CHEMICALLY MODIFIED CD4 PEPTIDE FRAGMENTS HAVING ANTI-RETROVIRAL PROPERTIES

Abstract: A general method of derivatizing and selecting fragments of CD4 peptides that exhibit enhanced (relative to the original fragment) capacity to modulate a CD4-dependent virus-induced cellular response. In one aspect, peptide compositions comprising a sequence of human CD4 which includes at least a core sequence of seven consecutive amino acids of the N-terminal proximal sequence or a sequence including the cysteine at position 84 (C84) are provided. At least one amino acid side chain is derivatized, such as the cysteine at position 84 (C84) or the glutamic acid at position 85 (E85). 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: CHEMICALLY MODIFIED CD4 PEPTIDE FRAGMENTS HAVING ANTI-RETROVIRAL PROPERTIES CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of Application Serial No. 346,159, filed May 2, 1989, which is a continuation-in-part of Application Serial No. 258,576, filed October 14, 1988, which application is a continuation-in-part of Application Serial No. 203,285, filed June 6, 1988, which is a continua¬ tion-in-part of Application Serial No. 108,160, filed October 13, 1987, which applications are incorporated herein by reference. INTRODUCTION Technical Field This invention relates to products, methods and compositions for modulating and blocking binding between proteins and their receptors, specifically viral effects on susceptible cells expressing the cell-surface antigen CD4, using polypeptides which interfere with binding interactions, e.g., between viral proteins and CD4. This invention is particularly directed to blocking virion infectivity and cytopathic effects of CD4- dependent retroviruses. Background of the Invention Many cellular responses are regulated by inter¬ action of a protein on the cell surface and a molecule in the local environment (such as a regulating hormone, interleukin, or virus). T cells regulate the immune response through "helper" and "suppressor" signals and also mediate part of the effector arm of cellular immunity. Human T lymphocytes were initially subdivided into two functionally distinct sublineages on the basis of expression of mutually exclusive cell surface proteins designated CD4 (also referred to as T4 or Leu3) and GD8 (also referred to as T8 or Leu2). T cells expressing CD8 mediate m jor-histocompatibility-cσmplex (MHC) class-I-restricted cytotoxic activity and non- cytotoxic antigen-specific suppressor function, while T cells expressing CD4 mediate helper function for B-cell growth and differentiation, proliferative responses, and inducer function for differentiation of CD8-expressing cytotoxic cells. CD4 is a 55-58 kD glycoprotein which, while origi¬ nally characterized as a T-lymphocyte-differentiation antigen found on a subset of mature T cells, is also found on cells of the mononuclear phagocyte lineage and on occasional B lymphocytes. In T-cell/target- cell interactions, the significance of the CD4 molecule can be demonstrated by studies with monoclonal antibodies. Antibodies directed against specific epitopes of the CD4 molecule inhibit MHC-Class-II-restricted functions, in¬ cluding antigen-induced T-cell proliferation, lyumpho- kine release, helper-cell function, and the cytotoxic activity of CD4-expressing cytotoxic cells. The CD4 molecule also serves as a receptor for the tetrovirus HIV-1 (also referred to as HTLV-III, LAV-1 or ARV), the etiologic agent for Acquired Immuno-deficiency Syndrome (AIDS), and other retroviruses such as the second AIDS- associated virus, HIV-2, and SIV (also referred 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-1- induced cytopathicity. Many of the clinical manifesta¬ tions of HIV infection appear to be caused by 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 protein gpl20. 