WO1989003420A1 - Anti-retroviral agent - Google Patents

Abstract

Peptide compositions comprising a sequence of human CD4 which includes at least a core sequence of seven consecutive amino acids including the cysteine at position 86 (C-86) are provided. The sulfur atom of C-86 may be derivatized, and at least one amino acid other than C-86 is derivatized, generally the glutamic acid at position 87 (E-87). Various groups may be used as derivatizing groups, including aryl-containing substituents, or in the case of cysteine, a thioether resulting from the reaction between the thio group of the cysteine and a maleimide. The resulting compositions may be used prophylactically or therapeutically for inhibiting interactions between CD4 and retroviral proteins.

Description

. ' . ^{■ "} , ^■ -

ANTI-RETROVIRAL AGENT

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Application Serial No. 108,160, filed October 13, 1987, which application is incorporated herein by reference.

INTRODUCTION

Technical Field

This invention relates to methods for modula¬ ting viral effects on susceptible cells expressing the cell surface antigen CD4 using polypeptides which in- terfere with interactions of viral proteins with CD4. This invention is particularly directed to blocking virion infectivity and cytopathic effects of CD4-depen- dent retroviruses.

Background of the Invention

T cells regulate the immune response through "helper" and "suppressor" signals and also mediate part of the effector arm of cellular immunity. Human T lym¬ phocytes were initially subdivided into two functional- ly distinct sublineages on the basis of expression of mutually exclusive cell surface proteins designated CD4 (also referred to as T4 or Leu3) and CD8 (also referred to as T8 or Leu2). T cells expressing CD8 mediate Ma¬ jor Histocompatibility Complex (MHC) Class I restricted cytotoxic activity and non-cytotoxic antigen specific suppressor function, while T cells expressing CD4 medi¬ ate helper function for B cell growth and differentia¬ tion, proliferative responses, and inducer function for differentiation of CD8 expressing cytotoxic cells. CD4 is a 55-58 kD glycoprotein which, while originally characterized as a T lymphocyte differentia¬ tion antigen found on a subset of mature T cells, is also found on cells of the mononuclear phagocyte line¬ age and on occasional B lymphocytes. In T cell-target cell interactions, the significance of the CD4 molecule can be demonstrated by studies with monoclonal anti- bodies. Antibodies directed against specific epitopes of the CD4 molecule inhibit MHC Class II restricted functions, including antigen-induced T cell prolifera¬ tion, lymphokine release, helper cell function and the cytotoxic activity of CD4 expressing cytotoxic T cells. The CD4 molecule also serves as a receptor for the ret- rovirus HIV-1 (also referred to as HTLV-III, LAV-1 or ARV) , the etiologic agent for Acquired Immunodeficiency Syndrome (AIDS) and other retroviruses such as the sec¬ ond AIDS-associated virus, HIV-2, and SIV (also re- ferred to as STLV-III, which is similar to or identical to a virus designated HTLV-IV) .

Interactions between one of the envelope glyco- proteins of HIV, gpl20, and CD4 underlie the binding of infectious HIV particles to susceptible cells. Thus, cells which express CD4 are the major targets of HIV infection and constitute the major in vivo reservoirs of virus in infected individuals. Interactions between gpl20 and CD4 are also critically involved in HIV- induced cytopathicity. Many of the clinical manifesta- tions of HIV infection appear to be due to effects of the virus or viral components on cells expressing CD4. It is therefore desirable to develop agents capable of blocking interactions between CD4 and the envelope glycoproteins of CD4-dependent retroviruses for pro- phylaxis and treatment of infection by these viruses. Preferably the agents will block interactions between CD4 and viral proteins without affecting the normal function of the CD4 molecule. Description of Relevant Literature

Two major functionally distinct T cell subsets in humans have been defined. CD4+.cells recognize MHC Class II determinants whereas CD8+ cells recognize MHC Class I determinants (Engleman e_t a^. , J. Immunol.

(1981) 122:2124-2129). Rao et al. , Cellular Immunol. (1983) 8_0:310-319, using monoclonal antibodies, showed that five epitopes of the CD4 antigen could be distin¬ guished on human T cells of the CD4+ subclass. The CD4 molecule is involved in a variety of crucial immunoreg- ulatory interactions between cells in the immune system. Maddon et al. (Cell (1985) 2:93-104) isolated and ob¬ tained a nucleotide sequence of a cDNA encoding the CD4 protein. In addition to T lymphocytes, anti-CD4 mono- clonal antibodies bind to the surface of human cells of the monocyte/macrophage lineage including circulating monocytes, tissue macrophages and Langerhans cells of the skin. Wood ej al. , J. Immunol. (1983) 131:212-216. The CD4 antigens on human T lymphocytes and monocytes are indistinguishable. Stewart e_t al.. , J. Immunol.

(1986) 13_6_: 3773-3778. Bank et al. (J. Exp. Med. (1985) 162:1294-1303) disclose that under some circum¬ stances the CD4 molecule may function as an independent transducer of negative signals inhibiting T cell acti- vation. Under some circumstances CD4 may play a role acting in concert with the T cell antigen receptor to facilitate antigen receptor dependent cellular activa¬ tion. Sleckman et al. , Nature (1987) 328:351-353; Rosoff et al.. Cell (1987) _4j):845-853; Rivas et al. , J. Immunol. (1988) 40:2912-2918; O'Neill et al. , Cell

(1987) _4^:143-151; Kupfer et al., Proc. Natl. Acad. Sci. USA (1987) 8_4: 5888-5892; Owens et al., Proc Natl. Acad. Sci. USA (1987) (3^:9209-9213.

HIV-1 has a selective tropism for CD4+ T cell lymphocytes. Klatzmann et al., Science (1984) 225:59- 63. CD4 tropism of SIV and HIV-2 has also been repor¬ ted. Kanki et al.. Science (1985) 231:951-954; Korn- feld et al. , Nature (1987) 326:610-613; Hirsch et al. , Cell (1987) 49:307-319; Guyader et al. , Nature (1987) 326: 662-669. The CD4 molecule on T lymphocytes be¬ haves as a receptor for the HIV-1 virus. Klatzmann e_t al., Nature (1985) 3_12_:767-768; Dalgleish et. al. , Na¬ ture (1984) 312:763-767. HIV-induced cell fusion, a characteristic manifestation of HIV-induced cytopathol- ogy, is dependent upon interactions between the viral envelope glycoprotein and CD4. Dalgleish e_t al. (1984) supra; Lifson e_t al. , Science (1986) 232:1123-1127;

Lifson et al. , Nature (1986) 3_23_:725-728; Sodroski et al., Nature (1986) 322:470-474.

When HIV-1 is bound to CD4+ T cells, detection of the CD4 antigen by a specific anti-CD4 antibody (OKT4a) is blocked. Although the CD4 antigen cannot be detected on the surface of HIV-1 infected cells, intra- cytoplasmic complexes of CD4 and the HIV envelope gly¬ coprotein (gpl20) have been demonstrated (Hoxie et al. , Science (1986) 227:1123-1127). Addition of monoclonal antibody to CD4 antigen during virus inoculation inhi¬ bits HIV-1 infectivity and replication. McDougal e_t al. , J. Immunol. (1985) 135:3151-3162; McDougal et al.. Science (1986) 2_31:382-385. Sera from HIV-1 infected subjects exhibit some capacity to inhibit (neutralize) HIV-1 infectivity _in vitro. McDougal e_t al. , J. Im¬ munol. Methods (1985) le_^'. lll ,' Weiss e_t al. , Nature (1985) 3_l :69-72; Robert-Guroff et al., Nature (1985) 316:72-74.

SUMMARY OF THE INVENTION

Methods and compositions are provided for modulating cellular responses induced in cells expressing the surface antigen CD4 as a result of interaction with a CD4-dependent retrovirus such as HIV. The compositions comprise derivatized peptides having substantially the same amino acid sequence as at least a portion of a consecutive amino acid sequence proximal to the N-terminus of the human CD4 antigen. Cellular responses which can be modulated include retrovirus-induced cell fusion and virion infectivity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a chromatographic fractionation of synthetic CD4(76-94). Bioactivity and mass spectra of fractionated UV-absorbing species were characterized by a standard HIV-induced cell fusion bioassay and FAB- mass spectrometry, respectively.

Figure 2 shows a chromatographic fractionation of synthetic CD4(83-94). Bioactivity and mass spectra of fractionated UV-absorbing species were characterized by a standard HIV-induced cell fusion bioassay and FAB- mass spectrometry, respectively.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Compositions comprising polypeptides, deriva¬ tives, fragments, or analogs thereof and formulations containing such compositions, as well as methods for their use are provided for inhibiting cellular respon¬ ses such as viral infection and virally-induced cyto- pathic effects which result from infection by CD4- dependent retroviruses such as HIV. The polypeptides are substituted derivatives of a naturally occurring polypeptide, CD4, and fragments thereof. The polypep¬ tides comprise at least a core sequence of seven amino acids including the cysteine at position 86 (C-86). The sulfur atom of C-86 may be substituted, and at least one amino acid in the polypeptide other than C-86 is derivatized.

