AU631357B2 - New anti-receptor peptides and therapeutic agents - Google Patents

Description

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INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 89/09782 CO7K 1/00, 7/06, 7/08 Al C07K 1/10, 7A6K 37/02 (43) International Publication Date: 19 October 1989 (19.10.89) (21) International Application Number: PCT/US88/03612 (74) Agent: OLIFF, James, Parkhurst Oliff, 277 S. Washington Street, Alexandria, VA 22314 (US).

(22) International Filing Date: 1\October 1988 (1 .10.88) i (81) Designated State: AU.

Priority data: 182,109 15 April 1988 (15.04.88) US 201,498 2 June 1988 (02.06.88) US Published 107I 9 i U- 147 7) US With international search report.

(71) Applicant: UNITED STATES OF AMERICA, represented by THE SECRETARY, UNITED STATES DE- PARTMENT OF COMMERCE [US/US]; Washington, DC 20230 (US).

(72) Inventors: EIDEN, Lee 4302 Kentbury Drive, Bethesda, MD 20814 NARA, Peter, L. 901 Pine Avenue, Frederick, MD 21701 FRASER, Blair 15224 Turkeyfoot Road, Darnestown, MD 20878 (US).

A%357 (54) Title: NEW ANTI-RECEPTOR PEPTIDES AND THERAPEUTIC AGENTS (57) Abstract A new concept of inhibiting formation of liquid-receptor complex by antireceptor m.iecule or derivative thereof is described. A new method of treating HIV infection is also disclosed.

*(Referred to in PCT Gazette No. 27/1989 Section II) r- t NEW ANTI-RECEPTOR PEPTIDES AND THERAPEUTIC AGENTS Technical Field: The present invention is related generally to synthesis of peptide based antireceptors. Antireceptors are fragments of receptor proteins, or derivative of such fragments, which include the ligand-binding region of the receptor protein, and which therefore act to block the interaction of ligands and their receptors by binding to the ligand and preventing its attachment to the native receptor molecule. More specifically, the present invention is related to fabrication by automated solid-phase peptide synthesis, and acid cleavage of the peptide from the solid-phase resin under controlled conditions, to produce a peptide mixture comprising authentic desired peptide, and deleted and/or derivatized (partially deprotected) congeners of these peptides which may, due to steric constraints or increased nonspecific binding, have a higher affinity for the receptor ligand than the unmodified peptide sequence itself. Thus, the process of fabricating antireceptors comprises synthesis of a series of peptides spanning the entire theoretical binding area of a given protein receptor molecule, and testing postresin peptide mixtures to identify and select, by further purification, peptide derivatives which can function as antireceptors.

Background of the Invention: A number of ligands including viruses, interact with cells via receptors. The formation of the ligandreceptor dyad is believed to be the first step in the initiation of biological response, such as viral infection, signal transduction, cell proliferation, cell S fusion ad the like. The present invention takes ad- Svantage of the proposition that synthetically designed iuumuirM^Mii- UI--CII I C -CII: WO 89/09782 PCT/US88/03612 I 2 molecules or agents which possess high affinity for binding to ligands, specifically at their receptor-binding epitopes, would block the interaction of the ligand to the receptor, thereby inhibiting the initiation of bioi 5 logical responses caused by the ligand.

It should be noted that this approach is quite distinct from the antigen-antibody mechanism where an antigenic ligand induces the production of antibodies in an immune-responsive system and then these antibodies bind to the epitopic site of the antigen to block the biologic response of the antigen. In contrast, the present invention is not dependent on the nature of the antigen at all. Rather, in accordance with the present invention, it is the receptor molecule which is analyzed and based on such analysis anti-receptor molecules are synthesized which block the receptor-ligand interaction.

It has previously been reported that synthetic peptides, which are derivatives of ligands, function more potently and/or efficaciously than their parent peptides as either agonists Rivier, J. Polrter, C.L. Rivier, M. Perrin, A. Corrigan, W.A. Hook, R.P. Saraganian and W.W. Vale, J. Med. Chem. 29, 1846 (1986) or antagonists Richardson, A. Scheid, and P.W. Choppin, Virology 105, 205 (1980) of ligand-receptor interactions. It was, therefore, appropriate to determine if derivatives of receptors might function more potently and/or efficaciously than their parent peptides in inhibiting ligandreceptor interactions.

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to synthesize modified or unmodified receptor fragments, or mixture thereof, which specifically block the interaction of the receptor with its ligand or ligands, that is to act as antireceptors.

It is a further object of the present invention that in cases where there are multiple ligands for a single receptor, which ligands may interact with dif- SUBSTITUTE SHEET i WO 89/09782 PCT/US88/03612 3 ferent specific regions of the receptor, that such antireceptors would inhibit selectively the ,interaction of one, or one class, of ligands for the receptor, without affecting the interaction between the receptor and other (or other classes) of ligands.

It is an additional object of the present invention to synthesize antiviral agents including modified or unmodified region(s) of a receptor which specifically block the receptor-ligand interaction without affecting other functions of said receptor, for example immunosuppression without antiviral effect and antiviral effect without immunosuppression.

It is a still further object of the present invention to provide a method for rational drug designing which spares the normal receptor function, but inhibits the binding of a ligand to the receptor. The drug design includes sparing class II cytotoxicity or deleting class II B-activation.

It is another object of the present invention to identify the anti-receptor structure and modify the same to increase its binding affinity by such methodology as addition, deletion, derivatization and the like.

It is yet another object of the present invention to provide unique anti-viral agents inhibiting viral infectivity, particularly of human immunodeficiency virus, HIV-1, HIV-II, SIV and the like.

It is yet another object of the present invention to provide isolated, substantially pure, antireceptor polypeptides or derivatives thereof, useful as therapeutic agents for those anomalous conditions which result from ligand-receptor interaction.

thr objects and advantages will beL.o e-evident from the lowing detailed description of the invention. in BRIEF DESCRIPTION OF T DRAWINGS These and other objects, featuire&and many of ;=he attendant dvan of the nentin will o-i b"tt-b SUBSTITUT E Sl -MT r Accordingly the present invention provides an antireceptor compound comprising fragments -of a 'receptor protein which comprise the ligand-binding region of said receptor protein or derivatives of said fragments and which inhibits the binding of a ligand to a receptor.

The present invention further provides an antireceptor compound comprising a sequence of at least seven amino acids of human CD4, which sequence includes the cysteine at position 86, wherein the sulfur atom of said cysteine is joined to a carbon atom or sulfur atom of a sulfur blocking group, said compound being an inhibitor of CD4-dependent virus-induced cell fusion.

The present invention further provides a compound comprising a sequence of at least 10 amino acids of human CD4, which sequence includes the sequence of amino acids 83 to including the cysteine at position 86, wherein the sulfur atom of said cysteine is joined to an aliphatic carbon atom or sulfur atom of a sulfur blocking group, said compound being capable of interfering with CD4-dependent virus-induced cell fusion.

The present invention further provides a composition of matter comprising a polypeptide comprising a sequence of at least seven amino acids of human CD4, which sequence includes the cysteine at position 86, wherein the sulfur atom of said cysteine is joined to a carbon atom of a sulfur blocking group, said composition prepared by the method comprising: S(a) combining in an aqueous solvent, said polypeptide with at least about a stoichiometric amount of a sulfur blocking group containing compound, which compound comprises an active halogen or active olefin functionality, at a temperature in the range of about 0° to 50 C for a time S sufficient for said sulfur blocking group containing compound to c'ovalently bond to said cysteine; neutralizing any acid formed; and 35 isolating said composition of matter.

The present invention further provides an antiviral pharmaceutical composition, comprising an effective amount of the antireceptor compound of claim 1 to inhibit viral -i 3a w^ 1 iinfection and pharmaceutically acceptable, non-toxic sterile carrier.

The present invention further provides a method of inhibiting viral infection, comprising administering to a host susceptible of viral infection an effective amount of the antireceptor compound of claim 1 to inhibit viral infection.

The present invention further provides a method of derivatizing a biologically active antiviral agent comprising the antireceptor compound of claim 1 from a purified inactive antireceptor compound comprising the step of benzylalkylating a purified inactive antireceptor compound obtained by solid phase process; and acid-cleaving the benzylalkylated product to obtain said bioactive antiviral agent.

Other objects and advantages will become evident from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better WO 89/09782 PCF/US88/03612 -4understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Fig. 1.A-C show the appearance of CEM-SS cells following inoculation with HTLV-IIIB, in the presence or absence of continuous treatment with peptide CD4(83- 94)BZL, and are photomicrographs (25X) of individual microtiter wells from a typical CEM-SS assay demonstrating HTLV-IIIB induced syncytium in the presence and absence of CD4(83-94)BZL. Fig. 1A illustrates microtiter well containing virus-induced syncytia after one hour inoculation with HTLV-IIIB in the absence of peptide followed by removal of virus and cell culture for five days. VO 150. Fig. 1.B illustrates virus surviving of fraction V n 10, at a final peptide concentration of 16 M. Fig. 1.C illus-trates virus surviving fraction, Vn O, at a final peptide concentration of 125 M. The peptide preparation CD4(83-94)BZL was incubated with the virus inoculum for 60 minutes after which the peptidevirus reaction mixture was incubated with the adherent CEM-SS cells for an additional hour. Virus-peptidecontaining medium was removed and replaced with fresh complete medium containing CD4(83-94)BZL at the same concentration. The number of syncytia listed above are counts taken from the entire microtiter well.

Fig. 2 represents chromatographic fractionation of synthetic CD4(76-94), including bioactivity, and UVabsorbing species characterized by FAB-Mass spectrometry.