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 (Engle an et al.., J. Immunol. (1981) 122:2124-2129). Rao et al.. Cellular Immunol. (1983) 80:310-319, using monoclonal antibodies, showed that five epitopes of the CD4 antigen could be dis¬ tinguished on human T-cells of the CD4+ subclass. The CD4 molecule is involved in a variety of crucial imrnu- noregulatory interactions between cells in the immune system. Maddon et al. (Cell (1985) 4^:93-104) isolated and obtained the nucleotide sequence of a DNA encoding the CD4 protein. In addition to T lymphocytes, anti-CD4 monoclonal 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 et al. , J. Immunol. (1983) 131:212-216). The CD4 antigens on human T lymphocytes and monocytes are indistinguishable. Stewart et al. , J. Immunol. (1986) 136: 3773-3778. Bank et al. (J. Exp. Med. (1985) 162:1294-1303) disclosed that under some circumstances the CD4 molecule may function as an independent transducer of negative signals inhibiting T-cell activation. Under some circumstances CD4 may play a role acting in concert with the T-cell-antigen receptor to facilitate antigen- receptor-dependent cellular activation. Sleckman et al.. Nature (1987) 3218:351-353; Rosoff et al.. Cell (1987) 49:845-853; Rivas et al., Cell (1987) 49:143-151; Kupfer et al. , Proc. Natl. Acad. Sci. USA (1987) , 81:5888-5892; Owens et al. , Proc Natl. Acad. Sci. USA (1987) 84:9209-9213. 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 reported. Kanki et al. , Science (1985) 231:951-954; Kornfield 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 behaves as a receptor for the HIV-1 virus. Klatzmann et al.. Nature (1985) 312:767-768; Dalgleish et al., Nature (1984) 312:763-767. HIV-induced cell fusion, a charac¬ teristic manifestation of HIV-induced cytopathology, is dependent upon interactions between the viral envelope glycoprotein and CD4. Dalgleish et al. (1984) supra; Lifson et al., Science (1986) 232:1123-1127; Lifson et al.. Nature (1986) 321:725-728; 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 (0KT4a) is blocked. Although the CD4 antigen cannot be detected on the surface of HIV-1 infected cells, intracytoplasmic complexes of CD4 and the HIV envelope glycoprotein (gpl20) have been demonstrated (Hoxie et al.. Science (1986) 227.:1123-1127). Addition of monoclonal antibody to CD4 antigen during virus inoculation inhibits HIV-1 infectivity and replication. McDougal at al., J. Immunol. (1985) 135:3151-3162; McDougal et al.. Science (1986) 231.:3822-385. Sera from HIV-1-infected subjects exhibited some capacity to inhibit (neutralize) HIV-1 infectivity in vitro. McDougal et al.,, J. Immunol. Methods (1985) 2i :171 ? Weiss et al. , Nature (1985) J16:69-72; Robert-Guroff et al. , Nature (1985) 316:72- 74. SUMMARY OF THE INVENTION Methods and compositions are specifically 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 an amino acid sequence proximal to the N- ter inus of the human CD4 antigen. The polypeptides preferably are substituted derivatives of a fragment of the naturally occurring polypeptide CD4 and in preferred embodiments comprise at least a core sequence of seven amino acids including the cysteine at position 84 (C84). Normally, the molecule will have two domains: a hydro- phobic domain, made increasingly hydrophobic by aryl substituents on oxygen and sulphur, and an anionic hydrophilic domain. Preferably, the hydrophobic domain will have at least two aryl substituents. The molecule may be further substituted with substituents on nitrogen. Cellular responses which can be modulated, particu¬ larly inhibited, include retrovirus-induced cell fusion and virion infectivity, as well as virus transmission following infection. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a chromatographic fractionation of synthetic CD4(74-92) including derivatized fragments. Bioactivity and mass spectra of fractionated UV- absorbing species were characterized by a standard HIV- induced cell-fusion bioassay and by FAB-mass spectro- metry, respectively. Figure 2 shows a chromatographic fractionation of synthetic CD4(81-92). Bioactivity and mass spectra of fractionated UV-absorbing species were characterized as described in Figure 1. DESCRIPTION OF SPECIFIC EMBODIMENTS In accordance with the subject invention, composi- tions and methods for their use are provided for inhibiting cellular responses such as viral infection, virally-induced cytopathic effects, and post-infection secondary viral infectivity consequent to infection with CD4-dependent retroviruses such as HIV. The active molecules comprise polypeptides, derivatives, fragments, or analogs thereof and formulations containing such molecules, wherein the polypeptide comprises at least a core sequence of 7 amino acids, including the cysteine at position 84 (C84). For the most part, a molecule of the invention is characterized by having at least two (preferably just two) domains, one being a hydrophobic domain and the other domain (an adjacent domain if more than two domains are present) being a hydrophilic domain (polar domain), where the hydrophobic domain will normally have more neutral and non-polar amino acids