The sequence of human CD4 is as follows (num¬ bering of residues is after the convention of Maddon e_t al., Cell (1985) 4_2:93-104). 10 20 30

Q G N K V V L G K K G D T V E L T C T A S Q K K S I Q F H W

40 50 60

K N S N Q I K I L G N Q G S F L T K G P S K L N D R A D S R

70 80 90

R S L T D Q G N F P L I I K N L K I E D S D T Y I C E V E D

100 110 120

Q K E E V Q L L V F G L T A N S D T H L L Q G Q S L T L T L

130 140 150

E S P P G S S P S V Q C R S P R G K N I Q G G K T L S V S Q

160 170 180

L E L Q D S G T W T C T V L Q N Q K K V E F K I D I V V L A

190 200 210

F Q K A S S I V Y K K E G E Q V E F S F P L A F T V E K L T

220 230 240

G S G E L W W Q A E R A S S S K S W I T F D L K N K E V S V

250 260 270

K R V T Q D P K L Q M G K K L P L H L T L P Q A L P Q Y A Q

280 290 300

S G N L T L A L E A-K T G K L H Q E V N L V V M R A T Q L Q

310 320 330

K N L T C E V T G P T S P K L M L S L K L E N K E A K V S K

340 350 360

R E K A V W V L N P E A G M T Q C L L S D S G Q V L L E S N 370 380 390

I K V L P T W S T P V Q P M A L I V L G G V A G L L L F I G

400 410 420

L G I F F C V R C R H R R R Q A E R M S Q I K R L L S E K K

430 435 T C Q C P H R F Q K T C S P I

The letters have the following meaning in accordance with convention: A = alanine; R = arginine; N = aspar- agine; D = aspartic acid; C = cysteine; Q = glutamine; E = glutamic acid; G = glycine; H = histidine; I = isoleucine; L = leucine; K = lysine; M = methionine; F = phenylaianine; P = proline; S = serine; T = threo- nine; W = tryptophan; Y = tyrosine; and V = valine. The sequence of the compositions of interest will usually be comparable to a sequence of a segment of the CD4 molecule, in particular a segment proximal to the N-terminus. The compositions will include a core sequence comprising substantially the sequence

T-Y-I-C-E-V-E, and may comprise the core sequence alone, or more usually will comprise at least one additional amino acid on at least one end of the core sequence. Compositions of particular interest include, those with the following sequences: T-Y-I-C-E-V-E-D-Q-K-E-E;

S-D-T-Y-I-C-E-V-E-D-Q-K-E-E; and L-K-I-E-D-S-D-T-Y-I-C- E-V-E-D-Q-K-E-E. The sequence of the composition 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 se¬ quence. The peptide sequences may be modified by terminal amino acylation, for example, acetylation; carboxy amidation, for example, with ammonia, methyl- a ine, and the like, to increase potency, decrease toxicity, increase stability, to improve the pharma- cologic characteristics, 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 4 conser- vative or non-conservative substitutions including de¬ letions and insertions usually involving not more than about 1 amino acid where the modification may include D-amino acids, without significantly affecting the activity of the product. Therefore, the subject poly- peptides may be subject to various changes, such as insertions, deletions, and substitutions, either con¬ servative or non-conservative, where such changes might provide for certain advantages in their use. By con¬ servative substitutions is intended combinations such as G, A; V, I, L; D, E; N, Q; S, T; K, R; and F, Y, W. Usually, the sequence of the composition will not dif¬ fer by more than 30% from the sequence of a segment of the CD4 molecule except where additional amino acids may be added at either terminus for the purpose of pro¬ viding an "arm" by which the peptides of this invention may be conveniently linked for immobilization for exam- pie to a carrier protein for use as an immunogen or to affinity groups, labels, etc. The arms will usually be at least about 5 amino acids and may be 50 or more amino acids.

The derivatization of the subject compositions will include derivatization of at least one, more usu¬ ally at least two heteroatoms, other than the peptide bond. By heteroatom is intended any atom including carbon; including atoms located in ring structures and non-ring structures; and including atoms in organic and non-organic compounds. In compositions in which at least two heteroatoms are derivatized, generally one of the heteroatoms is the sulfur of the cysteine at posi¬ tion 86 of the CD4 molecule (C-86). The remaining substitutions) may be on a heteroatom of any other amino acid in the composition, preferably a glutamic acid, more preferably the glutamic acid at position 87 of the CD4 molecule (E-87). The heteroatom to which the substituent is bound will depend in part upon the chemical structure of the amino acid derivatized, and may include any one of sulfur, oxygen, nitrogen and carbon. In compositions in which a single heteroatom is derivatized, the derivatizing group is on a hetero¬ atom of an amino acid other than C-86, usually a glutamic acid, more usually E-87. For i vivo use, the derivatizing groups should provide a physiologically acceptable product. Generally the derivatizing groups will have from about 1 to about 36 carbon atoms and may be aliphatic, ali¬ cyclic, aromatic, heterocyclic or combinations thereof. Usually, the derivatizing group will have from 0 to 10 heteroatoms, which may be in the longest chain, as a substituent on a chain or ring atom or the like. For the most part the heteroatoms of the derivatizing group are selected from halogen, nitrogen, oxygen or sulfur. The bulk of the derivatizing group is preferably less than that of a naphthyl group and greater than that of a linear lower alkanoic acid, most preferably approxi¬ mately the size of a phenyl group or similar cyclic or heterocyclic group (either aromatic or non-aromatic). Thus the derivatized peptides may have a 3-dimensional conformation, as determined by X-ray crystallography substantially similar to that of native CD4 bound to the CD4 binding domain on gpl20.

The group optionally may be further substitu¬ ted. Binding of the derivatizing group to the desired amino acid in the sequence of the composition will depend in part on the chemical structure of the deriva¬ tizing group and that of the amino acid. Thus the de¬ rivatizing group may be bound to the amino acid via a carbon atom, sulfur atom, nitrogen atom or oxygen atom on the derivatizing group. Various groups may be used as derivatizing groups, for example, an aryl-containing substituent or in the case of cysteine, a thioether resulting from the reaction between the thio group of the cysteine and a maleimide. The aryl group is preferably selected from 5- and 6-membered aromatic rings containing carbon and 0 to 1 oxygen or sulfur and 0 to 3 nitrogen atoms in the ring. Phenyl is a preferred aryl group, e.g., benzyl and naphthyl. The aryl-containing group may be substituted or unsubstituted. Substituents may include alkyl, particularly methyl, halogen, particularly chloro, nitro, hydroxyl, etc., where the substituents may be in any position, preferably at the ortho or para position. The aryl group may have from 0 to 3 substi¬ tuents, usually not more than 2 substituents, which substituents may be the same or different. For active olefins used as derivatizing groups, the olefin will usually be conjugated with a second site of unsaturation, e.g., a carbonyl group; acyclic groups; maleimido groups; conjugated polyole- fins; or the like may also find use.

Of interest will be to have a functionality present on the blocking group which allows for linking to another molecule, e.g., carboxy, carboxy ester, or the like. The carboxy may then be activated with a carbodiimide, carbonyl diimidazole, or the like for re¬ action 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 or other amino acid residues where derivatization occurs for bioactivity may be one of the following groups:

(a) alkyl and substituted alkyl compounds:

X-CH₂(CH₂)_n- where n = 0-20 and X is selected from H; OH; OCH₃; SH; SCH₃; NH₂; NHCH3; N(CH₃)₂; SO3H; S0₂CH₃; or halogen, being hetero only when n is other than 0.

(b) cycloalkyl and substituted cycloalkyl compounds:

CH₂-CH-X

(CH₂)_n where n = 1-10 and a hydrogen on any of the ring carbons is replaced by X as described in (a) above.

(c) aromatic or substituted aromatic compounds:

"(CH₂)_n-

X where n = 1-5 and X is as described in (a) above. (d) polyaromatic or substituted aromatic compounds:

(CH₂)_n-

X where n = 1-3 and X is as described in (a) above.

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

⁽i) -^(CH2>n- N

R where n = 0-3 and R is a pair of electrons; H; alkyl of

1-2 carbon atoms; or O.

^(U) " -<CH₂,_n-

N R where n = 0-3 and R is selected from C_gH₅; CH₃; or H.

(iii) OH

N R where R is a pair of electrons; H; or O.

(f) maleimide adducts such as m-maleimidoben- zoate, N-hydroxysuccinimide ester; m-maleimido-benzoyl- sulfosuccinimide ester; N-succinimidyl 4-(p-maleimido- phenyl)butyrate; N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; or sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate; bis-male- imidohexane; bis-maleimidomethyl ether; or N-male- imidobutyryloxysuccinimide.

(g) thio-containing compounds such as:

-S-

X where X is N₃; OH; OR; NH₂; NHR; N0₂; SH; SR; halogen; C0 H; or aryl of from 6 to 12 carbon atoms; and

R-S-

where R is alkyl or substituted alkyl.

(h) amino acids or oligopeptides,

(i) cytotoxic agents such as alkylating agents, for example pipobroman; thio-TEPA; chlorambucil; cyclo- phosphamide; nitrogen mustard; mephalan; or uracil mustard.

(j) membrane-perturbating agents, for example adriamycin; ionophores, such as valinomycin; or surface active agents, such as detergents.

(k) anti-retroviral agents such as 5-azido- thymidine (AZT); dideoxycytidine (DDC); dideoxyadeno- sine (DDA) ; or dideoxyinosine (DDI).

In some instances, particular derivatives may be found to be cytotoxic, which derivatives will usual¬ ly be modified to reduce cytotoxicity or substantially eliminate toxicity at pharmacologically active levels. The subject peptides may be employed for some applications linked to a soluble macromolecular carrier. Conveniently, the carrier may be a polypeptide, either naturally occurring or synthetic, antibodies to which 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, or the like. The choice is primarily one of convenience and availability. The manner of linking is conventional, employing such reagents as p_-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyde, etc. The linkage may occur at the

N-terminus, C-terminus, or at a site intermediate to the ends of. the molecule. The subject peptide may be derivatized for linking, may be linked while bound to a support, or the like. Any convenient techniques may be used for preparing the conjugates, including recombin- ant DNA methods and conventional chemical coupling.