A typical chromatogram of 1.8 mg of CD4(76-94) ,on a Vydac C8 (10 x 250 mm) bonded-phase semi-preparative column is shown. Material was post-resin CD4(76-94) dissolved in 10 mM ammonium acetate at pH 7.0. Mobile phase was ammonium acetate buffer and ammonium acetate buffer/80% acetonitrile. The percentage of B in the mobile phase was varied as shown (dashed line). Material eluting at retention times 2-3, 3-4.5, SUBSTITUTE

SHEET

WO 89/09782 PCT/US88/03612 5 4.5-5 and 5-8.5 minutes was pooled from several semipreparative runs, lyophilized, weighted and submitted to bioassay at nominal concentrations of 500 to 30 in the fusion assay. Bioactivity (hatched bar) is expressed as doses of anti-syncytial activity per fraction. One dose is the smallest amount of material necessary to completely inhibit fusion between 50,000 HTLVIIIB/H9 cells and 50,000 VB indicator cells over a twenty-four hour period under standard assay conditions. Aliquots of the major peak (3 to 4.5 minutes retention time) and the area of the chromatogram in which bioactive material eluted (5-8.5 minutes retention time) were submitted to fast atom bombardment-mass spectrometric analysis as described. The major peak (3-4.5 min retention time) gave a parent fragment mass (M+H 2287) consistent with the mass of the desired peptide LKIEDSDTYICEVEDQKEE as well as a fragment of mass 2269, the mass of the parent fragment minus H 2 0 (18 atomic mass units). The biologically active material eluting at 5-8.5 min retention time exhibited a complex mass spectrum containing the parent M+H (2287) and multiple higher-molecular weight peaks consistent with extensive derivatization of the parent peptide (dta not shown).

Fig. 3a is an FAB mass spectrograph of major peak No. 4 of the chromatogram of Figure 3.C, discussed below.

Fig. 3b is an FAB mass spectrograph of major peak No. 7 of the chromatogram of Figure 3.C, discussed below.

Fig. 3c represents chromatographic fractionation of S-benzylCD4(83-94).

Post-resin material from the synthesis of Sbenzyl-CD4(83-94) was employed. The desired peptide was TYICbzlEVEDQKEE where Cbzl indicates benzylation of cysteine 86 by insertion of 5-Boc-S-benzyl cysteine in place of 5-Boc-S-p-methylbenzyl cysteine in the solidphase automated synthesis sequence yielding a peptide SUBSTiTUTE SHEET WO 89/09782 PCT/US88/03612 6 derivatitad at cysteine with a benzyl moiety following HF cleavage of the peptide from the solid-phase resin. mg of the post-resin peptide mixture was applied to the semipreparative column under the conditions described in Fig. 2. Aliquots were taken for both FAB mass spectrometric analysis and bioassay (anti-syncytial activity).

Data shown are the absorbance profile at 225 nm (broken line), the anti-fusion activity in each fraction (hatched boxes; dose defined as in Fig. and the mass spectra of the major peak and the bioactive peak resolved by reverse-phase chromatography. The concentration of the seventh peak material required to completely inhibit syncytia formation in the standard fusion assay was 32 Am. This concentration was calculated from the weighed mass of the material collected from this region of the chromatograph, and the molecular weight of the desired peptide.

Fig. 4 shows the effects of CD4(1-25) on infection of CEM-SS cells in vitro by various HIV isolates.

Numbers in parenthesis are the number of syncytia per well in untreated wells.

DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by isolated, substantially pure anti-viral agents comprising anti-receptor polypeptides or derivatives thereof which inhibit viral infection and subsequent cell fusion and syncytia formation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

SUBSTTUTE S- ET WO 89/09782 PCT/US88/03612 7 The term "ligand" and "receptor" as used herein indicate two members of a binding dyad wherein the "ligand" is the component whose binding to the receptor is inhibitable by addition of the antireceptor peptide because the antireceptor peptide binds to the ligand, replacing receptor in the ligand-receptor dyad, and the "receptor" is that region of a molecule (before and after derivatization) which defines an epitope responsible for binding of the ligand and based on which the antireceptors are tailored. Thus a single macro molecule may have several epitopic sites, hence have several receptor subtype binding domains within the same molecular configuration for binding of several different ligands.

The term "anti-viral agent" as used herein means an agent which is at least in part a polypeptide or a derivative thereof (including conjugate, analog and the like) which inhibits either viral infection or viral induced cell fusion.

The term "substantially pure" as used herein means the product is as pure and homogeneous as can be obtained by employing standard techniques well known to one of ordinary skill in the art.

CD4 (Leu3A/T4) molecule is present on the surface of a subset of human T-lymphocytes which help cytotoxic- and B-lymphocytes during class II-restricted immune response to foreign antigen. The CD4 molecule is also the receptor by which the human immunodeficiency virus (HIV) binds to T-lymphocytes and infects these cells. Since the cloning and sequence of CD4 have been accomplished, this receptor was selected to illustrate the principles and the application of the present invention. Accordingly, several polypeptide fragments containing 7-25 amino acid sequences of the CD4 receptor extracellular domain were synthesized and tested for their ability to inhibit three CD4-mediated functions: Fusion of HIV-infected and non-infected CD4-positive T-lymphoma cells; Infection of CD4-positive lymphoma SUBSTITUTE

SHEET

i'C -r WO 89/09782 PCT/US88/03612 -8cells with HIV; and Proliferaton of T-helper-inducer cells in the presence of allogeneic irradiated stimulator cells (the mixed lymphocyte reaction). In addition, the ability of the peptide fragments to inhibit binding of a CD4 antibody, which neutralizes all three of these processes, to the CD4 molecule on the surface of peripheral blood leucocytes, has also been determined in order to map the binding epitope of this antibody.

For convenience, the synthetic peptides and peptide derivatives corresponding to regions of the deduced amino acid sequence of human CD4 have been assigned residue numbers based on the amino acid residue numbers of CD4 [Maddon et al, Cell 42, 93 (1985)]. Peptides and peptide derivatives in the region of CD4 spanning amino acid residues 76 to 94 are referred to herein as follows: CD4(76-94) re2ers to the desired 19 residue peptide LKIEDSDTYICEVEDQKEE. CD4(83-94) refers to the desired 12 residue peptide TYICEVEDQKEE. S-benzyl CD4(76-94) and S-benzylCD4(83-94) refer to the desired 19 residue peptide LKIEDSDTYICbzlEVEDQKEE, and its 12-residue congener, in which cysteine protection via benzyl, rather than methylbenzyl, derivatization during solidphase synthesis yields a final peptide product in which the cysteine residue remains protected (S-benzylated) after HF cleavage.

Material possessing ability to inhibit HIVinduced cell fusion generated from 1) authentic CD4(83- 94), authentic S-benzylCD4(83-94) or their 19-residue congeners, by liquid-phase benzyl or methylbenzyl alkylation, or 2) purified by HPLC of post-resin material from synthesis of S-benzylCD4(84-94) are designated CD4(83- 94)BZL or CD4(76-94)BZL. Unfractionated mixtures of the peptide material resulting from the solid-phase synthesis of the desired peptides are referred to as the "postresin peptide mixture"; for example CD4(76-94) post-resin peptide mixture, or CD4(76-94) peptide mixture.

Seven different polypeptide fragments, each SUBSTITUTE SHEET -r c ,.11lIII I_ t 1~1~1. WO 89/09782 PCT/US88/03612 9 containing 25-amino acid residues of the CD4 molecule, were first examined. None of these fragments, as the pure authentic peptide, inhibits HIV infection, cell fusion or the mixed lymphocyte reaction. However, one of the fragments, corresponding to amino acid residues 51-75 of the CD4, inhibited neutralizing antibody binding to CD4 on intact cells. A derivative of another fragment, corresponding to amino acid residues 76-94, inhibited HTLVIIIB infection of CEM cells, and fusion of HTLV-IIIBinfected and non-infected CD4-positive lymphoma cells, with an IC50 of 60 AM, with no effect on the proliferation of T-helper cells in the mixed lymphocyte reaction. Several other polypeptide fragments and various derivatives thereof were also prepared and tested. The methodology and the results obtained are now described.

MATERIALS AND METHODS Unless mentioned otherwise, all chemicals and reagents were of analytical grade and obtained from commercial sources.

The compounds of the present invention are characterized by having a sequence comparable to a sequence of the CD4 molecule, in particular a sequence distal to the N-terminus. The sequence includes the cysteine at position 86 of CD4 at which the sulfur on the cysteine is blocked.

In general, the compound is prepared by reacting underivatized peptide under mild conditions with reagents known to react with mercaptans. These reagents may be active halides, pseudohalides, active olefins, x,p-enones, such as maleimide, disulfides, or the like. For in vivo use, the derivatizing groups should provide a physiologically acceptable product.

The blocking groups will have from about 1 to about 36 carbon atoms and may be aliphatic, alicyclic, aromatic, heterocyclic or combinations thereof. Usually, the blocking group will have from 0 to 10 hetero-atoms, which may be in the longest chain, as a substituent cn a SUBSTITUTE SHEET i l~ l, i r +r:C c- -rr; -rh 7:1: ~-rll T-j i- r: C1 i' WO 89/09782 PCT/US88/03612 chain or ring atom or the like. For the most part the heteroatoms will be selected from halogen, nitrogen, oxygen or sulfur. Binding of the substituent to the sulfur of the cysteine residue may be via a carbon, or heteroatoms such as nitrogen, or sulfur atom. The bulk of the group immediately distal to the cysteine sulfur and attached directly to the sulfur is preferably less than that of a naphthyl group and greater than that of a linear lower alkanoic acid, most preferably approximately the size of a phenyl group or similar cyclic or heterocyclic group (either aromatic or non-aromatic). The group optionally may be further substituted.

Various groups may be used to block the sulfur, for example, an aryl-containing substituent or a thioether resulting from the reaction between the thio group of the cysteire and a maleimide. The aryl group is preferably selected from 5- and 6-membered aromatic rings containing carbon and 0-1 oxygen or sulfur and 0-3 nitrogen atoms in the ring. Phenyl is a preferred aryl group, benzyl and naphythyl. The aryl-containing group may be substituted or unsubstituted. Substituents may include alkyl, particularly methyl, halogen, particularly chloro, nitro, etc., where the substituents may be in any position, preferably at the ortho position. The aryl group may have from 0 to 3 substituents, usually not more than 2 substituents, which substituents may be the same or different.

For active olefins used as blocking groups, the olefin will usually be conjugated with a second site of unsaturation, a carbonyl group. Acyclic groups, maleimido groups, conjugated polyolefins, or the like may find use.

For disulfide formation, precursor disulfides will be employed which have a convenient leaving group, which is displaced by the cysteine to form a new disulfide bond. When using reagents known to react with mercaptans, intramolecular disulfides formed from another SUSTI'SiUTE SidiEZ7 r WO 89/09782 PCT/US88/03612 11 cysteine of a contiguous CD4 peptide chain are excluded.

It is also desirable to have a functionality present on the blocking group which allows for linking to another molecule, carboxy, carboxy ester, or the like. The carboxy may then be activated with a carbodiimide, carboxy diimidazole, or the like for reaction with an amine or alcohol, for example, a protein.