than charged amino acids, while the polar domain will have more charged amino acids then neutral or non-polar. Substi¬ tuents normally will be selected to enhance the hydro¬ phobic character of the hydrophobic domain. For the most part, the cysteine at position 84 (C84) will be substituted, particularly with a hydrophobic sub- stituent, more particularly an aryl group, e.g., phenyl. The phenyl group is desirably present as benzyl or chlorobenzyl. There will be at least one substituent, and usually not more than about 5 substituents, where the substituents can include chalcogen blocking agents, esterifying agents, acylating agents, nitrogen alkylating agents, or amidating agents. The sequence of the compositions of most interest are usually comparable to a sequence of a segment of the CD4 molecule (see Table 1), preferably segments proximal to the N-terminus. The most preferred compositions 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". The compositions of the subject invention generally have from 7 to 30, usually 7 to 20 amino acids. Compositions of particular interest include those of the CD4 amino acid sequences 81-92, 79-92, 81-94, and 74-92, particularly including 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 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 as normally found in humans. The peptide sequences may be modified by terminal amino acylation, for example, acetylation; carboxy amidation, for example, with ammonia or methylamine; cyclization; and the like to increase potency, decrease toxicity, increase stability, or otherwise improve the pharmaco- logic characteristics of the molecule. One particularly useful modification is to provide a polypeptide analog that consists of a backbone and sidechain groups that are otherwise identical to specific peptides or classes of peptides as described herein but in which at least one of the amide linkages (e.g., between individual amino acids in the main chain) have been replaced by a group isosteric to an amide, such as a -CH2-C0-, -NHNH-, -N » N-, -NHCH2-. -CH2CH2-, -0-C0-, and the like. The orientation of these groups in the amide chain is the same as is normally written (i.e., -NHCO-) , but reverse orientations are permitted for asymetrical groups. Such modifications are not peculiar to the present invention but have been previously used to increase the stability of peptides by reducing likelihood of amide cleavage. To improve bio- availability, the peptide sequences may also be modified by N-terminal methylation, esterification by addition of an alcohol, C-terminal carboxyl reduction to the corresponding alcohol, substitution of a d-amino-acid for an 1-amino acid, a peptide bond modification, or the like. 1 Table I 1 ' 2 1 1 N K V V L G K K G D T V E L T C T A S Q K K S 23 24 I Q F H W K N S N Q I K I L G N Q G S F L T K G P 48 49 S K L N D R A D S R R S L T D Q G N F P L I I K N 73 74 L K I E D S D T Y I C E V E D Q K E E V Q L L V F 98 99 G L T A N S D T H L L Q G Q S L T L T L E S P P G 123 124 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 148 149 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 173 174 I V V L A F Q K A S S I V Y K K E G E Q V E F S F 198 199 P L A F T V E K L T G S G E L W W Q A E R A S S S 223 224 K S W I T F D L K N K E V S V K R V T Q D P K L Q 248 249 M G K K L P L H L T L P Q A L P Q Y A Q S G N L T 273 274 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 298 299 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 323 324 A K V S K R E K A V W V L N P E A G M T Q C L L S 348 349 D S G Q V L L E S N I K V L P T W S T P V Q P M A 373 374 L I V L G G V A G L L L F I G L G I F F C V R C R 398 399 H R R R Q A E R M S Q I K R L L S E K K T C Q C P 423 424 H R F Q K T C S P I 434 The letters have the following meaning in accordance with convention: A = alinine; R s arginine; N » asparagin D - aspartic acid; C = cysteine; Q = gluta ine; E •* ** ■ glutam acid; G » glycine; H * histidine; I » isoleucine; L » leucine; K ■ lysine; M ■ methionine; F * phenylalanine ; P * proline;; S ■ serine; T ■ threonine; W =tryptophan; Y = tyros ine; and V = valine. 