Preparation of CD4 derivatives

The peptides can be prepared in a wide variety of ways. Methods for their preparation include the following. Because of their relatively short size, the peptides may be synthesized in solution or on a solid support. Additionally they may be prepared using recombinant DNA techniques. For solid phase synthesis, various automated synthesizers are commercially available and can be used in accordance with known protocols. See for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, 1984; and Tam et. al., J_^ Am. Chem. Soc. (1983) 105:6442. Various side-chain protecting groups can be used to block potentially reactive sites in the precursor amino acids. Choice of specific protecting groups will depend upon anticipated cleavage conditions and the desired product. When using a fluorenylmethoxycarbonyl (F-moc) method for preparation of the compositions, side-chain protecting groups may be as follows: t-butyl (D, E, S, T, Y); t-Boc (K); trityl (H) ; paratoluene sulfonyl (R) ; benzyl (C) . In many cases, protecting groups such as benzyl, chlorobenzyl, substituted benzyl, benzyla ide, chlorobenzamide can be employed for E, C, T and Y. When using a tertialbutyloxycarbonyl (t-Boc) procedure, side-chain protection used during synthesis can include Tos (R,H) , O-benzyl (D, E) , benzyl (S, T, in some cases C), Br-Z (Y), Cl-Z (K), or 4-MeO-benzyl (C) . Z repre¬ sents a benzyloxycarbonyl group. Choice of a method for removal of side-chain protecting groups and cleavage of synthetic peptides from the solid phase support resin will depend upon the nature of the protecting groups as well as the nature of the desired product. When using the F-moc method, harsh conditions which would remove the desired aryl- alkyl substituents on the peptide should be avoided. Generally, 50-80% trifluoroacetic acid (TFA) in di¬ chloromethane (DCM) is used, preferably 70%. Other organic acids such as 70% trifluoromethane sulfonic acid (TFMSA) , and hydrobromide (HBr) in TFA and a mix¬ ture of TFMSA, TFA and DCM may also find use. The TFA may additionally contain scavengers such as anisole, thioanisole, dimethylsulfide, and detergents such as sodium dodecyl sulfate (SDS). Following cleavage and deprotection, the resin and peptide are rinsed with ethyl ether and the peptide separated from the resin by dissolution in ammonium carbonate, and filtration. The resulting post-resin mixture is then lyophilized. When using a t-Boc method, the side-chain protecting groups of the synthetic peptide are depro- tected and the peptide cleaved from the resin simul¬ taneously by addition of, for example, hydrofluoric acid (HF) usually from about -20°C to 0°C which may contain scavengers such as anisole or thioanisole, p- thiocresol, and dimethylsulfide. After removal of HF and other volatile components under vacuum, the resin and the peptide are rinsed with an organic solvent such . ,

as ethyl ether and the peptide separated from the resin by dissolution in a carbonate containing buffer, pre¬ ferably ammonium carbonate or DMF and H₂0 (2:1 v/v) . Alternatively, the ethyl ether step can be omitted, and any residual scavengers, such as anisole and dimethyl sulfide, removed by chromatography on Sephadex G-10 or G-25.

Peptide compositions having the ability to inhibit HIV-induced cell fusion ("antisyncytial activ- ity") prepared by either the F-moc or t-Boc method are separated from inactive peptides in the post-resin mix¬ ture by conventional means, including high pressure liquid chromotography (HPLC) .

Peptides having antisyncytial activity also can be isolated from the post-resin mixture obtained from solid phase synthesis using the t-Boc method, in which C-86 in the core sequence is replaced with S-ben- zyl Cys by substitution of t-Boc-S-benzyl-cysteine as the precursor amino acid for t-Boc-S-para-methylbenzyl cysteine.

Compositions having antisyncytial activity can also be prepared using as a starting material a peptide comprising a purified, underivatized region of CD4 con¬ taining the core sequence CD4(83-89) or a purified de- rivative in which C-86 of the core sequence is derivatized particularly with an arylalkyl substituent having from 7-12 carbon atoms and from 0-2 heteroatoms such as a benzyl or chlorobenzyl group, or other substituted benzyl groups. The source of the starting materials, both underivatized and derivatized, may be the post- resin mixture obtained from solid phase synthesis of the underivatized sequence, from which the peptides are isolated by for example HPLC.

Depending upon the nature and location of the desired substitutions, derivatized starting materials may additionally be prepared by reaction of purified underivatized or derivatized peptide under mild condi- tions with reagents known to react with nucleophilic groups such as mercaptans, active halides, pseudo- halides, active olefins, e.g., α,ρ-enones, such as maleimide, disulfides, or the like. Peptides prepared from these starting materials will generally comprise at least two derivatizing groups, where one derivatiz¬ ing group is attached to C-86.

Derivatized peptides comprising at least two derivatizing groups can also be synthesized in solid phase using the F-moc procedure and acid-resistant side chain protecting groups. For example, a peptide com¬ prising benzyl substitutions on C-86 and E-87 can be synthesized by the F-moc method using N-F-moc-S-benzyl- L-cysteine and N-F-moc-L-glutamic acid-α-benzyl ester as a precursor. The remainder of the precursor amino acids and the procedure of cleavage, extractions and purification of peptides are similar to those described.

Alternatively, hybrid DNA technology may be employed for preparation of the subject compositions where a synthetic gene may be prepared by employing single strands which code for the polypeptide or sub¬ stantially 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 expression vectors, which are readily available. See for example, Maniatis et al. , Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. Or, the region of the genome coding for the peptide may be cloned by conventional recombin- ant DNA techniques and expressed (see Maniatis, supra) . DNA coding sequences based upon the known se¬ quence for CD4 may be used. Fragments from these se- quences may be employed for expression of peptide frag¬ ments, conservative base changes can be made, where the modified codon(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 conserva¬ tive or non-conservative change.

The coding sequence may be extended at either the 5'- or 3'-terminus or both termini to extend the peptide, while retaining the region of interest. The extension may provide for an arm for linking, for pro¬ viding antigenic activity, or the like.

For expression, the coding sequence will be provided with start and stop codons, promoter and ter¬ minator regions and usually a replication system to provide an expression vector for expression in a cellu¬ lar host, for example prokaryotic or eukaryotic, bac¬ teria, yeast, mammal, or the like. Once the peptide has been expressed by recom- binant DNA methods, and purified to a suitable degree, the thio group may be blocked with any convenient re¬ agent which replaces the hydrogen of the mercaptan of the cysteine. As indicated for thioethers, thioesters-, thioamides, active halogens, active pseudohalogens, or active olefins may find use. The reaction temperature will generally be mild, 0-50°C, usually 10-30°C, with reaction time ranging from about 0.5 to 24 hours. Polar solvents, particularly aqueous solvents, may be employed, where organic solvents may be present up to about 60 volume %. Organic solvents include aceto- nitrile, acetone, diethyl ether, dimethylformamide, dichloromethane etc. With active halides, a mild basic acid acceptor will be present, conveniently, carbonate, bicarbonate, or the like. Usually an excess of the blocking agent will be employed.

To block with a thio group, various disulfides may be employed, such as methyldithio, p-nitrophenyldi- thio, 2-pyridyldithio, etc., where the other sulfur may be joined to a methylcarboxy ester, aryl or other con¬ venient group. The conditions for displacement are well known and need not be described here. After the reaction is complete, the product may be isolated and purified according to conventional techniques.

Use of CD4 Derivatives

The subject compounds and compositions have a number of uses and may be used in vitro and in vivo. In vitro, the subject compounds and compositions may be employed for detecting the role of CD4 in viral infec- tion, preventing infection of CD4-bearing cells includ¬ ing T cells and macrophages susceptible to HIV, inhi¬ biting CD4-dependent viral cytopathic effects and the like. In vivo, the subject compounds and compositions may be used prophylactically or therapeutically for preventing infection or inhibiting propagation of CD4- dependent retroviruses such as HIV and infection of or cytopathic effects on additional T cells or other CD4- bearing cells by inhibiting HIV envelope glycoprot-ein- CEt4 interactions related to clinical manifestation of viral disease. The term "cells" is intended to include a plurality of cells as well as single cells. The cells isolated include isolated cells and cells which form a part of a larger organization of cells such as an organ, and may be _in vivo or in vitro. The com- pounds additionally may be used, for example, during pregnancy or at the time of delivery to prevent maternal-fetal transmission of CD4-dependent retroviruses.

The subject compounds and compositions will generally be administered so as to enter the blood stream, being administered parenterally, for example intramuscularly, intraperitoneally, intravenously, in- tranasally, topically, or the like. Any physiological¬ ly acceptable medium may be employed, such as deionized water, saline, phosphate buffered saline, aqueous eth¬ anol, and the like. The concentration of the active ingredient of the subject composition will vary, depen- i i / K . ,' i ^{■ ■ ■}

19 ding upon the solubility, use, frequency of administra¬ tion, and the like. The amount used will depend upon a number of factors, including the route of administra¬ tion of the composition, the number and frequency of treatments, the formula weight of the active component of the composition, and the relative biological activ¬ ity (for example, anti-syncytial activity or anti- infectivity activity) of the formulation employed. Where the biologically active component has a formula weight of about 1600, the amount of the biologically active component of the composition administered per treatment to a 70 kg man generally will be not more than about 1 g, more usually in the range of about 200 mg to about 500 mg, and may be in the range of about 10 mg to about 100 mg for compounds having a high relative biological activity, for example a composition which is capable of blocking HIV-induced cell fusion in vitro at a concentration of about 1 uM to about 10 μM. The subject compositions may also find use as competitive inhibitors of binding interactions between HIV envelope glycoproteins and CD4, both _in vivo and in vitro. There is a CD4 binding domain on the envelope glycoprotein (gpl20) of HIV which appears to be a con¬ served region. The compositions of the subject appli- cation may act by interferring with binding between CD4 and gpl20. Thus the subject derivatized peptides may also find use in binding studies with purified gpl20 followed by cross-linking and biochemical analysis to localize the CD4 binding domain on the gpl20. The subject compositions may also find use in the design and development of vaccines to protect against HIV infection, for example knowledge of the se¬ quence of the CD4 binding domain on gpl20 may be used to design a vaccine to protect against HIV infection. The gpl20 or a fragment thereof comprising at least the binding domain can be used in a suitable diluent as an immunogen, or if not immunogenic per se, can be bound to a carrier to make the protein immunogenic. Carriers include bovine serum albummin, keyhole limpet hemocya¬ nin and the like. Suitable diluents are water, saline, buffered salines, complete or incomplete adjuvants and the like. The immunogen is administered using standard techniques for antibody induction.