Examples of reaction compounds for preparing derivatives of the CD4 molecule and fragments thereof include the following, wherein the group bound to the sulfur of cysteine 86 may be one of the following groups: alkyl and substituted alkyl compounds: X-

CH

2

(CH

2 )n where n 0 20 and X is selected from

H;OH;OCH

3

;SH;SCH

3

;NH

2

;NHCH

3

;N(CH

3 2

;SO

3

H;SO

2

CH

3 or halogen, being hetero only when n is other than 0.

cycloalkyl and substituted cycloalkyl compounds:

CH

2

-CH-

(CH

2 )n where n 1-10 and a hydrogen on any of the ring carbons is replaced by X as described in above.

aromatic or substituted aromatic compounds: S(CH2)n-

X,

where n 1-5 and X is as described in above.

polyaromatic or substituted aromatic compounds: (C a where n 1-3 and X is as described in above.

heterocyclic or substituted heterocyclic compounds such as substituted pyridyl, (ii) imidazole or (iii) quinoline: SUBSTITUTE

SHET

WO 8909782PCT/US88/03612 -12-

IN

Itwhere n =0-3 and R is a pair of electrons; H; alkyl of 1-2 carbon atoms; or 0.

(CH2 where n =0-3 and R -is selected from COS 5

CH

3 or H.

(j)OH where R is a pair of electrons; H; or 0.

maleimide adducts such as m-maleimidobenzoyIN-hydroxysuccinimide ester; m-maleimido-benzoylsulfosuccinimide ester; N-succinimidyl4-(p-maleimidophenyl )-butyrate; N-succinimidyl4- (N-maleimido-methyl) cydlohexane-1-carboxylate; or sulfosucci" imidyl-4- (Nmaleimidomethy'.) cyclohexane-1-carboxylate; bismaleimidohexane; bismaleimidomethyl ether; or N-Y-maleimidobutyryloxysuccinimide.

thio-containing compounds such as 14where X is N 3 OH; OR; NH 2 NHR; NO 2 SH; SR; halogen;

CO

2 H; or aryl of from 6 to 12 carbon atoms; and hi~ R-Swhere R is alkyl or substituted alkyl.

amino acids or oligopeptides.

cytotoxic agents such as alkylating agents, f or example pipobroman; thio-TEPA; chlorambucil; cyclophosphamide; nitrogen mustard; mephalan; or uraci.

3 5 mustard.

membrane-perturbating agents, for example SUBSTITUTE SHEET WO 89/09782 PCT/US88/03612 13 adriamycin; ionophores, such as valinomycin; or surface active agents, such as detergents.

anti-retroviral agents such as thymidine (AZT); dideoxycytidine (DDC); dideoxyadenosine (DDA); or dideoxyinosine (DDI).

In some instances, particular derivatives may be cytotoxic, in which case these derivatives may be modified to reduce cytotoxicity or substantially eliminate toxicity at pharmacologically active dosage levels.

A preferred peptide in accordance with the present invention comprises substantially the same sequence as CD4 amino acids of the sequence 85 to 92, usually 83 to 94, and more particularly 81 to 94, conveniently 76 to 94 where the sequence may be further extended by as many as 10 amino acids or more at either terminus, where the extension amino acids may be the same or different from the CD4 sequence. The sequence will usually have at least greater than 2 amino acids of the natural sequence on each side of the cysteine. The numbering of the amino acids is as set forth in Maddon et al., Cell (1985) 41: 93-104. The peptide sequences may be modified by terminal amino acylation, for example, acetylation; carboxy amidation, for example, with ammonia, methylamine and the like.

It will be appreciated that the amino acid sequence need not correspond exactly to the sequences given above, but may be modified by from 1 to 3 conservative or non-conservative mutations, including deletions and insertions involving not more than about 1 amino acid, without significantly affecting the activity of the product. Therefore, the polypeptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes provide for advantages in their use.

Conservative substitutions include combinations such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr, trp. Usually, the sequence will SUBSTITUTE

SHEET

WO 89/09782 PC'/US88/03612 14 not differ by more than 20% from the sequence of the epitope of the CD4 molecule except where additional amino acids may be added at either terminus for the purpose of providing an "arm" by which the peptides of this invention may be conveniently linked for immobilization. The arms will usually be at least about 5 amino acids and may be 50 or more amino acids.

The peptides of the present invention may also be conjugated with or linked to a soluble macromolecular entity. Conveniently, the macromolecular entity may be a polypeptide, either naturally occurring or synthetic, to which antibodies are unlikely to be encountered at high levels in human serum. Illustrative polypeptides include poly-L-lysine, bovine serum albumin, keyhole limpet hemocyanin, bovine gamma globulin and the like. The choice is primarily one of convenience and availability.

The conjugates will generally comprise at least one molecule of the peptide of the present invention per macromolecule and usually not more than about 1 per kDal and preferably not more than about 1 per 2 kDal of the macromolecule. Of course, one or more different peptides may be linked to the same macromolecule.

Conjugation or linking may be accomplished by any conventional method employig such reagents as pmaleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyde and the like. The linkage may occur at the N-terminus, C-terminus, or at a site intermediate to the ends of the molecule. Furthermore, peptide may be derivatized for linking or linked while bound to a support, or the like.

The peptides can be prepared in a wide variety of ways. The peptides, because of their relatively short size, may be synthesized in solution or on a solid support in accordance with standard techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See for example, Stewart and Young, Solid Phase Peptide Synthe- SSUeSI STIr SHE WO 89/09782 PCT/US88/03612 sis, 2nd Ed., Pierce Chemical Company, 1984; and Tam et al., J. Am. Chem. Soc. (1983) 105:6442.

Alternatively, hybrid DNA technology may be employed where a synthetic gene may be prepared by employing single strands which code for the polypeptide or substantially complementary strands thereof, where the single strands overlap and can be put together in an annealing medium so as to hybridize. The hybridized strands may then be ligated to form the complete gene and by choice of appropriate termini, the gene may be inserted into any suitable and readily available expression vector. See for example, Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982. Alternatively, the region of the genome coding for the peptide may be cloned by conventional recombinant DNA techniques and expressed (see Maniatis, et al, supra).

DNA coding sequences based upon the known sequence for CD4 may also be used to obtain the peptide. Fragments from these sequences may be employed for expression of peptide fragments, conservative base changes may also be made, where the modified sequence(s) code for the same amino acid(s), or non-conservative changes in the coding sequence may be made, where the resulting amino acid may be a conservative or nonconservative entity.

The coding sequence may be extended at either the or terminus or both termini to extend the peptide, while retaining its epitopic site. The extension may provide for an arm for linking, for providing antigenic activity, or the like.

For expression, the coding sequence is provided with start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a cellular host, for example prokaryotic or eukaryotic, bacteria, yeast, mammal and the like.

SUBSTITUTE

SHEET

WO 89/09782 PCT/US88/03612 16 Once the peptide has been expressed by recombinant DNA methods, and purified to a suitable degree, the thio group may be blocked with any convenient reagent which replaces the hydrogen of the mercaptan of the cysteine. As indicated for thioethers, active halogens, active pseudohalogens or active olefins may be used. The reaction temperature may range from about 0 to about 500 C, usually 10-300 C, with the reaction time ranging from about 0.5 to 24 hours. Polar solvents, particularly aqueous solvents, may be employed and organic solvents may be present up to about 60 volume Organic solvents include acetonitrile, acetone, diethyl ether, dimethylformamide and the like. With active halides, a mild basic acid acceptor is usually required such as carbonate, bicarbonate and the like. Generally an excess of the blocking agent is employed.

To block with a thio group, various disulfides may be employed, such as methyldithio, p-nitrophenyldithio, 2-pyridyldithio and the like and the second sulfur may be joined to a methylcarboxy ester, aryl or other convenient group. The conditions for displacement are well known to one of ordinary skill in the art and need not be illustrated here.

After the reaction is complete, the product is isolated and purified according to standard conventional techniques.

The peptides of the present invention and compositions may be used in vitro and in vivo. In vitro, the compounds or compositions may be employed for detecting the role of CD4 in viral infection, preventing infection of CD4-bearing cells including T cells and macrophages susceptible to HIV, inhibiting CD4-dependent viral cytopathic effects and the like. In vivo, the compounds or compositions of the present invention may be used prophylactically or therapeutically for preventing infection or inhibiting proliferation of the virus and infection of or cytopathic effects on additional T cells or SUBSTITUTE SHEET r WO 89/09782 PCr/US88/03612 17 other CD4-bearing cells by inhibiting HIV-CD4 interactions related to clinical manifestation of viral disease. The composition can be made with any suitable pharmaceutically acceptable carrier and can be administered in any suitable anti-viral amount by any suitable route such as intramuscularly, intraperitoneally, intravenously, parenterally, intranasally, topically, orally and the like. Any physiologically acceptable medium may be employed, such as deionized water, saline, phosphate buffered saline, aqueous ethanol, and the like. The effective anti-viral amount of the active ingredient(s) will depend upon the solubility, particular use, route and frequency of administration, and the like. The amount used will also depend upon the relative antisyncytial activity of the composition employed. Generally, the dosage will be in the range of about 0.2 mg to about 500 mg, preferably in the range of about 10 mg to 100 mg.

The following specific examples are illustrative.

A. Preparation by Peptide Synthesizer: Synthesis of target peptides and their derivative products was carried out on an Applied Biosystems, Inc., Model 430A Peptide Synthesizer, essentially as. described in the User's Manual supplied by the manufacturer of the machine.

Synthesis begins with the alpha-amino deprotecti~n of the first (C-terminal) amino acid in the chain, which is linked to the polystyrene/divinylbenzene-cross-linked resin via a 4 (oxymethyl) phenylacetamidomethyl bridge.

Activation occurs in trifluoroacetic acid/dichloromethane (TFA/DCM), followed by neutralization in N,N-diisopropylethylamine/N,N-dimethylformamide (DIEA/DMF), and washing in DMF. Addition of the next amino acid residue occurs by mixing in the reaction vessel (RV) the deprotected resin-linked growing chain, and activated incoming amino acid and R-blocked) in DMF. The activated incoming amino acid is prepared by dissolution of the amino acid with alpha amino protected with t-butyloxycarbodi- SU0STITUTE

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WO 89/09782 PCT/US88/03612 18 imide (DCM); addition of 0.5 equivalents of dicyclohexylcarbo-diimide (DCC) to form the symmetric anhydride; and transfer to the concentrator and DCM/DMF exchange.