2 The numbering of residues is after the convention of Littman et al.. Cell (1988) 5_5:541. It will be appreciated that the amino acid sequence of a polypeptide of the invention need not correspond exactly to fragments of the sequence shown in Table 1, but may be modified by from 1 to 4 conservative or non- conservative substitutions including deletions and insertions usually involving not more than about 1 amino acid, where the modification may include D-aπtino acids. without significant y a ecting the activity of the product. Therefore, the subject polypeptides may be subject to various changes, such as insertions, deletions, substitutions, and the like, either conservative or non-conservative, where such changes may provide for certain advantages in their use. By conservative substitutions is intended a substitution within one of the following groups or other recognized groupings of amino acids: G, A; V, I, L; D, E; N, Q; S, T; K, R; and F, Y, W. See, e.g., Lehninger, Biochemistry, Worth Publishers, New York, 1970, pp. 68<- 71. Usually, the sequence of the composition will not differ 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 example, to a carrier protein, for use as an immunogen, or to affinity groups, labels, etc. The arms will usually be at least about 3 amino acids and may be 50 or more amino acids. The derivatization of the subject compositions includes derivatization of at least one, more usually at least two, preferably three heteroatoms other than heteroatoms in the peptide bond. By heteroatom is intended any atom excluding carbon, including atoms located in ring structures and non-ring structures. In compositions in which at least two heteroatoms are derivatized, generally one of the heteroatoms is the sulfur of the cysteine at position 84 of the CD4 molecule (C84). The remaining substitutions) may be on a heteroatom of any other amino acid in the composition, preferably on a side-chain oxygen of a glutamic acid residue, more preferably the glutamic acid at position 85 of the CD4 molecule (E85). 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, and nitrogen. In compositions in which a single heteroatom is derivatized, the derivatized group is usually the heteroatom of the amino acid C84, although a glutamic acid, usually E85, may alternatively be derivatized. In compositions in which more than one heteroatom is derivatized, conveniently two adjacent chalcogens having an atomic number of 16 or less are derivatized. By chalcogen having an atomic number of 16 or less is intended oxygen and sulfur. The chalcogens are generally present on threonine (T81), tyrosine (Y82), cysteine (C84) and glutamate (E85). Preferred compositions include those in which at least one heteroatom in addition to the two adjacent chalcogens is derivatized. Preferred multisubstituted compositions are compositions in which (a) T81, Y82 and C84 or (b) T81, C84 and E85 are derivatized. The derivatizing groups generally have from about 1 to about 36 carbon atoms and may be aliphatic, alicyc- lic, aromatic, heterocyclic or combinations thereof. Usually, the derivatizing group has from 0 to 10 heteroatoms, which may be in the longest chain, as a substituent on a chain, as a ring atom, or the like. For the most part the heteroatoms of the derivatizing group are 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 approximately the size of a phenyl group or similar cyclic or heterocyclic group (either aromatic or non-aromatic). The over-all effect of the derivitizing group will be to increase the hydrophobicity of the derivitized fragment, as measured, for example, by the partitioning coeffecient between ether and water or by retention time in reverse phase HPLC (e.g., using C g or C.g columns eluted by a gradient consisting of (1) water and 0.1% TFA and (2) acetoni- trile and 0.1% TFA). Bonding 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 derivatizing 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, such as, an aryl-containing substituent or an activated (particularly carbonyl-activated) olefin which forms a thioether resulting from the reaction between the thio group of a cysteine and the C-C double bond, as for example, a maleimide. The aryl group is preferably selected from 5- and 6-membered aromatic rings contain- ing carbon and 0 to 1 oxygen or sulfur and 0 to 3 nitrogen atoms in the ring. A benzene-containing substituent is a preferred aryl group; e.g., benzyl or naphthyl. The aryl-containing group may be substituted or unsubstituted. Substituents may include alkyl, particularly methyl; halogen, particularly chloro; nitro; hydroxyl; and the like, where the substituents may be in any position, preferably at the ortho and/or para positions. The aryl group may have from 0 to 3 substituents, usually not more than 2 substituents, which substituents may be the same or different. When more than