishable has 21 been determined. Coronal sections of rat brain revealed 22 a very similar cortex/hippocampus-rich pattern of recep- 23 tor distribution whether OKT4 or 125 I-gp 120 was used 24 for visualization. Furthermore, this pattern was not apparent when incubation occurred in the presence of 26 unlabelled gp 120 (1 aM), OKT4A (10 ig/ml) or OKT4 27 sg/ml). Other mouse Mabs directed against other human 28 T cell surface antigens including OKT8 and OKT11 gave 29 no detectable pattern on rat brain when visualized by 125 I-goat anti-mouse IgG secondary antibody just as 31 there was no reproducible, detectable antigen/receptor 32 with secondary antibody alone.

P, I

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Claims (19)

1. A peptide selected from the group consisting of the peptides having the formula I: Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb, peptides having the formula II: R 1 -R 2 -R 3 -Tyr-R peptides having the formula I or II with an additional Cys residue at the amino terminus, peptides having the formula I or II with an additional Cys residue at the carboxy terminus, peptides having the formula I or II with an additional Cys residue at the amino terminus and at the carboxy terminus, peptides having the formula II, and physiologically acceptable salts of any of the preceding peptides, wherein 0ee* *@0e S 0* 55 S 555' 0* Ra is an consisting of Rb is an consisting of R 1 is an consisting of D-Ser, D-Asn, R 2 is an consisting of R 3 is an consisting of R 5 is an consisting of amino acid residue selected from the group Ala and D-ala; amino acid residue selected from the group Thr, Thr-amide and Thr-Cys-amide; amino acid residue selected from the group Thr, Ser, Asn, Leu, Ile, Arg, Glu, D-Thr, D-Leu, D-Ile, D-Arg and D-Glu; amino acid residue selected from the group Thr, Ser and Asp; amino acid residue selected from the group Thr, Ser, Asn, Arg, Gln, Lys and Trp; and amino acid residue selected from the group Thr, Arg, Gly, Thr-amide, Arg-amide, Gly-amide, D-Thr, D-Arg and D-Gly.

2. A peptide selected from the group consisting of the peptides having the formula II: R 1 -R 2 -R 3 -Tyr-R peptides having the formula II with an additional Cys residue at the amino terminus, peptides having the formula II with an additional Cys d I 1 a 1 i 1 L C -sq i 'i i- I 1 I-- 19 residue at the carboxy terminus, peptides having the formula II with an additional Cys residue at the amino terminus and at the carboxy terminus, and physiologically acceptable salts of any of the preceding peptides, wherein R 1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, D-Thr, D-Ser and D-Asn; R 2 is an amino acid residue selected from the group consisting of Thr, Ser and Asp; R 3 is an amino acid residue selected from the group consisting of Thr, Ser, Asn and Arg; and R 5 is an amino acid residue selected from the group consisting of Thr, Arg, Gly, Thr-amide, Arg-amide and Gly-amide.

3. A peptide selected from the group consisting of the peptides having the formula III: R1-R2-R3-R4 wherein :o R 1 is an amino acid residue selected from the group consisting of Thr, Ser, Asn, Glu, Arg, Ile and Leu; R 2 is an amino acid residue selected from the group consisting of Thr, Ser, Asp; R 3 is an amino acid residue selected from the group consisting of Thr, Set, Asn, Arg Gin, Lys, and Trp; and S* R 4 is Tyr or a derivative of Tyr.

4. A peptide of claim 1 selected from the group consisting of peptides having the formula: Cys-R1-R 2 -R 3 -R 4 -R 5 -Cys.

5. A peptide selected from the group consisting of Ala-Ser-Thr-Thr-Thr-Asn-Thr, Thr-Thr-Asn-Tyr-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.

6. A composition of matter containing as an active ingredient at least one peptide of claim 1 in a pharmaceutical carrier.

7. A composition of matter containing as an active ingredient at least one peptide of claim 2 in a pharmaceutical carrier.

8. A composition of matter containing as an active ingredient at least one peptide of claim 3 in a pharmaceutical carrier.

9. A composition of matter containing as an active ingredient at least one peptide of claim 4 in a pharmaceutical carrier. A composition of matter containing as an active ingredient at least one peptide of claim 5 in a pharmaceutical carrier.

11. A method of preventing binding of antigen to cells of eoee mammals which comprises administering an effective T4 see* receptor blocking amount of a composition of claim 6.

12. A method of claim 11 wherein the antigen is a virus.

13. A method of claim 12 wherein the virus is the causative agent of AIDS.

14. A method of preventing binding of virus to cells of mammals which comprises administering an effective T4 S* 5. receptor blocking amount of a composition of claim 7. A method of preventing binding of virus to cells of mammals which comprises administering an effective T4 receptor blocking amount of a composition of claim 8.

16. A method of preventing binding of virus to cells of mammals which comprises administering an effective T4 receptor blocking amount of a composition of claim 9.

17. A method of preventing binding of virus to cells of 0@5@@5 Smammals which comprises administering an effective T4 receptor blocking amount of a composition of claim

18. A composition of matter comprising a peptide of claim 1 conjugated to a protein, wherein said protein enhances the stability of said peptide, whereby said peptide i 37 tively block receptor sites of cells and to prevent I I I ICI r ii I I" 21 retains its ability to bind to T4 receptors.

19. A composition of claim 18 wherein the protein is human serum albumin. A test kit for detecting antibodies to antigen which bind to the T4 receptor containing a peptide of claim 1 bound to a porous surface or solid substrate.

21. A a kit of claim 22 wherein the antigenic peptide is bound to wells of a microtiter plate.

22. A composition of matter of claim 6, wherein said compositions contain more than one peptide of claim 1. DATED this 14 day of September 1990 THE UNITED STATES OF AMERICA o* *AS REPRESENTED BY THE •SECRETARY, U.S. DEPARTMENT OF COMMERCE Patent Attorneys for the Applicant: F.B. RICE CO. O 9. r