Dicyclohexylurea formed in the reaction is left in the activation vessel and is dissolved in methanol and discarded prior to the activation of the next amino acid in that vessel. Some fragments are prepared as esters of 1-hydroxybenzotriazole (HOBT) in the activation vessel prior to the concentration and coupling. Each step ends with N-deprotection, neutralization and washing of the resin. Functional groups on the amino acid side chain are blocked during synthesis by side-chain derivatization of each amino acid and esters or amides of: Tos O-benzyl benzyl in some cases C) Br-Z Cl-Z or 4-Me-benzyl Following synthesis, the peptide is cleaved from the resin by treatment with HF at 0 C for two hours with stirring, with the addition as specified of anisole, thioanisole, p-cresol, dimethylsulfide (DMS). HF and other volatile components were removed under vacuum, resin and peptide rinsed with ethyl ether, peptide dissolved in ammonium acetate, and resin removed by filtration. After lyophilization, the peptide mixture, including side-chain protected and unprotected fulllength peptide deletion sequences, anisolated peptide, and rearranged and oxidized peptide was designated "post resin peptide After dissolution in 10% acetic acid or ammonium acetate and passage over G-10 Sephadex and G- 25 Sephadex, respectively, to remove low-molecular weight side products including trace amount of scavengers, the mixture was designated "post-G-10 peptide After dissolution in 0.1% TFA or ammonium acetate and chromatography on reverse phase HPLC, the individual components were designated "post-HPLC peptide X peak (or fraction) or simply "post-HPLC peptide X" if a peak comprised a single pure authentic peptide by amino acid analysis.

usriFrut Sr 8ff r r WO 89/09782 PC/US88/03612 19 Edman degrading sequencing and/or fast atom bombardment mass fragmentography (see Appendix A for specific conditions).

Peptides were then submitted to one or all of the following four tests. Some of the peptides were also derivatized, after purification, by incubation with alpha-bromo-toluene or alpha-bromo-xylene to obtain the benzylated adducts of cystein residues within the peptide (Erickson et al, J. Amer. Chem. Soc. 95:11, 1973). A typical protocol for derivatization (post-synthesis alkyl benzylation) is as follows: About 5 mg (2.2 mol) of peptide of interest (such as H, post HPLC shown to be authentic human CD4 76-94 by FAB mass fragmentography and Edman sequencing) was placed in the flask, and about ml triethylamine and about 1.22 mg 4-methylbenzyl bromide (6.6 mmol) was added. Reaction mixture was stirred for sufficient time (6-16 hrs.) at 25 0 C, vacuum evaporated for about 1.5 hrs., re-suspended in 0.01 mM ammonium acetate, pH 7.0, extracted with one volume of chloroform if necessary, and the resultant aqueous phase lyophilized. The lyophilized material was reconstituted in PBS and tested for anti-fusion activity in the assay of Lifson et al, (Nature 323:725-728, 1986).

B. Fusion assay: Post-resin, post-G-10 or post-HPLC peptides were dissolved it phosphate-buffered saline to a nominal concentration (based on the molecular weight of the desired underivatized peptide) of 1000 uM and two fold dilutions of this concentration down to 10 L M.

Peptides were added, in a volume of 50 4l, to 25 ul of RPMI 1640/20% fetal calf serum containing 50,000 H9/HTLVIIIB cells in 96 well flat-bottomed culture dishes and incubated at 37 0 C for 30 minutes, after which time 50,000 VB cells in 25 Ul culture medium were added. Cell fusion was scored at 1-24 hours as described by Lifson et al, supra.

C. Syncytial Center Assay: 50 ,l of each peptide in PBS was mixed with 50 j l of 50-280 syncytial-forming units SUBSTITUTE SHEET WO 89/09782 PCT/US88/03612 20 (SFUs) of HTLV-IIIB, RF or CC virus in RPMI 1640. Tubes were incubated at room temperature (about 220-25 0 C) for minutes, and 40 .1 aliquots of each used to inoculate CEM cells. After an hour at 37 0 C, inoculum ws removed, minitray well fed 100 A1 of fresh RPMI mediums, and syncytial centers scored at day 2 post-infection. In some experiments, supernatants were harvested at day two for measurement of the viral antigen p24 as an additional confirmation of blockage of infection.

D. Inhibition of antibody binding to CD4+ cells estimated by FACS: VB CD4+ cells in 10 j41 of medium are added to 100 al of the anti-CD4 antibody S3.5 labeled with FITC, and previously incubated at 37 0 C for one hour with 500 SM peptide. S3.5 is a murine IgGI monoclonal antibody which binds to the CD4 molecule within the Leu3A/OKT4A epitope cluster and block HIV infection as well as other CD4-dependent T-cell functions. Sufficient is added to cells to saturate their CD4 antigen.

Cells are incubated an additional 45 minutes at 37 0 C and analyzed by flow cytometry.

E. Mixed Lymphocyte Reaction (MLR): Peripheral blood mononuclear leucocytes were separated from heparinized blood from healthy donors by Ficoll-Hypaque centrifugation. Stimulator cells from donor 1 were irradiated to 3000 rad and 50,000 cells mixed with 50,000 nonirradiated responder cells from donor 2, in 200 /l pooled male human AB serum. Peptides were added to a final concentration of 100 Am. Reactions were performed in triplicate for each stimulator-responder pair, in the presence and absence of pnptides. Mixed cells were cultured at 37 0 C for 6 days in 10% CO 2 One ACi of H-thymidine was added, the cells cultured overnight (about 12-16 hours at 37 0 C) and harvested on glass fiber filters and counted.

The results are summarized in Tables I, II and III. In Table I, the structure of the peptide synthesized, its degree of purity upon testing, and its t WO 89/09782 PCT/US88/03612 -21 activity in each of the four assay systems examined are given. In Table II, the activities of various deletions from the 19 mer which were active in the initial screen to inhibit fusion of HTLVIIIB infected and noninfected CD4-positive T-lymphoma cells are given. Table III shows the effect of anti-receptor polypeptide of the present invention on syncytium formation by three different infectious viral stocks of HIV.

SU~ITUTE SHEET -P 7 WO 89/09782 PC/US88/03612 22 Table I Structure/ Synthesis a CD4, Y1-25/E CD4, Y26-50/F CD4, Y51-75/G CD4, 76-94/H CD4, 76-94/H(r) Purity Fusionc Infectiond MLRe Ab Decor f G-10 n.a. n.a. n.a. n.a.

G-10 n.a.

G-10 n.a.

G-10 125 G-10 500 n.a. n.a. n.a.

n.a.

50 n.a. 100 n.a. n.a.

CD4, 76-94/H(rr) resin CD4, 76-94/H(rrr) resin 500 125 CD4, 76-94/H CD4, 261-285/K CD4, 331-355/L HPLC n.a.

G-10 n.a.

G-10 n.a.

n.a.

n.a.

n.a.

a) structure given by amino acid residue numbers in CD4.

If modified, preceded by one letter symbol for the added amino acid. Letter which follows slash is the code letter for a unique synthetic preparation.

b) all post-G-10 preparations give a predominant peak on HPLC. H post HPLC is a single peptide of sequence CD476-94 without derivatization, as confirmed with amino acid composition, sequence (Edman), single peak on HPLC, and FAB mass fragmentography.

c) dose given is that required to completely inhibit fusion at 24 hours in the standard assay. n.a. not active at 500AM, the highest dose tested.

d) dose given is that required to decrease the number of syncytial-forming centers two days after infection by more than 90%. not active at 500AM, the highest dose tested.

e) all compounds tested at 100 AM, none inhibit MLR at this dose.

f) dose given is that required to decrease S3.5 binding to CD4-positive cells more than 50%, measured by flow cytometry. n.a. not active at 100A M, the highest dose tested.

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WO 89/09782 PCT'/US88/03612 WO 89/09782 PCT/US88/03612 23- Table II Anti-syncytial activity of deletants and altered-sequence variants of CD4cys-benzyl (75-94) define a core anti-synctyial peptide.

Sequence LKIEDSDTYICbzlEVEDQKEE KIEDSDTYICbz 1

EVEDQKEE

EDSDTYI CbzlEVEDQKEE SDTYICbzlEVEDQKEE TYICbZlEVEDQKEE YICbZlEVEDQKEE LI(IEDSDTYICbz EVEDQKE TYICbzlEVEDQKE TYICbzlEVEDQK TYICbzlEVEDQ TYICbzlE-VE YICbzlEVE

TYIAEVEDQKEE

LKI EDSDTYIAEVEDQKEE

TYISEVEDQKEE

LKIEDSDTYI SEVEDQKEE Anti-syncytila] Potency 125 aM 125,&M 125 ,uM 125 aM not active 125 ,gM 125 AX 250,aM 250 aM 500 41

M

not active not active not active not active not active Peptides were tested as post-resin peptide mixtures as described. Anti-syncytial activity was assessed as described in the text. Not active indicates no anti-syncytial activity at early (6 hours) or late (24 hours) time points at the highest dose tested, 500,aM.

SUBSTiTUTE SHEET WO 89/0982 PCT/U88/0361 WO 89/09782 PCTUS88/03612 24 TABLE IIA Post-synthesis benzylation of S--enzyl-CD4(83-94) 4 imparts anti-syncytial activity to the peptide.

Material Method of Nominal Assayed Preparation Potency A. CD4(83-94) solid phase, 250 M BZL[TYICbzlEVEDQKEE] post resin B. Peak 4 solid phase, [S-benzyl-CD4(83-94)] post-HPLC not active C. Peak 4 BZL liquid phase [CD4(83-94)BZL] derivatization, of peak 4 post-HPLC 250 M The anti-syncytial activity of three preparations of CD4(83-94)BZL were compared. The three preparations were A. TYICblEVEDQKEE, the peptide mixture obtained as described in Figure 3 by solid phase synthesis of the desired peptide TYIC(S-benzyl)EVEDQKEE, B. The purified 1 peptide S-benzyl-CD4(83-94) obtained by HPLC fractionation of the peptide mixture described in C. The peptide mixture obtained by liquid-phase derivatization of B. afforded by addition of 7.1 mol of alpha-bromoxylene to 2.2 mol of S-bentyl-CD4(83-94) dissolved in ml of triethylamine followed by stirring at room temperature for 16 hours, removal of volatile material under vacuum, dissolution in 10 mM ammonium acetate, pH extraction with one volume of chloroform, and repeated lyophilization of the resultant aqueous phase.