one heteroatom in the polypeptide is deriva¬ tized, the derivatizing group on each heteroatom may be the same or different. Preferred derivatizing groups include benzyl and chlorobenzyl. For active olefins used as derivatizing groups, the olefin will usually be conjugated with a second site of unsaturation; e.g., a carbonyl group, acyl group, malei- mido group, conjugated polyolefin,.or he like. Also of interest is having 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 carbo- diimide, carbonyl diimidazole, or the like such as anhydride for reaction with an amine or alcohol, for example, in a protein. Examples of substituents present in the claimed derivatives of the CD4 molecule and fragments thereof include the following, wherein the group bound to the sulfur of cysteine 84 or to the heteroatom of another amino acid residue (where derivatization occurs for bioactivity according to the present invention) is one of the following groups: (a) alkyl and substituted alkyl compounds: X-CH 2 (CH 2 ) n - where n = 0-20 and X is selected from H, OH, OR, SH, SR, C0 2 H, C0 2 R, NH 2 , NHR, N(R) 2 , S0 3 H, S0 2 CH 3 , or halogen, being other than H only when n is other than 0, in which R is lower alkyl (especially C.-C 4 , most preferably CH^-. (b) cycloalkyl and substituted cycloalkyl compounds: -CH-CH 2 \T " """ X < CH 2>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: where n = 0-5, m - 1-3, and X is as described in (a) above. (d) polyaromatic or substituted aromatic compounds: (CH 2 ) n - where n = 0-3 and X (present in either ring) is as described in (a) above. (e) heterocyclic or substituted heterocyclic compounds such as (i) substituted pyridyl, (ii) imidazole where n = 0-3 and R is a pair of electrons, H, alkyl of 1-2 carbon atoms, or 0. where n = 0-3 and R' is a pair of electrons, H, or 0. (f) maleimide adducts such as m-maleimidobenzoate, N-hydroxysuccinimide ester; m-maleimidobenzoylsulfo- βuccinimide ester; N-succinimidyl-4-(p-maleimidophenyl) butyrate; N-succinimidyl-4-(N-maleimidomethyl)cyclo- hexane-1-carboxylate; sulfosuccinimidyl-4-(N-male- imidomethyl)cyclohexane-1-carboxylate; bis-maleimido- hexane; bis-maleimidomethyl ether; or N-maleimido- butyryloxysuccinimide. (g) thio-containing compounds such as: where m is 1-3 and X is N 3 ; OH; OR; NH 2 ; NHR, N0 2 ; SH; SR; halogen; CO..H; or aryl of from 6 to 12 carbon atoms; or R-S- where R is alkyl or substituted alkyl (especially C1-C4 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-azidothy- midine (AZT); dideoxycytidine (DDC); dideoxyadenoβine (DDA); or dideoxyinosine (DDI). (1) toxins, such as ricin A chain, diphtheria toxin A chain, abrin, trichosanthin, etc. In some instances, carbon atoms in the basic deri¬ vatizing group, particularly aryl carbon atoms, may themselves be substituted, where the substituents may be those described above in the definition of X, as well as alkyl groups of from 1 to 6, usually 1 to 3 carbon atoms. Compounds of interest include multisubstituted compounds, where the multiplicity of substituents is in both the hydrophobic and hydrophilic domain. For the most part in a CD4 fragment, the hydrophobic domain extends to V86, and the hydrophilic domain begins with D88. There will generally be from 2 to 5, usually 2 to 4 substituents in the hydrophobic domain, where the total effect of the substituents is to enhance hydro- phobicity. Therefore, these substituents usually have from 3 to 12, more usually from 3 to 9 carbon atoms, preferably including a phenyl ring, and have fewer than 3, preferably fewer than 2 heteroatoms, particularly. oxygen and nitrogen. Usually, the ratio of carbon to heteroatom will be >_4:1. A large number of different functional groups may be employed which may enhance or diminish the antiviral activity of the initial reactant. Furthermore, the various substituents may have different effects upon the different roles the subject compounds may play in modulating viral activity. Thus, in some instances it may be desirable to use combinations of compounds, where the combination may provide for improved results as to the different roles the subject compounds may play. A wide variety of substituents may be employed to increase or decrease water solubility and/or reduce cyctotoxicity and/or to vary the half-life of the active compound. Groups of interest include amide groups, which may be formed as a result of acylation of amino groups, such as a lysine side chain or terminal amino group; by having substituents which