The binding domain for CD4, which is apparent¬ ly involved in interactions between CD4 and gpl20 cru¬ cial for HIV infectivity and cytopathic consequences of infection, does not appear to be involved in at least one CD4-dependent immune function, namely the mixed leukocyte reaction. The binding domain on CD4 which interacts with HIV appears to be distinct from the MHC site. Thus immunization with this binding domain on CD4 may protect against HIV without compromising the physiologic immunoregulatory functions of CD4.

In addition to use as a vaccine, the composi¬ tions can be used to prepare antibodies to the binding domains on CD4 and gpl20 involved in the interaction ^" between CD4-bearing cells and HIV. The antibodies can be used directly as antiviral agents. To prepare anti¬ bodies, a host animal is immunized using the binding domain itself, or, as appropriate, bound to a carrier as described above. The host serum or plasma is col- lected following an appropriate time interval to pro¬ vide a composition comprising antibodies reactive with the relevant domain. The gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. To enhance the specificity of the anti-binding domain antibody composition, the composition can be purified by adsorbing the preparation with gpl20 fragments or CD4 (as appropriate) lacking the binding domain of interest. The antibodies are substantially free of many of the adverse side effects which may be asso¬ ciated with other anti-viral agents such as drugs. ^■

21

The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of anti-binding domain antibodies from human/human hybridomas (see below).

Anti-binding domain antibodies can also be used as a means of enhancing the immune response since antibody-virus complexes are recognized by macrophages. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is admin- istered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis to interfere with viral entry into cells. Thus, antibodies reactive with the CD4/ gpl20 interaction domains can be passively administered alone or in conjuction with another anti-viral agent to a host infected with a CD4-dependent retrovirus to en¬ hance the immune response and/or the effectiveness of the antiviral drug.

Alternatively, anti-binding domain antibodies can be induced by administering anti-idiotype anti¬ bodies as immunogens. Conveniently, a purified anti- binding domain antibody preparation prepared as de- scibed above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies pro¬ duced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-binding domain antibodies, or by affinity chromatography using anti-binding domain antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are specific for the region of gpl20 implicated in inter¬ actions with CD4.

When used as a means of inducing anti-binding domain antibodies in a patient, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutane- ously or the like in an effective concentration in a physiologically suitable diluent with or without adju¬ vant. One or more booster injections may be desirable. The induction of anti-binding domain antibodies can alleviate problems which may be caused by passive ad¬ ministration of anti-binding domain antibodies, such as an adverse immune response, and those associated with administration of blood products, such as infection. In addition to being useful as anti-vira^'l agents, antibodies to proteins comprising the binding domain and anti-idiotype antibodies may also find use in diagnostics.

For both _in vivo use and in diagnostics it may be preferable to use monoclonal antibodies. Monoclonal anti-binding domain antibodies or anti-idiotype anti¬ bodies can be produced as follows. The spleen or lym¬ phocytes from an immunized animal are removed and im¬ mortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human- human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a CD4-dependent retro- virus (where infection has been shown for example by the presence of viral antibodies in the blood or virus culture) may serve as a suitable lymphocyte donor. Lymphocytes may be isolated from a peripheral blood sample or spleen cells may be used if the donor is sub- ,

23 ject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary _in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies. Antibodies secreted by the immortalized cells are screened to determine the clones that secrete anti¬ bodies of the desired specificity. For monoclonal anti-binding domain antibodies, the antibodies must bind to gpl20 so as to inhibit binding of gpl20 to CD4. For monoclonal anti-idiotype antibodies, the antibodies must bind to the binding site region of anti-binding domain antibodies. Cells producing antibodies of the desired specificity are selected. The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Table of Contents

Example 1: Automated Synthesis and Bioassay of Synthetic CD4(76-94) Peptides and Fragments Thereof

A. Synthesis by t-Boc Procedure

B. Bioassay of Synthetic Peptides

(a) Effect on HIV-induced, CD4-dependent Cell Fusion

(b) Effect on Mixed Leukocyte Reaction

(c) Effect on Viral Infectivity

C. Isolation and Identification of Ac¬ tive Component of Post-Resin Mixture

(a) Fractionation and Identification of Bioactive Fractions

(i) CD4(76-94) (ii) CD4(83-94)

(b) Analysis of Bioactive Peak by FAB-MS

(i) CD4(76-94) (ii) CD4(83-94)

(c) Effect of Bioactive Fraction on Viral Infectivity of Heterogeneous HIV Isolates

Example 2: Post-synthesis Derivatization and Bioassay of Analogs of Parent CD4(76-94)

A. Preparation of Derivatives

B. Effect on Cell Fusion

Example 3 Comparison of Bioactivity of S-benzylated Analogs of CD4(76-94) and Fragments Thereof Prepared by Solid Phase Synthesis and by Post-synthesis Modification

A. Solid Phase Synthesis of S-benzylated Analogs by t-Boc Procedure

(a) Synthesis

B. Solid Phase Synthesis by F-moc Procedure

(a) Synthesis Table of Contents

(Cont'd)

Example 3: C. Post-synthesis Modification of (Cont'd) CD4(76-94) Analogs, Derivatives and Fragments

(a) Post-synthesis Benzylation of CD4(76-94)

(b) Post-synthesis Benzylation of S-benzyl CD4(83-94)

(c) Post-synthesis Benzylation of S-benzyl CD4(83-94)

D. Comparison of Effect on Cell Fusion of S-benzylated Analogs

(a) CD4(76-94)

(b) CD4(83-94)

^■E. Analysis of Analogs by FAB-MS

Example 1 Automated Synthesis and Bioassay of Synthetic CD4(76-94) Peptides and Fragments Thereof

A. Synthesis by t-Boc Procedure

A series of substituted peptides derived from the sequence of the extracellular portion of the CD4 molecule proximal to the N-terminus were synthesized using automated solid phase peptide synthesis methodol- ogy (Applied Biosystems, Inc., Foster City, CA, Model 430A Peptide Synthesizer) according to the principles of Merrifield (G. Barany and R.B. Merrifield, in The Peptides: Analysis, Synthesis, Biology, Vol. 5, ed. by E. Grass and J. Meinhofer, Academic Press, New York, pp. 1-284, 1979). Side-chain protection in the t-Boc method was: Tos (R, H) , O-benzyl (D, E) , benzyl (S, T, in some cases C) , Br-Z (Y) , Cl-Z (K) , or 4-MeO-benzyl (C) .

Side-chain deprotection-of the peptide at the end of the synthesis and cleavage of the peptide from the resin were accomplished by addition of 20 ml HF containing 1 ml anisole or thioanisole, p-thiocresol, and 200 μl of dimethylsulfide to resin containing about 300 mg of peptide, and reaction at 0°C with stirring for 60 min. The material obtained following cleavage of the peptide for the resin (post-resin mixture) , comprises about 65-95% inactive underivatized peptides as well as other peptide species having antisyncytial activity.

After removal of HF and other volatile compo- nents under vacuum, the resin and peptide were rinsed with ethyl ether and the peptide separated from the resin by dissolution in 0.1 M ammonium carbonate, and filtration on a frittered filter disk, followed by lyophilization. Alternatively, the ethyl ether step was omitted, and any residual scavengers, such as anisole and dimethylsulfide, were removed by Sephadex G-10 chromatography. The lyophilized post-resin mix- l i

27 ture was dissolved in 10% acetic acid or ammonium acetate and chromatographed on G-10 Sephadex or G-25

Sephadex, respectively, to remove low-molecular weight species. Where indicated, the preparations were fur- ther purified by HPLC. The post-resin mixtures were heterogeneous as assessed by HPLC.