Table IIB.

Anti-syncytial activity of CD4(76-94) as Benzyl or Xyly Derivatives.

Preparation Activity CD4(76-94) not active Xylyl CD4(76-94) 125 M Benzyl CD4(76-94) 60 M mg of CD4(76-94) purified by HPLC as described in Figure 2 was submitted to chemical aQFivatization as described. Resultant peptide derivatives were evaporated to dryness, reconstituted in water, extracted with one volume of chloroform, and the aqueous phase lyophilized and tested for anti-syncytial activity in the standard assay.

Potency is expressed as the lowest concentration of the peptide mixture (nominal .oncentration based on mass of the input peptide and f .mula weight of the parent peptide .'KIEDSDTYICEVEDQKEE) capable of complete inhibition of HIVHxB- 2induced cell fusion. Not active, no anti-syncytial activity at 1500, M.

SSU TiTOT Sx T >2 WO 89/09782 PCr/US88/03612 25 Table III Inhibition of Viral Infection of CEM cells by anti-receptor Polypeptide Isolatea

IIIB

RF-2 inhibition syncytiun formationb 500 ,M H 50 AM H 5,M H 100 95 37 a) each fresh (CC) or frozen (IIIB, RF-2) viral stock was brought to room temperature, diluted 1:1 with peptide in PBS, and allowed to stand for 60 min at room temp. This mixture then added to CEM cells in micro-titre wells. Incubation at 37 0 C for 60 min was followed by a rinse. Cells then allowed to grow to confluent monolayer over next 48 hours. Number of syncytial centers per well counted.

b) inhibition syncytia formation equals 1, minus the number of syncytium counted in the presence of the peptide divided by the number of syncytia in the control (untreated, infected) wells, times 100.

SUesmiTUTE SHEET WO 89/09782 PCT/US88/03612 26 The chemical purity, activity, stability and other properties of the synthetic peptides prepared in accordance with the present invention are now described. The amino acid sequences are shown in single letter code.

It is noted that a number of various fragments of CD4 receptor did not show any activity at all (data not shown) indicating that a specific molecular structure and configuration are required. As shown hereunder, two preparations (post-G10 H and peptide E) were found to be effective as the inhibitor of ligand-receptor interaction (vide infra).

LKIEDSDTYICEVEDQKEE (CD76-94) Peptide H Cleavage conditions were 1 ml thioanisole, about 20 ml HF. Tested as a post G-10 (one major peak on analytical HPLC, no other peaks visible).

This material is active in the fusion assay. It has the correct sequence by Edman degradation sequencing (note no cys in this sequencing method).

Peptide Resynthesis of peptide H.

Cleavage conditions were 1 ml thioanisole, about 20 ml HF, same as for H. Post resin mass spec shows molecular ion within a complex spectrum, and -HOH (dehydro) ion as well. Post G-10 (100 mg on, 14 mg recovered from acetic acid chromatography). Mass spec post G-10 shows dehydro peak, no sign of parent peak. The post G-10 H(r) showed complete inhibition of fusion at 100 M at 2 and 4 hours but some breakthrough overnight, and complete inhibition of fusion at 500 AM 2, 4 and 24 hours.

Peptide Bzl H: On-line benzylation, with tBoc- Cys-bz. Taken from resin with HF in two batches. First batch gave about 40 mg, active in fusion assay at JM. Second batch gave about 270 mg, active in fusion assay at 120; M.

TYICEVEDQKEE (CD83-94) Peptide Bzl H8-19: Synthesis with bzl Cys instead of methylbzl Cys. Potent as crude resin at 120 SUBSTTUTE SHEET iiC C~i sl~a~irlur y T WO 89/09782 PCT/US88/0361 2 WO 89/09782 PCr/US8/03612 27gM in fusion assay.

Synthesis of Peptide H Analogs A series of peptide H analogs including those with substitutions other than a benzyl group on cysteine residue number 86 were also synthesized. The following methods were employed.

Method 1. One and one-half mg of purified peptide H was dissoved in 120 Al acetonitrile plus 150 Ul deionized water and 60 L1 sodium bicarbonate (0.5 N).

Acetonitrile, 200 a 1 was added followed by a 40 molar excess of benzyl bromide or other reaction compound (see Table The mixture was incubated at room temperature for 1 hr, then 2 Al of triethylamine was added to the reaction mixture which was further reacted at room temperature (about 22 0 -25 0 C) for 1 hr. Forty microliters of ammonium bicarbonate (1 M) was added, then 1 hr later the reaction mixture was reduced to dryness in a centrifugal vacuum concentrator. The desired powder was reconstituted in PBS and used directly in the cell fusion assay (vide supra).

Method 2. This method is identical to Method 1 except that the addition of triethylamine was avoided in the reaction. The dry powder was dissolved in PBS plus tetrahydrofuran and an equal volume of chloroform.

The mixture was vortexed, then the water layer and interface were collected and used for bioassay.

Method 3. One mg of pure peptide H was dissolved in 400 I 1 L of 60% acetonitrile and 40-80 1l sodium bicarbonate (0.05 An eight molar excess of benzyl bromide or other reaction compound (see Table 4) was then added to the peptide H solution and reacted at room temperature for 6 hrs. After completion of the reaction, the product was dried by centrifugal vacuum concentration and dissolved in 5 mM sodium bicarbonate. Then one volume of PBS was added. The solution was further mixed with an equal volume of chloroform and allowed to partition by mixing of the solution. The chloroform layer was v, U SU~J3TITU 2 WO 89/09782 P~r/US88/03612 28 removed and the aqueous phase used for bioassay.

results are shown in Table IIIA.

The SU3BS1TTUE SHET WO, 8909 8 2 WO 89/09782 PCF/US88/0361 29 Table IIIA New Derivatives of Peptide H 1 at Cys 86 Reaction Compound Method of Preparation Titer 2 1. Benzyl bromide 1,2,3 60-120,aM 2. Xylyl bromide 1 cytotoxic 3. 2-chlorobenzyl bromide 3 60-120 :M 4. 4-(N-Maleimidomethyl) cyclohexane-lcarboxylic Acid N-Hydroxysuccinimide Ester 1,2,3 <8 /,M 3-(2-Pyridyldithio) propionic Acid N-Hydroxysuccinimide Ester 2,3 32 ,M 6. None >5001M 1 Preparations tested were not homogeneous by HPLC.

The sequence shown is the nominal peptide sequence expected for the dominant synthetic product.

2 500 LM was the highest concentration tested.

The most effective derivatives of peptide H were those prepared using benzyl bromide, 2-chlorobenzyl bromide, 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester or 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester. The two derivatives prepared using naphthyl reaction compounds were ineffective at the concentrations tested. Benzyl cysteine, including "N-term"-T-BOC-blocked and CBZblocked "N-term" blocked benzyl cysteine, had no effect on HIV-induced cell fusion at any concentration tested (up to 500M).

Demonstration that multiple derivatization of the parent 12-mer peptide structure produces biologically active (anti-viral) material.

Biologically inactive, pure TYIC(benzyl)- EVEDQKEE was solution derivatized as follows: Purified S-benzyl-TYICEVEDQKEE 2 .2.p mol) was dissolved in 1.5 ml triethylamine to which was added 7.12 SUBSTITUTE SHEET WO 89/09782 PCT/US88/03612 43 Table V Relative anti-viral efficacy of CD4(83-94)BZL after infection of CEM-SS cells with multiple isolates of HIV-1 WO 89/09782 PCT/US88/03612 30 nmol of alphabromoxylene. The solution was stirred at room temperature for 16 hours, and volatile material removed under vacuum. Remaining material was dissolved in 10 mM ammonium acetate, pH 7.0, extracted with one volume of chloroform, and repeatedly lyophilized. The resultant material was active at a nominal concentration of 250 gM in the standard fusion assay.

Synthesis of Peptide E.

The peptide of sequence corresponding to CD4(1- 25) and including an N-terminal tyrosine residue (YQGNKVVLGKKGDTVELTCTASQKKS) was synthesized exactly as described for peptide H, and the post-resin peptide mixture (exactly as described for peptide H) was dissolved in phosphate-buffered saline and tested for anti- V 15 syncytial and anti-infection activity in two standard assays described herein supra. The peptide mixture was without effect to inhibit HIV-induced cell fusion, but the mixture inhibited infection of CEM-SS cells as measured in the syncytial-forming quantitative microtiter assay, using four isolates of HIV-1 as shoim in Figure 4. The data are for peptide incubation with virus minutes before inoculation, peptide presence during the minute inoculation, and no peptide present during the following 5-6 days prior to counting syncytia in each cell culture well.

As shown herein, cysteine is one candidate for a residue within the sequence of CD4(76-94) that could be benzylated in the reaction performed. The importance of the cysteine residue, whether derivatized or free, in generation of biological activity upon synthesis of CD4(76-94) was confirmed by the synthesis of serinyl and alanyl congeners of CD4(83-94) and the phenylalanyl congener of CD4(76-94)amide. Substitution of the amino acids serine or alanine for cysteine in otherwise identical solid-phase syntheses of CD4(76-94) or CD4(83-94) resulted in peptide mixtures without detectable antisyncytial activity (Table The phenylalanyl congener SUBSTITUTE SHEET i WO 89/09782 PCT/US88/03612 -31 of CD4(76-94) amide was likewise inactive. In view of the comparable activity of the CD4(76-94) synthetic mixture and the benzylated peptides, several peptide mixtures were synthesized as S-benzyl cysteine congeners of CD4(76-94). In these syntheses, the cysteine residue is added to the growing peptide chain as t-Boc-S-benzylcysteine rather than t-Boc-S-methylbenzyl-cysteine, and remains largely S-protected after HF cleavage, compared to complete or nearly complete removal of the S-methylbenzyl block group used in the previous syntheses of CD4(76-94). In several independent syntheses, the peptide mixture obtained after automated synthesis of the desired peptide S-benzylCD4(83-94) was approximately three-fold more potent to inhibit HIV-induced cell fusion than the peptide mixture obtained after synthesis of CD4(83-94) using t-Boc-S-methylbenzyl-cysteine. A series of deletion peptides were synthesized using t-Boc-Sbenzyl-protected cysteine, and tested as inhibitors of HIV-induced cell fusion. The results indicate that the core sequence for this biological activity is CD4(83-89) (Table Furthermore, it was found that the biological activity requires the correct sequence of the core peptide, as well as derivatization, since a nineteen-residue peptide with the same composition but slightly altered sequence compared to CD4(76-94) had no activity as an anti-syncytial agent. The activity was attributable to peptide material, since proteolytic digestion completely abolished the anti-syncytial activity of the active peptide preparation. Furthermore, a core peptide [SbenzylCD4(83-89)] retaining significant biological activity was defined. Syntheses of CD4(84-94) or CD4(84- 89) yielded material with no anti-syncytial activity, while addition of N-terminal threonine 83 to either sequence restored anti-syncytial activity. Non-specific effects of threonine addition were considered unlikely since a threonine-rich control peptide synthesized under identical conditions was completely devoid of bio- SUBSTITUTE SHEET X 1: c~c. t 89/978 PCT/S88/36 WO 89/09782 PC~r/US88/0361 32 logical activity, and the threonine-containing nineteen-residue peptide DQKEEELKIEDSDTYICEV of identical composition to the CD4(76-94) peptide was also without detectable anti-syncytial activity.

These results indicate that anti-syncytial activity could be generated by two independent routes, both requiring the existence of a parent peptide. Thus biologically active material could be recovered either from the peptide mixture after solid-phase synthesis of the CD4(76-94) peptide, by xylyl- or benzyl-derivatization of inactive authentic CD4(76-94), or by xylyl derivatization of inactive authentic S-benzylCD4(83- 94). The S-benzylCD4(83-94) material partially purified by HPLC was stable to heating at 65 0 C and the biological activity was destroyed by digestion with Pronase.

As indicated herein supra the anti-viral activity of these CD4 peptide preparations is specific and restricted to CD4-dependent virally induced cell fusion. CD4(76-94) at concentrations up to 500 M had no effect on HTLV-I-induced cell fusion in vitro. Likewise, activity appears to be restricted to inhibition of CD4 function related to viral interaction, since complete inhibition of HIV-induced cell fusion could be observed at a dose of the CD4(76-94) peptide which did not affect the MLR response (vide supra). Without being bound to any theory, it is postulated that the mechanism of action of these CD4-derived peptides may involve competitive blockade of viral attachment to CD4 via peptide binding to the CD4-combining region of the HIV gpl20 glycoprotein. Consistent with this proposed mechanism of action, the partially purified S-benzyl-CD4(83-94) peptide blocked fusion between HIV-infected T cells and CD4expressing T-cell lines or CD4-expressing peripheral blood-derived cells, independent of the isolate of HIV used in the assay and also blocked the CD4-dependent fusion induced by the structurally variat simian immunodeficiency virus.

S3U

T

i IL WO 89/09782 PCT/US88/03612 33 In summary, several 12-25 amino acid residue containing fragments of the T4 molecule, the receptor for the human immunodeficiency virus, were tested for their ability to inhibit fusion of HTLV-III-infected and noninfected CD4+ human lymphoma cells, to inhibit CD4 antibody binding to CD4+ cells by various isolates of HIV, and for their ability to block the CD4+ CEM cells by various isolates of HIV, and for their ability to block the CD4-dependent mixed lymphocyte reaction in human peripheral blood leucocytes. It is noted that none of the fragments tested were active as pure peptides, but an unfractionated preparation of one of the syntheses, that of the peptide corresponding to residues 76-94 of the T4 antigen (Post G-10 H) inhibited fusion and infection at concentrations between 125 and 500/.M. This effect was reproducible across several separate syntheses of the peptide tested in three different laboratories. An Sbenzyl and an S-methylbenzyl derivative of this peptide was produced by two separate laboratories using similar but distinct published protocols for adding the benzyl moiety to a sulfhydryl group in the polypeptide without derivatization of other weaker nucleophilic attaching groups found in the peptide. Each of these preparations was active in inhibiting giant cell formation between infected and non-infected CD4+ cells in vitro. An active peptide could also be produced by modifying the synthesis of the authentic peptide such that a benzyl instead of a methylbenzyl protecting group was incorporated into the growing chain of cystein, affording a peptide with cystein derivative more stable to HF cleavage than the original compound. This material was also efficacious in inhibiting fusion. In addition, chloroform extraction of an aqueous solution of the original synthesis of peptide H afforded at the interface of the organic/aqueous layers material which was potent and completely efficacious at M to inhibit CD4+-dependent fusion between HTLV-IIIB infected and non-infected human lymphoma cells. This SUBSTITUTE

SHEET

p; WO 89/09782 PC/US88/03617 34 represented about 3-fold increase in the specific activity of the derivatized peptide H material.

It was further found that a shorter, benzylated version [CD4(83-94)BZL] was a potent inhibitor of HIVinduced cell fusion. This material was prepared and partially purified by collection of a single UV-absorbing peak after fractionation using reverse-phase (C8) chromatography. CD4(83-94)BZL was tested for its ability to inhibit infection of CEM-SS cells by HTLV-IIIB (Fig.

When CD4(83-94)BZL was present only during the sixty minute period of viral inoculation, the IC 50 (the dose required to achieve a 50% reduction in the number of syncytia formed in peptide-treated versus virus-infected, non-treated controls) for this peptide preparation was approximately 63 AM (Fig. 1A). Inhibition of infection rather than inhibition of cell fusion following infection appears to be the mechanism of action of CD4(83-94)BZL as well as the CD4(76-94) peptide mixture, since decreased numbers of syncytia were observed five to six days after viral inoculation, despite the fact that peptide was present only during the initial one-hour viral absorption step of the assay. When the peptide was present during the one-hour inoculation period, and thereafter in the culture medium until syncytia were scored five days later, an eight-fold increase in CD4(83-94)BZL anti-viral potency resulted [a shift in the IC 50 from 63 to 8 aM (Fig. This experiment also demonstrated that the peptide derivative at concentrations up to 125,aM had no apparent toxic effects on CEM-SS cells even during five days' exposure in vitro (Fig. 1C).

The findings, presented herein now provide a "process principle" for obtaining an antiviral agent.

The principle is that the viral receptor is fractionated into various smaller entities. These entities are then separated and purified. The purified entities, if inactive as antiviral agent, are then derivatized as described in detail herein and thus biologically active -SUBSTITUTE SHEET

I

WO 89/09782 PCr/US88/3612 35 antiviral peptide derivatives are obtained. In the present instance, a 19mer pure peptide was reduced to a 12mer pure peptide which was then converted to a S-benzyl 1?mer. An inactive peak 4 was then converted by solution derivatization to an active peptide peak 7 which was active as an antiviral agent at the nominal concentration of 32 aM. Thus a synthetic, isolated, substantially pure, biologically active product is obtained by the process of the present invention.

In accordance with the principle enunciated herein, the following are noted. It was found that peptide G (25mer comprising CD4 (51-75)) blocks antibodies directed against CD4 molecule. A 19mer molecule (such as 71-89, 72-90, 73-91, 74-92, 75-93, 76-94) could partially inhibit HIV infection; 12mer (83-94) shows antiviral property and 7mer (83-89) is the core peptide; an extension of N-terminus of CD4 providing improvement in biological activity to inhibit infection.

It should be noted that no strain of HIV or SIV has been identified which does not employ the CD4 molecule to gain entry into T-lymphocytes. To further determine that the benzylated CD4 derivatives acted as competitive blockers of the CD4 binding site of retroviruses, the efficacy of the peptides to inhibit infection of CEM-SS cells by other structurally variant HIVisolates was also tested. CD4(83-94)BZL inhibited infection of CEM-SS cells by HIVcc, HIVmn and HIVRFII, as well as HTLV-IIIB. Inhibition was virtually complete at concentrations less than 125 aM (Table IV). CD4(83-94)BZL also -effectively inhibited HIV-induced cell fusion regardless of the isolate of HIV-1 used in the assay.

Inhibition of fusion by CD4(83-94)BZL occurred whether fusion was induced using a CD4-positive lymphoid indicator cell line, or antigen-activated freshly isolated human peripheral blood leucocytes.

CD4(83-94)BZL is a potent and efficacious inhibitor of cell fusion induced by simian immunodefi- SUBSTITUTE

SHEET

WO 89/09782 PCT/US88/03612 36 ciency virus as well (Table IIIA). The amino acid sequence of the large envelope glycoprotein of SIV is quite different from that of HIV-1, and both envelope proteins are significantly structurally different from that of HIV-2. The ability of CD4(76-94)BZL to inhibit infection by HIV-2 was also tested and the peptide was found to inhibit infection of CEM-SS cells by both viruses with similar potency (Table V).

All of these data provide strong, direct and indirect evidence that the anti-viral activity of the original synthetic preparation of peptide h resides in a side fraction (most likely an S-derivatized side fraction), which is completely efficacious at nominal concentration of 30 aM, as an HIV-specific antiviral agent without activity to disrupt endogenous CD4-dependent immune functions in vitro. "On-line" S-benzyl-containing syntheses of peptide H lacking one or more residues at the C- or N-terminus of the molecule have been constructed, and serve to demonstrate that the N-terminal seven residues of the peptide, and at least one of the Cterminal glutamic acid residues, are not essential for anti-viral activity. On the other hand, this core undecapeptide is responsible for anti-viral activity in a sequence dependent way, since removal of the eighth Nterminal residue, removal of the C-terminal hexapeptide, or removal of the C-terminal hexapeptide and replacement in a different order at the N-terminus of the molecule, result in peptides totally without anti-viral activity.

Since H23V is known to have a high degree of variability in its structure, a multivalent groupspecific vaccine will be required if the vaccination is chosen as the method of combating HIV infection. The present invention avoids this problem because the conservation of the receptor for the virus is exploited. For, although the virions may be slightly different, they still share the property of binding to the CD4 molecule and thus share the property of inhibition by the binding SUBSTITUTE

SHEET

A, 61- WO 89/09782 PPr/S88/03612 37 epitope of the CD4 molecule. Data with two other isolates of HIV, besides HTLVIIIB, which differ significantly in their sequence in the envelope region, indicate that infection of CD4-positive cells by all three isolates is inhibited by peptide H, but to different degrees, reflecting different affinities of each virion for the CD4 molecule (Table III).

Because of their anti-receptor property, the polypeptide derivatives of the present invention could function as a short-acting immunosuppressant and be useful in transplantation and grafting.