B. Bioassay of Synthetic Peptides (a) Effect on HIV-induced, CD4-dependent Cell Fusion

The ability of the compositions synthesized as described above to block CD4-dependent, HIV-envelope induced cell fusion was assessed as follows. The cell fusion assay was performed essentially as described in Lifson et al. , Nature (1986) 3_23_:725-728. Briefly

5xl0^{4 H}9«^HIV_HXB_₂ cells were preincubated with varying concentrations of the heterogeneous peptide composition, obtained as described in Example 1, in the wells of flat-bottomed 96-well microtiter plates for 30 minutes at 37°C. 5xl0⁴ CD4+ VB cells were then added. The total volume was 100 μl. After culture at 37°C for 2-24 hours, syncytia (defined as at least four nuclei within a common cell membrane) were scored by inverted phase contrast microscope (xlOO) as described (Lifson et. al., supra) . The results are shown in Table 1, where titer indicates the nominal peptide concentration required for complete blockade of HIV-envelope induced CD4-dependent cell fusion after an overnight incubation in a standard assay. Nominal peptide molarities are calculated based on the formula weight for the dominant synthetic product, namely the peptide having the indi¬ cated sequence but in which only C-86 is derivatized. Table 1

Inhibition of HIV Induced Cell Fusion by

CD4 Derived Synthetic Peptide Compositions

Peptide Sequence^1,3 Titer²

LKIEDSDTYIC_bzlEVEDQKEE⁴ 60 μM

KIEDSDTYIC_b2τ_EVEDQKEE 125 μM

EDSDTYIC_bzlEVEDQKEE⁴ 125 μM

SDTYIC_bzχEVEDQKEE 125 μM

TYIC_bzχEVEDQKEE⁴ 125 μM

YIC_bzlEVEDQKEE Not Active

LKIEDSDTYIC_bzlEVEDQKE 125 μM

TYIC_bzlEVEDQKE 125 μM

TYIC_bzlEVEDQK 250 μM

TYIC_bzlEVEDQ 250 μM

TYIC_bzlEVE 500 μM

^YICbzl^EVE Not Active

1 Preparations tested were post-resin peptide mixtures prepared by the t-Boc method, and were not homogene¬ ous by HPLC.

2 Not active indicates no anti-syncytial activity at the highest concentration tested, 500 μM.

3 Designation C_bzτ_ indicates synthesis conducted using as a side protection group benzyl rather than para- methyl benzyl. The sequence shown is the nominal peptide sequence expected for the dominant synthetic product.

4 FAB-MS analysis of HPLC fractionated post resin mix¬ tures demonstrated that the indicated mixtures com¬ prised peptides in which C-86 and at least one other amino acid residue were derivatized.

Purified nonderivatized CD4(76-94), which has the se¬ quence LKIEDSDTYICEVEDQKEE, was inactive (at <500 μM, the highest concentration tested) as measured by abil- ity to inhibit HIV-induced cell fusion, showing that derivatization is essential for antisyncytial activity. The purity of the nonderivatized peptide was confirmed by FAB-MS and sequencing. The post-resin mixture from synthesis of a peptide in which the amino terminal 7 residues of CD4(76-94) were deleted had a slightly reduced ability to block HIV-induced cell fusion. The post-resin mix- ture from synthesis of a derivatized peptide in which the amino terminal 8 residues of CD4(76-94) were dele¬ ted had no fusion inhibiting activity at <500 μM. The post-resin mixture from synthesis of a peptide in which the carboxy terminal 6 amino acids were deleted also had no fusion inhibiting activity at <500 μM. These results demonstrated a minimum requirement of a core sequence of amino acids T-Y-I-C-E-V-E, at least one of the amino acids in the composition being derivatized.

The post-resin mixture from synthesis of a de- rivatized peptide in which the carboxy terminal 6 amino acids of CD4(76-94) were deleted from the carboxy ter¬ minal end of the peptide then added back to the amino terminal end of the molecule in other than the order fourid in native CD4, had no activity, further demon- strating the requirement for sequence specificity of the peptide to obtain inhibition of HIV-induced cell fusion. Furthermore, the post-resin mixtures from syn¬ thesis of CD4(76-94) in which C-86 was replaced with alanine, serine or phenylalanine were not active; like- wise, post-resin mixtures from synthesis of CD4(83-94) in which C-86 was replaced with alanine, methionine or serine were not active.

The ability of the post-resin mixture from synthesis of CD4(83-94) (t-Boc synthesis, using S- benzyl protected cysteine) to inhibit HIV-induced cell fusion using both transformed T-cells and peripheral blood mononuclear cells as the CD4-expressing component in the fusion assay was also evaluated. Cells were pre-incubated for 30 min at 37°C with H9-HIV (HXB2) post-resin peptide mixtures of the nominal concentra¬ tions shown. Either VB cells or peripheral blood mono¬ nuclear cells (PBMCs) stimulated with phytohemaggluti- nin (1 μg/ml, Burroughs, Beckenham, England) were then added to the H9-HIV (HXB2) cells and peptide for 48 hrs, and cultured at 37°C for 24 hrs, at which time syncytia were scored. The results shown in Table 2 indicate that the peptide blocked CD4 dependent fusion of both primary and transformed CD4-expressiήg cells.

Table 2 Comparison of CD4(83-94) Post Resin Mixture to Inhibit HIV-induced Cell Fusion of

VB Indicator Compared to PHA Activated, Fresh Human Peripheral Blood Mononuclear Cells

Dose PHA PBMCs VB

500 μM -

250 μM -

125 μM - 4

63 μM - 4

32 μM 3 4

1 166 μμMM 4 4

8 μM 4 4

To assess the ability of peptides to block cell fusion induced by additional HIV isolates and SIV, H9 cells (50,000) infected with the viral isolates HIV- l_Tj, HIV-1_DV, HIV-l_HχB2 or ^SIVr_jCDavjL_S were preincubated for 1 hr at 37°C with CD4(83-94)BZL in 96-well icro- titer plates. Levels of viral expression in each in¬ fected cell line were sufficient to allow formation of syncytia upon co-culture with 50,000 VB cells (HIV-1 infected cells) or 50,000 HUT-78 cells (SIV-infected cells) in a volume of 50 μl 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 as previously reported. Results shown are for 24 hrs after initiation of co- culture: -, no visible syncytia or pre-syncytial ag¬ gregates observed in duplicate wells; 4, maximum number of syncytia, comparable to number observed in the ab- sence of treatment with peptide. As shown in Table 3 below, the post-resin mixture from synthesis of CD4(83- 94) (t-Boc synthesis, S-benzyl protected cysteine) also completely inhibited fusion induced by SIV and two additional HIV isolates at nominal peptide concentra- tions for the dominant synthetic product of between

63 and 250 μM, but did not show any inhibition of cell fusion induced by HTLV-I,. a non-CD4-dependent human retrovirus (not shown).

Table 3

Ability of Post-Resin Mixture of CD4(83-94) to Inhibit Fusion Induced by Several Isolates of HIV and by SIV

Dose HIV-TJ HIV-DV HIV-HXB2 SIV(UC Davis)¹

125 μM — — —

63 μM 4 4 -

32 μM 4 4 4 4

16 μM 4 4 4 4

1 Obtained from Dr. Preston Marx, California Regional Primate Center, Davis, California.

(b) Effect on Mixed Leukocyte Reaction

Post-resin mixtures having antisyncytial ac¬ tivity were analysed to determine if they also affected normal functions associated with the CD4 antigen using a mixed lymphocyte reaction assay. Peripheral blood mononuclear cells were isolated by density centrifuga- tion over Ficoll-Hypaque from heparinized whole blood obtained from healthy volunteer donors at the Stanford University Blood Bank. Cells were suspended in RPMI- 1640 supplemented with 10% (v/v) heat-inactivated human male AB serum 1-glutamine and antibiotics. One-way mixed leukocyte cultures were performed in triplicate in round bottom 96-well microtiter plates, using 5x10 responder cells and an equal number of irradiated (3000R) stimulator cells/well in a total volume of 200 μl. Assays were performed using post-resin mixtures at a nominal concentration (for the dominant synthetic pro- duct) of 125 μM. Cells were cultured for 6 days at 37°C in humidified 5% C0₂ in air, then pulsed with [³H]-thymidine (1 μCi/well) and harvested on glass fiber filters on day 7. Labeled thymidine incorporated into DNA was then measured by scintillation counting as an index of alloantigen-induced cellular proliferation. None of the post-resin peptide mixtures tested, including a post-resin mixture from the synthesis of CD4(76-94) which completely blocked HIV-induced cell fusion, had significant inhibitory effects on the mixed leukocyte reaction.

(c) Effect on Viral Infectivity

The ability of the post-resin peptide mixtures to inhibit infectivity of cell-free HIV virions using the assay of Nara e_t aJL. , AIDS Research and Human Retro¬ viruses (1987) 3_:283, was also assessed. One hundred to two hundred syncytial-forming units of HTLV-IIIB or HIV-2 (pre-titered frozen stock from infected H9 cells) in phosphate-buffered saline were preincubated for 60 min at 25°C with the indicated concentrations of post- resin peptide mixture or HPLC fractionated post-resin mixture. The virus-peptide mixture, or virus alone was then incubated with CEM-SS cells for 60 min at 37°C to allow viral adsorption to the cells. Medium was re- moved and replaced with RPMI 1640/10% fetal calf serum with or without additional peptide to maintain the de¬ sired concentration. Cells were re-fed with fresh [ : I " 33 ^f medium on day three, and syncytia counted when cell confluency was reached 5-6 days later. These data are shown in Table 4.

Table 4

Inhibition of HIV- -1 and HIV-2 Infection of

CEM- -ss Cells by CD4(76- ^•94) Post-resin Peptide Mixture

Peptide Concentration¹ Vn/Vo² HTLV -IIIB Vn/Vo HIV-2

10 250 μM 0.003 0.10

125 μM 0.003 0.34

63 μM 0.003 0.60

32 μM 0.003 0.71

16 μM 0.15 0.79

15 8 μM 0.33 0.80

4 μM 0.47 0.95

2 μM 0.78 >1.00

1 Peptide was present at the indicated concentration

20 during both the viral inoculation phase of the assay and the subsequent culture period up to scoring of syncytia.

2 (Vn/Vo): the number of syncytia/well at confluence (5-6 days after viral infection) in the presence of peptide (Vn) divided by the number of syncytia/well at confluence in the absence of peptide (Vo).