The results presented herein also indicate that hybrid molecule of two separate epitopes could also be combined for example with a disulfide bond or a flexible polymethylene linker to give a more potent inhibitor if more than one receptor epitope is found to be involved in ligand binding such as peptides H and E. Accordingly, peptides E and H are joined to make a peptide E/H heterodimer by disulfide bond formation between the two purified peptides. Without being bound to any theory, it is postulated that since peptides E and H are both active, and are joined by a disulfide bond in the native molecule (CD4), it is reasonable that the peptide E and H mixtures may become active because they contain derivatives of the respective authentic peptides which are conformationally restricted by derivatization, and therefore have a higher affinity for the HIV envelope glycoprotein than their underivatized, conformationally flexible parent peptides. In this case, the conformation of both peptides would most closely approximate their conformation in the native molecule, and be relatively restricted to this conformation by disulfide bond formation Classon, J. Tsagaratos, I.F.C. McKenzie, I.D. Walker, Proc. Natl.

Acad. Sci. USA 83, 4499 (1986)]. Thus a peptide E/H heterodimer is produced by disulfide bond formation between the two purified peptides, CD4(1-25) and CD4(76- 94). Accordingly, these peptides are synthesized by SUBSTITUTE SHEET i' i WO 89/09782 PCT/US88/03612 38 solid-phase methodology as described herein and the desired peptides QGNKVVLGKKGDTVELTCTASQKKS and LKIEDSDTYICEVEDQKEE combined in water and allowed to stand overnight at room temperature with slow oxygen bubbling, to effect dimerization. Dimers are purified by high pressure molecular sieving chromatography and tested for biological activity.

The biological activity of peptides E and H (peptide mixtures) to inhibit infection of CD4-positive cells, and HIV-induced fusion of CD4-positive cells, was then compared. Peptide H and peptide E [YCD4(1-25)] were tested for their ability to inhibit HIV-induced cell fusion or in.fction of CEM-SS cells by HIV (HXB-2 isolate) by the tests described herein. The results are as follows: Peptide Fusion Assay Infection Assay E not active 63 /M H 63 ,M 31 4M The above results are expressed as concentration required to completely inhibit HIV-induced cell fusion after a 24 hour co-cultivation experiment (fusion) and the concentration required to reduce the number of infectious centers (syncytia) present after six days of culture, following viral inoculation of the cultures in the presence of the peptide for one hour and incubation in peptide-free culture medium thereafter, by more than Further data show that antireceptors in the CD4(76-94) region can be structurally refined to distinguish not only between ligands of the CD4 receptor antigen in the presence of class II molecules vs.

HIV envelope glycoprotein) but also between structural aspects of the same ligand when presented in different biological contexts (here, HIV envelope glycoprotein in intact infectious viral particles versus HIV envelope glycoprotein in cell membrane during cell-cell fusion).

These data are presented below.

SUBSTITUTE SHEET WO 89/09782 PCT/US88/03612 39 Peptide Mixture Vn/Vo. 1 hour using HTLV-IIIB peptide H 250 M 0.01 125 M 0.08 (peptide H mixture 63 M 0.09 represents CD76-94) 31 M 0.14 peptide T4DTE (S-benzyl 12mer fraction, CD83-94) 250 M 0.57 125 M 0.91 63 M 0.95 31 M z 1.00 The above data show that the syncytia-forming quantitative micro,'ssay can be used to distinguish effects of the peptides on infection alone (the 19mers are much more active than the 12mer after 1 hour exposure to peptide) compared to effects on HIV-induced cell fusion in which the 12 and 19 mers are about equal.

Of course, the finding that a fragment of a peptide or protein receptor can inhibit, with great efficacy albeit relatively low potency, the binding of I 20 the ligand to its receptor, opens a new vista in the design and synthesis of therapeutic agents. When peptide fragments of a receptor are found to inhibit ligandreceptor interaction at a low affinity, the possibility exists of derivatizing these fragments to increase their affinity for ligand by restricting their conformational flexibility, or increasing their non-specific binding to the ligand by altering hydrophobicity or adding covalent modifying agents and the like. Such derivatization of the core active peptide includes substitution, addition of various substituents on the cysteine sulfur, methylation of glutamic acid, addition of alkylating agents, addition of hydrophobic side groups and the like in order to increase the potency and duration of action of the S compound.

Without being bound to any specific theory, it is proposed that in the ligand-receptor interaction the ligand binds to a small numb7i' 1) of continuous oligo- SUJsTI si-EET WO 89/09782 PCT/US88/03612 peptide sequences in the receptor. The free energy of this reaction is comprised of both the entropy and enthalpy of binding, the former does not prohibit binding because of the restriction of conformer flexibility of the binding epitopes by the non-binding portions of the receptor molecule and within the epitope itself. It is further postulated that restriction of conformer flexibility contributes to the free energy of binding, and therefore to the affinity of binding of ligand to receptor, largely by decreasing delta S. Therefore, fragments (or even a fragment) of the receptor involved in binding is identifiable by synthesis from the receptor sequence and subsequent assay for inhibition of ligand-receptor interaction. The potency to inhibit should be many orders of magnitude less than the ligandreceptor Kd since there is no restriction on conforner flexibility by the rest of the molecule. To accomplish this, synthesis is done on 430A with subsequent controlled HF cleavage to give a mixture of authentic and protected groups to take advantage of conformer flexibility restriction within the peptide sequence. Derivatives of 25 mers based on S-S, hydrophilicity and the like are thus prepared and the crude mixtures are tested for their anti-receptor activity. Then active preparations are purified to homogeneity by standard methods.

Then, purification of the specific inhibiting molecule to homogeneity (after test of crude mixture) is accomplished by standard purification techniques.

A pharmaceutical anti-viral composition in accordance with the present invention comprises an effective amount of the anti-receptor compound of the present invention to inhibit viral infection, and pharmaceutically acceptable, non-toxic sterile, carrier.

The present invention also provides a method of inhibiting viral infection comprising administering to a host susceptible of viral infection an effective (antiviral) amount of the active ingredient (anti-receptor H -USS r T

I

WO 89/09782 PCT/US88/03612 41 molecule including derivatives of analogs thereof) to inhibit viral infection.

It has been suggested that a mirror-image molecule of a receptor, made on the principles of molecular complementarity, may also inhibit ligand-receptor interaction. However, the amino acid sequence of the peptide of the present invention demonstrates that this theory is not workable in this case.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in lii'ht thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

SUBSTITUTE

SHEET

1 i WO 89/09782 PCT/US88/03612 42 Table IV Inhibition of HIV-2 infection of CEM-SS cells by CD4(76-94) post-resin peptide mixture.

Peptide concentration 250 AM 125)LM 63AM 32AcM 16 IM 8AM 4 AM 2 /M Vn/Vo. HTLV-IIIB Vn/Vo. HIV-2 0.003 0.10 0.003 0.34 0.003 0.60 0.003 0.71 0.15 0.79 0.33 0.80 0.47 0.95 0.78 1.00 Virus inoculation of CEM-SS monolayers was carried out in the test. Viral inocula (HIV-lHTLV-IIIBor

HIV-

2

NIHZ)

were preincubated in the presence or absence of the nominal concentrations shown of the post-resin peptide mixture from the automated solid-phase synthesis of the desired peptide LKIEDSDTYICEVEDQKEE. Peptide, when present, was added during viral inoculation and also during subsequent growth to confluence of the CEM-SS cell monolayer.

SUBSTITUTE

SHEET

j- *--LI WO 89/09782 PCT/US88/03612 43 Table V Relative anti-viral efficacy of CD4(83-94)BZL after infection of CEM-SS cells with multiple isolates of HIV-1 Dose IIIB RF MN CC 125 #M 0.005 0.003 0.003 0.003 63,M 0.005 0.43 0.54 0.05 32, M 0.005 0.69 1.03 1.01 16 M .0.04 0.84 >1.00 >1.00 8pM 0.31 >1.00 >1.00 >1.00 4 pM 1.06 >1.00 >1.00 >1.00 Viral stocks of HIV-1HTLV-IIIB, HIV-1RF-II, HIV- 1 MN and HIV-lcc were prepared as either fresh or frozen cell culture supernatants from HIV infected cells. Viral inocula were pre-treated with varying nominal concentrations of peptide in PBS or complete medium were incubated with DEAE-dextran pre-treated CEM-SS cells for one hour at 37 0 C. Inocula were removed from the cultures by aspiration and replaced with fresh medium or medium containing the nominal concentrations of CD4(83-94)BZL shown. CD4(83-94)BZL was prepared as described in detail in the legend to Figure 1A, and represents an HPLCpurified biologically active fraction (peak 7) from the i automated solid-phase synthesis of the desired peptide TYICbzlEVEDQKEE. Results are the averages of duplicate determinations (all within 30% of the mean values) in a single experiment repeated at least once with similar results.

SUBSTITUTE SHEET I -1 WO 89/09782 PCT/US88/03612 44 Table VI Potency of CD4(83-94)BZL to inhibit fusion induced by several isolates of HIV and by SIV Dose TJ DV HXB2 SIV-UC 125,M 6 3 ,M 4 4 32,M 4 4 4 4 16 M 4 4 4 4 CD4(83-94)BZL prepared as described in the legend to Figure 1 was pre-incubated with 50,000 H9 cells infected with the viral isolates HIV-1Tj, HIV-DV, HIV-1HTLV-IIIB (HXB2) or SIVUC for one hour at 37 0 C. Levels of viral expression in each cell line were sufficient to allow formation of syncytia upon co-culture with 50,000 VB cells in a volume of 50 ul RPMI 1640 supplemented with heat-inactivated 10% fetal calf serum at a rate and frequency similar to that previously reported for the reference isolate HXB2 scored at four, six and twenty-four hours after co-culture: no visible syncytia or presyncytial aggregates observed in duplicate wells, 1-4, graded increase in syncytia to the maximum seen in the absence of treatment with peptide or anti-Leu 3A CD4 antibody. Syncytial scores shown are for the end of a twenty-four hour observation period.

SUBSTITUTE SHEET "t i WO 89/09782

D

2 1 1 PCT/US88/03612 45 Table VII Comparison of CD4(83-94)BZL potency to inhibit HIV induced cell fusion of VB indicator compared to acutely activated, fresh human peripheral blood mononuclear cells ose 00M 631M 32VM 16a M 8 M PHA PBMCs VB The post-resin peptide mixture obtained from the synthesis of the desired peptide TYICbzl EVEDQKEE was preincubated for 30 minutes at 37 0 C at the nominal concentrations shown. Cells and peptide were combined with either VB cells or phytohemagglutinin (PHA)-stimulated PBMCs and cultured at 37°C for 24 hours, at which time syncytia were scored.