25

To confirm that the post-resin peptide CD4(76- 94) mixture inhibited infection of CEM-SS cells as well as the formation of syncytia, HIV-1 viral antigen p24

30 was measured (as described by Arthur ejt al. , Proc. Natl. Acad. Sci. USA (1987) 4:8583) in culture super- natants on day 6 post-infection, at the time cells were observed and quantitated for syncytia. Duplicate cul¬ tures inoculated with HTLV-IIIB in the absence of virus

35 contained an average concentration of p24 of 68 ng/ml. Duplicate cultures inoculated with HTLV-IIIB in the presence of 500 μM CD4(76-94) peptide mixture (without further exposure to peptide) contained an average con¬ centration of p24 of 4.6 ng/ml when the supernatents were analyzed 6 days following inoculation.

C. Isolation and Identification of

Active Component of Post-Resin Mixture (a) Fractionation and Identification of Bioactive Fractions (i) CD4(76-94) ♦ A representative chromatogram of 1.8 mg of CD4(76-94) (t-Boc synthesis; S-methyl- benzyl protected cysteine) on a Vydac C8 (10x250 mm) bonded-phase semi-preparative column is shown in Fig¬ ure 1. Material was post-resin CD4(76-94) dissolved in 10 mM ammonium acetate at pH 7.0. Mobile phase was

(A) ammonium acetate buffer and (B) 20% ammonium ace¬ tate buffer/80% acetonitrile. The percentage of B in the mobile phase was varied as shown (dashed line). Material eluting at retention times 2 to 3, 3 to 4.5, 4.5 to 5, and 5 to 8.5 minutes was pooled from several semi-preparative runs, lyophilized, weighed and submit¬ ted to bioassay at nominal concentrations of 500 to 30 μM, in the fusion assay described above. Bioactiv¬ ity (hatched bar) is expressed as doses of antisyn- cytial activity/f action. (One dose is the smallest amount of material necessary to completely inhibit fusion between 50,000 HTLV-IIIB/H9 cells and 50,000 VB indicator cells over a 24-hr period under standard assay conditions.) (ii) CD4(83-94) . Ten mg of CD4(83-94) dis¬ solved in 10 mM ammonium acetate at pH 7.0 and prepared using the t-Boc method using S-benzyl protected cysteine was chromatographed on a Vydac C8 (10x250 mm) bonded- phase semi-preparative column. A representative chro- matogram is shown in Figure 2. The mobile phase was (A) ammonium acetate buffer and (B) 20% ammonium ace¬ tate buffer/80% acetonitrile. The percentage of B in the mobile phase was varied as shown (dashed line). Bioactivity (hatched bar) is expressed as doses of antisyncytial activity per fraction. (One dose is the smallest amount of material necessary to completely inhibit fusion between 50,000 HTLV-IIIB/H9 cells and 50,000 VB indicator cells over a 24-hr period under standard assay conditions.)

(b) Analysis of Bioactive Peak by FAB-MS (i) CD4(76-94) . As shown in Figure 1, all of the bioactivity (antisyncytial activity) present in the post-resin mixture eluted in peak 4, with a retention time of 5 to 8.5 min. Aliquots of the major peak (3 to 4.5 min retention time) and the area of the chromato- gram (peak 4) in which bioactive material eluted (5 to 8.5 min retention time) were submitted to fast atom bombarbment-mass spectrometric analysis as described by Lee ("Fast atom bombardment and secondary ion mass spectrometry of peptides and proteins" in Methods of Protein Microcharacterization, J.E. Shively, ed., The Humana Press, Clifton, New Jersey, pp. 403-441, 1986) using a JOEL HX-100 HF instrument with a double focus¬ ing magnetic sector of normal geometry, operating at 5 KV acceleration potential and a nominal resolution of 3000. The major peak gave a parent fragment mass (M+H = 2287) consistent with the mass of the desired peptide LKIEDSDT ICEVEDQKEE as well as a fragment of mass 2269, the mass of the parent fragment minus H₂0 (18 atomic mass units). The major peak had no measur- able antisyncytial activity. The material in peak 4 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. (ii) CD4(83-94) . As shown in Figure 2, the biologically active peptide(s) eluted in peak 7, having a retention time of 8 to 11 min. Aliquots of the major protein peak (4.5 to 6.0 min retention time) and peak 7 were submitted to FAB-MS as described by Lee, see supra. The major peak, which had no measurable antisyncytial activity gave a parent fragment mass (M+H = 1576) con- sistent with the mass of a peptide having the sequence TYICEVEDQKEE wherein the C residue has a benzyl substi¬ tuent. The biologically active material eluting at 8 to 11 min retention time exhibited a complex mass spectrum containing the parent M+H (1576) and multiple higher-molecular weight peaks consistent with extensive derivatization of the parent peptide.

(c) Effects of Bioactive Fraction on Viral

Infectivity of Heterogeneous HIV Isolates The ability of a peptide preparation to inhi¬ bit infectivity of multiple defined heterogeneous HIV isolates was also evaluated. An HPLC fractionated preparation (peak 7; see Figure 2) of the post-resin mixture from the_*automated solid phase synthesis of CD4(83-94) (t-Boc synthesis; S-benzyl protected cyste¬ ine), with demonstrated antisyncytial activity, was tested for its effect on infectivity of multiple HIV isolates.

The kinetics of infection of different HIV isolates were standardized by the use of DEAE-dextran pre-treated CEM-SS cells. To determine the effect of addition of DEAE-dextran on the anti-viral efficacy of CD4(76-94)BZL, parallel experiments were carried out in the presence and absence of DEAE-dextran, using a single stock of HTLV-IIIB as the virus input and the

CD4(76-94) post-resin peptide mixture as the prototype anti-viral compound. In the absence of DEAE-dextran, a 1:2 dilution of the virus stock produced 158 ± 38 (S.E.) syncytia/well, and Vn/Vo values of 0.77, 0.10 and 0.003 for 5, 50 and 500 μM nominal concentrations of peptide, respectively (average of duplicate determinations). In the presence of DEAE-dextran, a 1:2 dilution of the I ^' ' I . ^' i ' ^; 37 ' ^{' :} ' ' ^''

virus stock produced 270 + 16 (S.E.) syncytia/well, and Vn/Vo values of 0.93, 0.19 and 0.006 for 5, 50 and 500 μM nominal concentrations of peptide, respectively (average of duplicate determinations). Viral stocks of HIV-1 strains HTLV-IIIB,

RF-II, MN and CC were prepared as either fresh or frozen cell culture supernatants from HIV-infected H9 cells. Viral inocula pretreated with varying nominal concentrations of peptide in PBS or complete medium were incubated with DEAE-dextan pretreated CEM-SS cells for 1 hr at 37°C. Inocula were removed from the cul¬ tures by aspiration and replaced with fresh medium or medium containing the nominal, concentrations of CD4(83-94)BZL shown. Results (shown in Table 5) are the averages of duplicate determinations (all within

30% of the mean values) in a single experiment repeated at least once with similar results.

Table 5 Relative Anti-viral Efficacy of

CD4(83-94) Peak 7 on Infection of CEM-SS Cells with Multiple Isolates of HIV-1¹

Dose IIIB RF MN CC

125 μM 0.005 0.003 0.003 0.003

63 μM ^• 0.005 0.43 0.54 0.05

32 μM 0.005 0.69 1.03 1.01

16 μM 0.04 0.84 ≥l.OO ≥l.OO

8 μM 0.31 ≥l.OO ≥l.OO ≥l.OO

4 μM 1.06 ≥l.OO ≥l.OO ≥l.OO

1 See Footnote 1, Table 4. Example 2

Post-synthesis Derivatization and Bioassay of

Analogs of Parent CD4(76-94)

A. Preparation of Derivatives

A series of derivatives and related analogs of CD4(76-94) were synthesized, using benzyl bromide and other reaction compounds as indicated in Table 6 (below) using one of the following methods. Method 1. One and one-half mg of HPLC-puri- fied CD4(76-94) was dissolved in 120 μl acetonitrile- plus 150 μl deionized water and 60 μl sodium bicarbon¬ ate (0.5 N) . Acetonitrile, 200 μl, was added followed by a 40 molar excess of benzyl bromide or other reac- tion compound (see Table 6). The mixture was incubated at room temperature (about 20-25°C) for 1 hr, then 2 μl of triethylamine was added to the reaction mixture which was further reacted at room temperature 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 powder was reconstituted in PBS and used directly in the cell fusion assay (described above in Example l.B) . 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 10% tetrahydrofuran and an equal volume of chloro¬ form. The mixture was vortexed, then the water layer and interface were collected and used for bioassay. Method 3. One mg of HPLC purified CD4(76-94) was dissolved in 400 μl of 60% acetonitrile and 40-80 μl sodium bicarbonate (0.05 M). An eight molar excess of benzyl bromide or other reaction compound (see Table 6) was then added to the CD4(76-94) solution and reacted at room temperature for 6 hrs. After com¬ pletion of the reaction, the product was dried by cen¬ trifugal vacuum concentration and was then dissolved in 5 mM sodium bicarbonate. One volume of PBS was then added. The solution was further mixed with an equal volume of chloroform then allowed to partition after mixing. The chloroform layer was removed and the aqueous phase used for bioassay.

B. Effect on Cell Fusion

The derivatives synthesized as described above were evaluated for their ability to inhibit syncytium formation using the cell fusion assay described in Ex¬ ample l.B(a). The following were the results obtained.