SUBSTITUTE SHEET Fil r.FI 1 -r -Cli-~ WO 89/09782 PCT/US88/03612 46 APPENDIX "A" Synthesis of peptide H: The peptide mixture was synthesized on an Applied Biosystems, Inc. 430A Automated Peptide Synthesizer. The synthesizer was programmed to couple to a PAM-glutamic acid resin (0.5 mmol glutamic acid equivalents) the amino acids E,K,Q,D,E,V,E,C,I,Y,T,D,S,D,E,I,K, and L as the tBoc, R-blocked derivatives shown in the dynamic run file attached. Double-coupling cycles were run for S,D,E,I, K and L (the last six.amino acids of the synthesis). Activation, coupling, washing, deprotection, washing cycles were as described in the 430A User Manual. The resultant resin-coupled peptide mixture from the complete run (approximately 1.5 gm) was air-dried, and placed in the HF cleavage apparatus. 1 ml anisole and 1 ml dimethylsufide, and approximately 100 Ig of p-thiocresol were added, and the mixture placed under vacuum. 18 ml of HF were added under vacuum, and the mixture stirred for one hour at 00 C. Volatile materials were removed under vacuum over a one-hour period. The resin-peptide mixture was suspended in approximately 30 ml of ethyl ether, and allowed to stir at room temperature for 30 min. The slurry was vacuum-filtered, and saspended in approximately 250 ml of 100mM ammonium bicarbonate pH 6.5. This suspension was vacuum-filtered to remove resin, and lyophilized overnight. This material constitutes postresin peptide H, or peptide H mixture, comprising 1) the desired peptide sequence LKIEDSDTYICEVEDQKEE, 2) derivatives of this sequence including R-protecting groups not removed during HF cleavage, R-protecting groups or scavengers obtained upon re-adduction during cleavage, 3) deletion peptides generated by premature chain termination during synthesis, 4) re-arrangements of R-groups glutamine deamidation, pyroglutamyl ring formation, beta-elimination, etc.) and peptide backbone isopeptide formation) and 5) peptide products resulting from SUBSTITUTE SHEET 2 WO 89/09782 PC/US88/03612 47 combinations of the above as well as other uncharacterized intra-molecular and inter-molecular reactions and rearrangements occuring during synthesis, cleavage, or purification.

Synthesis of peptide T4DTEbzl: Synthesis was as described above, except the sequence TYIC(benzyl)EVEDQKEE was the desired peptide, and the corresponding input protected amino acids were the same except for substitution of N-tBoc-S-methylbenzylcysteine by N-tBoc-S-benzylcysteine, and omission of p-thiocresol during the cleavage reaction.

iS SUBSTITUTE SHEET /1 WO 89/09782 PTU8/31 PCr/US88/03612 48

CYCLE

NUMBER

1 2 3 4 6 7 8 9 11 12 13 14 16 17 18

AMINO

ACID

BEG

Glu Lys Gin Asp Giu Val Glu Cys Ile Tyr Thr Asp Sar Asp Glu Ile Lys Leu

END

REACTION CONCENTRATOR VESSEL VESSEL rbeg 1 cbeg 1 TEMP ACTIVATOR

VESSEL

RBOC 1 RBOC 2 RBCC 10 RBOC 1 RBOC 1 REOC 2 RBOC 1 REOC 2 1BOC 2 RBOC 1 RBOC 2 RBOC 1

DF-DM-VR

DF-DM-VR

DF-DM-VR

DF-DM-VR

DF-DM-VR

DF-DM-VR

rend 1 cboc 2 cboc 4 cboc id cboc 2 cboc 2 cboc 2 cboc 2 cboc 2 cboc 1 cboc 4 cboc 1 cboc 2 CBC1 2DDM CBC1 2DDM CBC12DDM CBC12DDM CBC 15DDM CBC1 2DDM cend 1 lOc 25c 25c loc 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c 25c loc abeg 1 aboc 1 aboc 2 aboc 2d aboc 1 aboc 1 aboc 1 aboc 1 aboc 1 aboc 1 aboc 1 aboc 1 aboc 1 AB30C 21D ABOC 21D ABOC 21D ABOC 21D ABOC 22D ABOC 22D laend 1 SUBST1,TU.TrEET WO 89/09782 P~U8/31 PCr/US88/03612 49 RES.i AMINO NUM1BER ACID TOTAL WEIGHT BOC/MW. OF PEPTIDE OF ACID RESIN

SUBSTITUTION

-(mmoi/q)

COM~MENT

INITIAL

SUB

19 18 17 16 15 14 13 12

I

10 09 08 07 06 05 04 03 02 01 Giu Giu Lys Gin Asp Glu Val Giu Cys Ile Tyr Thr Asp Ser Asp Glu Ile Lys Leu 000 337 116 216 323 337 217 337 325 231 191 309 323 295 323 337 231 116 231 00. 696 00.805 00. 951 01. 018 01. 120 01. 230 01. 279 01. 389 01. 192 01. 519 01. 737 01. 832 01. 935 02.023 02.126 02.235 02.292 02. 110 02.197 0.720 0.720 0.:1 0.521 0.191 0.116 0.107 0.391 0.360 0.335 0.323 0.299 0.273 0.258 0.217 0 .235 0.221 0.218 0.205 0.200 (OBzi) (Ci-Z) (OBzi) (OBzl) (OBzl) (1-MeBzi) (Br-Z) (Bzi) (QBzl) (Bzi) (OBzl) (OBzl) (C1-Z) SU T;T S TiT1%17-EE

Claims (28)

1. Antireceptor compound comprising fragments of a receptor 'protein which comprise the ligand-binding region of said receptor protein or derivatives of said fragments and which inhibits the binding of a ligand to a receptor.

2. An antireceptor compound according to claim 1 wherein said compound, at least in part, is a polypeptide.

3. An antireceptor compound according to claim 1 or 2 wherein said receptor comprises human CD4 molecule or a portion thereof.

4. An antireceptor compound according to any one of claims 1 to 3 wherein said ligand is a virus.

An antireceptor compound according to claim 4 wherein said virus is human immunodeficiency virus (HIV).

6. An antireceptor compound according to any one of claims 1 to 5 having amino acid sequence expressed in single letter code of LKIEDSDTYICEVEDQKEE.

7. An antireceptor compound comprising a sequence of at i least seven amino acids of human CD4, which sequence includes the cysteine at position 86, wherein the sulfur atom of said t cysteine is joined to a carbon atom or sulfur atom of a sulfur blocking group, said compound being an inhibitor of S CD4-dependent virus-induced cell fusion.

8. A compound according to claim 7, wherein said i :25 sequence includes a sequence of at least 5 amino acids of the i CD4 and at least one of the C-terminal or N-terminal side of i said cysteine.

S9. A compound according to claim 7 or 8 wherein said sequence comprises amino acids 76 to 94 of CD4. i 30

10. A compound according to any one of claims 7 to 9 wherein said cysteine sulfur atom is bonded to a carbon atom of a sulfur blocking group.

11. A compound according to any one of claims 7 to wherein said carbon atom is a member of a succinimidyl ring.

12. A compound according to any one of claims 7 to 9 wherein said cysteine sulfur atom is bonded to a sulfur atom of a sulfur blocking group.

13. A compound comprising a sequence of at least 10 amino (I acids of human CD4, which sequence includes the sequence of amino acids 83 to 90 including the cysteine at position 86, wherein the sulfur atom of said cysteine is joined to an aliphatic carbon atom or sulfur atom of a sulfur blocking group, said compound being capable of interfering with CD4-dependent virus-induced cell fusion.

14. A compound according to claim 13 wherein said aliphatic carbon atom is a ring carbon atom of a succinimidyl group.

15. A compound according to claim 14 wherein said succinimidyl group nitrogen atom is substituted with a substituent comprising a carboxyl carbonyl.

16. A compound according to claim 7 or 8 wherein said sequence comprises amino acids 79 to 94 of CD4.

17. A compound according to claim 7 or 8 wherein said sequence comprises amino acids 81 to 94 of CD4.

18. A compound according to claim 7 or 8 wherein said sequence comprises amino acids 83 to 94 of said CD4.

19. A compound according to claim 7 or 8 wherein said carbon atom is the methylene of a benzyl group. S

20. A composition of matter comprising a -olypeptide comprising a sequence of at least seven amino acids of human S CD4, which sequence includes the cysteine at position 86, S: wherein the sulfur atom of said cysteine is joined to a carbon atom of a sulfur blocking group, said composition prepared by the method comprising: combining in an aqueous solvent, said polypeptide with at least about a stoichiometric amount of a sulfur blocking group containing compound, which compound comprises 30 an active halogen or active olefin functionality, at a temperature in the range of about 0 to 50°C for a time sufficient for said sulfur blocking group containing compound to covalently bond to said cysteine; neutralizing any acid formed; and isolating said composition of matter.

21. An antiviral pharmaceutical composition comprising an effective amount of the antireceptor compound of claim 1 to inhibit viral infection and pharmaceutically acceptable, I 51- I -P 1 I i non-toxic sterile carrier.

22. A method of inhibiting viral infection, comprising administering to a host susceptible of viral infection an effective amount of the antireceptor compound of claim 1 to inhibit viral infection.

23. A method according to claim 22 wherein said viral infection is caused by human immunodeficiency virus.

24. A method of derivatizing a biologically active antiviral agent comprising the antireceptor compound of claim 1 from a purified inactive antireceptor compound comprising the step of benzylalkylating a purified inactive antireceptor compound obtained by solid phase process; and acid-cleaving the benzylalkylated product to obtain said bioactive antiviral agent.

25. An antireceptor compound according to claim 1 being a heterodimer of two or more antireceptor compounds.

26. An antireceptor compound according to claim 1 being a derivative of a peptide containing amino acid residues 1 to of CD4 molecule.

27. An antireceptor compound according to claim 26 being a heterodimer of peptide H containing amino acid residues S 76-94 of CD4 molecule and peptide E containing amino acid S residues 1-25 of CD4 molecule.

28. A compound according to claim 1, 7 or 13 substantially as hereinbefore described with reference to any one of the examples. 4: I 1, I g O 0 t~~ 52