Table 6

Derivatives of CD4(76-94)

Method of Reaction Compound Preparation Titer²

1. Benzyl bromide 1,2,3 60-120 μM

2. Xylyl bromide 1 cytotoxic

3. 2-chlorobenzyl bromide 3 60-120 μM

4. 4-(N-Maleimidomethyl)cyclo- hexane-1-carboxylic acid

N-Hydroxysuccinimide ester 1,2,3 <8 μM

5. 3-(2-Pyridyldithio)propionic acid N-Hydroxysuccinimide ester 2,3 32 μM

6. None - not active

1 Preparations tested were not homogeneous by HPLC.

2 500 μM was the highest concentration tested. All con¬ centrations are nominal concentrations based upon the amount of peptide used in the reaction. Titers shown indicate the concentration required for complete blockade of HIV-induced cell fusion, after overnight incubation.

As shown in Table 6 (above), derivatives of CD4(76-94) which inhibited HIV-induced cell fusion included those prepared using benzyl bromide, 2-chloro- benzyl bromide, 4-(N-Maleimidomethyl) cyclohexane-1- carboxylic acid N-Hydroxysuccinimide ester or 3-(2-Py- ridyldithio)propionic acid N-Hydroxysuccinimide ester. Two derivatives prepared using naphthyl reaction com¬ pounds were ineffective at the concentrations tested. Benzyl cysteine, including "N-term"-t-Boc-blocked and CBZ-blocked "N-term"-blocked benzyl cysteine, had no effect on HIV-induced cell fusion at all concentrations tested (up to 500 μM) .

Example 3 Comparison of Bioactivity of S-benzylated Analogs of CD4(76-94) and Fragments Thereof Prepared by Solid Phase Synthesis and by Post-synthesis Modification

A. Solid Phase Synthesis of S-benzylated Analogs by t-Boc Procedure (a) Synthesis

Derivatives of CD4(76-94) in which C-86 was replaced with S-benzyl cysteine were synthesized by substituting t-Boc-S-benzyl-cysteine for t-Boc-S-methyl- benzyl-cysteine in the method outlined in Example 1A. Following HF cleavage, 10 mg of S-benzyl CD4 dissolved in 10 mM ammonium acetate, pH 7.0, was chromatographed by HPLC on a Vydac C8 (10x250 mm) bonded phase semi- preparative column eluted with a mobile phase compris- ing a linear gradient of 10-80% acetonitrile in 20% ammonium acetate buffer. Aliquots were taken for both FAB mass spectrometric analysis and assay of antisyn¬ cytial activity. Material isolated as "peak 4" (see Figure 2) was confirmed by amino acid sequence and FAB- MS analysis to be pure polypeptide having the structure TYIC_bzlEVEDQKEE. This material had no antisyncytial activity at <500 μM, the highest concentration tested. B. Solid Phase Synthesis by F-moc Procedure (a) Synthesis

A series of CD4-derived synthetic peptide de¬ rivatives were also synthesized using the F-moc method. Side-chain protection in the F-moc method was t-butyl

(D, E, S, T, Y), t-Boc (K), trityl (H), DMTR [WHICH IS?] (R), benzyl (C); in some cases benzyl (E, C, Y, T), chlorobenzyl (E, C, Y, T), benzylamide (E, C, Y, T), and chlorobenzylamide (E, C , Y, T) . At the end of the synthesis, the peptide was side-chain deprotected and cleaved from the resin by addition of 70% trifluoroacetic acid containing 30% di¬ methylsulfide with or without anisole (0.1 volume of TFA) . The remainder of the procedure was similar to that described in Example l.A for the t-Boc procedure. Derivatized peptides having the following sequences TYIC_bzlE_bzlVEDQKEE, TYIC_bzlEVE_bzlDQKEE, TY_bzlIC_bzlEVEDQKEE, and T IC_bzlE_bzlVE_bzlDQKEE were pre¬ pared using the F-moc technique. As shown in Table 7, the heterogeneous, post-synthesis mixture obtained from synthesis of TYIC_bzlE_bzlVEDQKEE possessed antisyncytial activity; mixtures obtained from the synthesis of the other two multiply-derivatized peptides did not. The authentic peptide TYIC_bzlE_bzlVEDQKEE was purified to homogeneity by HPLC and the structure and purity of the purified composition confirmed by FAB-MS. This compo¬ sition, in purified form, possessed antisyncytial activity, with an estimated potency of 100-200 μM re¬ quired to completely block HIV-1-induced cell fusion in the standard fusion assay. Table 7

Inhibition of HIV-induced Cell Fusion by

CD4-derived Synthetic Peptide Composition

Peptide Sequence Titer¹

TYIC_bzlE_bzlVEDQKEE² 200 μM

TY_bzlIC_bzlEVEDQKEE² Inactive

TYIC_bzlEVE_bzlDQKEE² Inactive

TYIC_bzlE_bzlVE_bzlDQKEE² Inactive ^". TYIC_bzlE_bzlVEDQKEE³ Active⁴

1 The concentration required for complete blockade of HIV-induced cell fusion after overnight incubation under standard assay conditions (Lifson e_t al_. , supra. ) .

2 Preparations tested were post-resin peptide mixtures and were not homogeneous, by HPLC.

3 Preparation tested was HPLC purified peptide deriva¬ tive with purity and structure confirmed by HPLC, FAB-MS, and amino acid sequence. 4 The concentration of peptide present in this pure antisyncytial preparation was not precisely quanti- tated, but was estimated at <200 μM.

C. Post-synthesis Modification of-CD4(76-94) Analogs, Derivatives and Fragments (a) Post-Synthesis Benzylation of CD4(76-94)

Purified non-derivatized CD4(76-94), prepared as described in Example l.A, was derivatized by incuba¬ tion with α-bromotoluene or α-bromoxylene according to the method of Erickson et al., J. Amer. Chem. Soc. (1973) 95.:11. Briefly, 5 mg (2.2 μmol) of CD4(83-94) was dissolved in 1.4 ml triethylamine (11 mmol) and 1.22 mg 4-methylbenzyl bromide (7.1 μmol) added. The mixture was allowed to react, with stirring, for 6 hrs at 25°C. The resulting product was vacuum evaporated for 1.5 hrs, re-suspended in 0.01 mM ammonium acetate, and lyophilized. The lyophilized material was recon¬ stituted in phosphate-buffered saline and tested for fusion-inhibiting activity.

(b) Post-synthesis Benzylation of S-benzyl CD4(83-94)

Seven and one-tenth μmole of α-bromoxylene was added to 2.2 μmole of CD4(83-94) (peak 4, see Figure 2) prepared as described above in 3.A and purified by HPLC was dissolved in 1.5 ml of triethylamine followed by stirring at room temperature for 16 hrs. Volatile material was removed under vacuum, and the residue dissolved in 10 mM ammonium acetate, pH 7.0, extracted with one volume of chloroform, and the resultant aque- ous phase lyophilized repeatedly.

(c) Post-synthesis Benzylation of S-benzyl CD4(83-94)

Five mg of CD4(83-94) (peak 4, see Figure 2) purified by HPLC as described in Example l.C was sub¬ mitted to chemical derivatization as described in Example 2, Method 1. The resultant peptide derivative was evaporated to dryness, reconstituted in water, ex¬ tracted with one volume of chloroform, and the aqueous phase lyophilized and tested for anti-syncytial activ¬ ity as described in Example 1. Potency is expressed as the lowest concentration of the peptide mixture (nominal concentration based on mass of the input peptide and formula weight of the parent peptide TYICEVEDQKEE) capable of complete inhibition of HIV_HχB -induced cell fusion. I

44

D. Comparison of Effect on Cell Fusion of S-benzylated Analogs (a) CD4(76-94)

The antisyncytial activity of derivatives of CD4(76-94) prepared by solid phase synthesis (t-Boc synthesis; S-paramethyl-benzyl protected cysteine) and derivatized post-synthesis by benzylation or xylylation (Example 3.C(a) above) were compared (see Table 8).

Table 8

Comparison of Anti-syncytial Activity of CD4(76-94) as Benzyl or Xylyl Derivatives

Preparation Activity¹ CD4(76-94)² Not Active

Xylyl CD4(76-94) 125 μM

Benzyl CD4(76-94) 63 μM

1 Not Active = No anti-syncytial activity at <500 μM.

2 HPLC purified authentic CD4(76-94) with purity and structure confirmed by HPLC, FAB-MS and amino acid sequence.

Thus both the post synthesis derivatized peptides were active. Underivatized purified CD4(76-94) was not active.

(b) CD4(83-94)

The anti-syncytial activity of three prepar¬ ations of derivatized CD4(83-94) were also compared. The three preparations were: (A) TYIC_bz-LEVEDQKEE, the peptide mixture obtained as described in Example l.C by solid-phase synthesis of TYICEVEDQKEE, using the t-Boc method with Srbenzyl protected cysteine; (B) the puri¬ fied peptide S-benzyl-CD4(83-94) obtained by HPLC fractionation of the peptide mixture described in (A) ι ^{■ •}

45 (peak 4, see Figure 2); and (C) the peptide mixture obtained by post-synthesis derivatization of (B), using the method in Example 3.C(b) above. The results are shown in Table 9 below.

Table 9

Comparison of Anti-syncytial Activity of

Solid-Phase and Post-Synthesis

Analogs of CD4(83-84)

Method of Nominal Material Assayed Preparation Potency

A. CD4(83-94)

Solid phase, 250 μM post-resin

B. Peak 4

CD4(83-94) Solid phase, Not Active post-HPLC

C. Peak 4 BZL

CD4(83-94) Post-synthesis 250 μM modification of peak 4 post-HPLC

As shown, only the post-resin peptide mixture and CD4(83-94) benzylated post-synthesis were biologi¬ cally active. HPLC-purified S-benzyl CD4(83-94) pre¬ pared by the t-Boc method using S-benzyl protected cysteine (peak 4) was inactive at <500 μM.

E. Analysis of Analogs by FAB-MS

The post-resin mixtures obtained from automa¬ ted synthesis were analyzed by Fast Atom Bombardment- Mass Spectrometry (FAB-MS) to determine the identity of the component peptide(s). The post-resin mixtures were separated into component peptides by HPLC using a C8 reverse-phase column then further purified by Vydac C18 reverse-phase column. Active fractions were identified using the cell fusion assay described above. Fractions so identified were then analyzed by FAB-MS (Lee, supra) . The samples were ionized by means of 6 Kev Xenon using different matrixes. Positive ion spectra were obtained using a matrix of dithiothreitol:dithioerythreitol (5:1) in camphor sulfuric acid. For negative ion spectra, a glycerol matrix was used. Spectra were scanned and collected under computer control using a JEOL DA5000 data system. The structural interpretations were based on both positive and negative fragment ion series. In those fractions having antisyncytial activity, cysteine and glutamic acid or aspartic acid residues of the pep¬ tide were modified by benzyl or chlorobenzyl as shown. These data are summarized in Table 10.

Table' 10

FAB-MS Analysis of Compositions Having Anti-syncytial Activity

Synthesis Composition Substitution Derivatized Amino Acids

TYICEVEDQKEE di(chlorobenzyl) Cys_8β(Cl-Bzl)-Glu₈₇(Cl-Bzl) 83 94 di(benzyl) Cys_Rfi(Bzl)+Glu_fl7(Bzl)

TYICEVEDNKEE dibenzyl Cys₈₆(Bzl)+Glu₈₇(Bzl) 83 94 Cys₈₆(Bzl)+Asp_go(Bzl)

LKIEDSDTYICEVEDQKEE benzyl and Cys₈₆(Bzl)+Glu₈₇(Bzl) 76 94 chlorobenzyl Cys₈₆(Bzl)+Glu₈₇(Cl-Bzl) Cys_β6(Bzl)+Glu₇₉(Cl-Bzl)

43

It is evident from the above results, that novel compositions have been provided which inhibit cell fusion induced by CD4 dependent retroviruses as well as infectivity of such viruses. The compositions are effective against multiple distinct isolates with documented heterogeneity including both numerous iso¬ lates of HIV-1 as well as HIV-2. Thus, the subject compositions may provide for protection against infec- tion and progression of symptoms associated with AIDS- related complex and from AIDS.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising: a sequence of at least seven consecutive amino acids of human CD4, wherein at least one heteroatom of other than a peptide bond of at least one amino acid in said sequence is joined to a derivatizing group and wherein said polypeptide is characterized as capable of modulating at least one CD4-dependent virus-induced cellular response.

2. A polypeptide according to Claim 1, wherein at least one heteroatom is sulfur.

3. A polypeptide according to Claim 2, wherein said derivatized amino acid is the cysteine at position 86 of human CD4.

4. A polypeptide according to Claim 1, wherein at least one heteroatom is oxygen.

5. A polypeptide according to Claim 4, wherein said derivatized amino acid is the glutamic acid at position 87 of human CD4.

6. A polypeptide according to Claim 1, wherein at least one derivatizing group is an aralkyl group having from 7 to 12 carbon atoms and from 0 to 2 heteroatoms.

7. A polypeptide according to Claim 6, wherein said aralkyl group is a benzyl or a chlorobenzyl group.

8. A polypeptide according to Claim 1, wherein said sequence includes a core comprising:

86 87 T—Y—I—C—E--V—E wherein said 86 and 87 refer to amino acid positions in said human CD4.

9. A polypeptide according to Claim 8, wherein said sequence is T-Y-I-C-E-V-E.

10. A compound comprising: a sequence of at least 10 consecutive amino acids of human CD4, which sequence includes

86 87 T—Y—I—C—E—V—E

wherein said 86 and 87 refer to amino acid positions in said human CD4 and wherein the oxygen atom of the glut¬ amic acid at position 87 is joined to an aliphatic car¬ bon atom of a first derivatizing group, said compound being -characterized as capable of inhibiting CD4-depen- dent virus-induced syncytium formation.

11. A compound according to Claim 10, wherein the sulphur atom of the cysteine at position 86 is joined to an aliphatic carbon atom or sulfur atom of a second derivatizing group.

12. A compound according to Claim 11, wherein at least one of said first derivatizing group and said second derivatizing group is a benzyl group or a chlorobenzyl group.

13. A composition made by a method comprising: synthesizing on a solid support a polypeptide having a sequence of at least eight consecutive amino acids of human CD4, which sequence includes a core comprising:

86 87 T—Y—I—C—E—V—E . ( . , ■

51

wherein numerals 86 and 87 refer to amino acid posi¬ tions in said human CD4 and wherein said synthesizing is by the t-Boc method using a benzyl or 4-methoxy 5 benzyl group for side-chain protection for cysteine and an oxybenzyl group for side-chain protection for glutamic acid; cleaving said polypeptide from said solid support whereby a post-resin mixture is obtained; and 0 isolating at least one polypeptide character¬ ized as inhibiting CD4-dependent virus-induced cell fusion from said mixture, whereby said composition is obtained.

5 14. A composition according to Claim 13, wherein said cleaving comprises contacting said solid support with hydrofluoric acid at a temperature of from about -20°C to about .0°C.

0 15. A composition made by a method comprising: synthesizing on a solid support a polypeptide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising: 5 86 87

T—Y—I--C—E—V—E

wherein said 86 and 87 refer to amino acid positions in said human CD4 and wherein said synthesizing is by the 0 F-moc method using S-benzyl or chlorobenzyl cysteine as a substitute for cysteine and using a benzyl group for side-chain protection for said cysteine substitute and a t-butyl group for side-chain protection for glutamic acid; 5 cleaving said polypeptide from said solid support whereby a post-resin mixture is obtained; and isolating at least one polypeptide character¬ ized as inhibiting CD4-dependent virus-induced cell fusion from said mixture, whereby said composition is obtained.

16. A composition according to Claim 15, wherein said cleaving comprises contacting said solid support with from about 50% to about 80% trifluoroacetic acid.

17. A composition according to any one of Claims

13-16, wherein said composition consists essentially of a substituted polypeptide wherein at least one of the sulfur atom of the cysteine at position 86 and a hetero¬ atom of other than a peptide bond is joined to an ali- phatic carbon atom of an aralkyl group.

18. A composition for use in inhibiting CD4- dependent virus-induced cell fusion consisting essentially of: a polypeptide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising:

86 87 T—Y—I—C—E—V—E wherein said 86 and 87 refer to amino acid positions in said human CD4, wherein the sulfur atom of the cysteine at position 86 of CD4 is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group and the oxygen atom of at least one other amino acid in said sequence is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group, and wherein said composition is characterized as capable of inhibiting CD4-dependent virus-induced cell fusion.

19. A method for interfering with at least one cellular response resulting from interaction between CD4-dependent retroviruses and cells comprising a CD4 surface antigen, said method comprising: i ^■

53 contacting said cells with a compound compris¬ ing a polypeptide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising:

86 87 T—Y—I—C—E—V—E

wherein said 86 and 87 refer to amino acid positions in said human CD4, wherein the sulfur atom of the cysteine at position 86 is joined to an aliphatic carbon atom of either a benzyl or a chlorobenzyl group, and the oxygen atom of the glutamic acid at position 87 is joined to an aliphatic carbon atom of either a benzyl or a chlorobenzyl group, in an amount sufficient to interfere with said cellular response.

20. A method according to Claim 19, wherein said CD4-dependent retrovirus is HIV-1 or HIV-2.

21. A method according to Claim 19, wherein said cellular response is fusion of CD4-bearing cells or virion infectivity.

22. A pharmaceutical composition useful for inter¬ fering with a CD4-dependent virus-induced cellular response consisting essentially of a polypeptide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising:

86 87 T—Y—I—C—E—V—E

wherein said 86 and 87 refer to amino acid positions in said human CD4, wherein the sulfur atom of the cysteine at position 86 of CD4 is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group and the oxygen atom of at least one other amino acid in said sequence is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group, and wherein said composition is characterized as capable of interfering with said CD4- dependent virus-induced cellular response.

23. A pharmaceutical composition according to Claim 22, wherein said CD4-dependent virus is HIV-1 or HIV-2.

24. A composition of matter comprising a polypep¬ tide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising: 86 87

T—Y—I--C—E—V--E

wherein said 86 and 87 refer to amino acid positions in said human CD4, wherein the sulfμr atom of the cysteine at position 86 of CD4 is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group and the oxygen atom of the glutamic acid at position 87 is joined to an aliphatic carbon atom of a benzyl or a chlorobenzyl group, said composition prepared by the method comprising: combining in an aqueous solvent, said polypep¬ tide with at least about a stoichiometric amount of a sulfur blocking group containing compound, which com¬ pound comprises an active halogen or an active olefin functionality, at a temperature in the range of about

0° to 50°C for a time sufficient for said sulfur block¬ ing group containing compound to covalently bond to said cysteine; neutralizing any acid formed; and isolating said composition of matter. - ' i ^■ i , ^{■ i}

55^'

25. Use of a polypeptide for preparation of a pharmaceutical composition for treatment of infection by a CD4-dependent retrovirus, said polypeptide having a sequence of at least seven consecutive amino acids of human CD4, which sequence includes a core comprising:

86 87

T—Y—I—C—E—V—E

wherein the sulfur atom of the cysteine at position 86 is joined to a derivatizing group.

26. A use according to Claim 25, wherein said derivatizing group is a benzyl or a chlorobenzyl group.

27. A use according to Claim 25, wherein said CD4- dependent retrovirus is HIV-1 or HIV-2.