include amide or imide groups, such as malemide, benzomaleimide or succimide; or the like. Other substitutions of interest include derivatizing a carboxy group with an amino group, derivatizing a hydroxyl or amino group with a cyclic anyhydride, or derivatizing of an amino group with various hydrocarbon or substituted hydrocarbon groups. If desired, a cysteine may be reacted with a second cysteine to form a eyetine, where the second cysteine may be by itself (preferably) or serve as a member of an oligopeptide generally of not more than 8, usually not more than 4, amino acids. The total number of carbon atoms added to the core sequence by side-chain substitution (including the terminal functionalities of the amino acids) will generally be at least 1, more usually at least about 7 carbon atoms, and not more than about 60 carbon atoms, more usually not more than about 45 carbon atoms. Desirably, the substituents will have from about 0 to 15, more usually from about 0 to 6 heteroatoms present. primarily selected from chalcogen (oxygen and sulfur), halogen, and nitrogen. In addition to the types of derivatives described above, cyclized versions of such CD4 fragments have proven to be especially effective. Several different cyclic derivatives have been made linking the amino terminus of a derivatized CD4 fragment to a functional group on an amino acid proximal to the carboxy terminus of the fragment. These cyclized fragments have exhibited enhanced anti-syncytial activity. It appears that by cyclizing the derivatized peptide a conformation is obtained that is more restricted compared with the linear analog. This is similar to the effect that has been obtained in other peptides in which cyclization of the peptide increases the peptide stability (as measured by its half life) relative to the uncyclized version. Particularly preferred cyclic derivatives are those in which an amino acid at or near (preferably at) the amino terminal of the fragment is covalently joined to either the amino acid at the carboxy terminal or a side group on an amino acid proximal to the carboxy terminal. For example, an amide bond can be formed between the amino group of the amino terminus and a side-chain or terminal carboxy group. Alternatively, a side-chain carboxy group in the amino-terminus amino acid (in an aspartate or glutamate) can be linked to an amino group in a side chain in the carboxy-proxxmal half of the molecule. Other types of direct amino acid to amino acid links are possible, such as formation of ester or disulfxde linkages. Alternatively, a non-amino-acid linking group can be used to link the two regions of the molecule together. Such linking groups are generally bi-functional reagents containing functional groups that can react with both the amino acids at the indicated positions above. Selective reaction at particular locations can be achieved using standard techniques of peptide chemistry in.which reactive side-chains are blocked except at the location where reaction is desired. Preferred linking groups are aryl or alkyl groups (as defined above) substituted with carboxylate groups. The reactive function in such a case is typically the anhydride of two adjacent carboxylate groups or an acid halide. The dianhydride molecules can have all four carboxylates on one phenyl ring, or adjacent carboxylate groups can be present in two phenyl rings that are joined together, either directly or by a linking functionality such as an alkyl chain or a carbonyl group. Specific examples of such compounds are set forth in the examples that follow. In some instances, particular derivatives may be found to be cytotoxic for in vivo use. It will then be necessary to modify the derivatives to reduce cyto- toxicity or substantially eliminate toxicity at pharmacologically active levels by using derivatizing groups which provide for a physiologically acceptable product. Modifications which can be used include coupling with a polymer such as polyethylene glycol or polypropylene glycol, as described in U.S. Patent Serial No. 4,179,337, issued December 18, 1979. The subject peptides may be employed for some applications linked to a soluble macromolecular carrier. Conveniently, the carrier may be a polypeptide, a polysaccharide, or a combination thereof, either naturally occurring or synthetic. Antibodies to proteins which are unlikely to be encountered at high levels in human serum can be used as carriers. Illustrative polypeptides include poly-L-lysine, bovine serum albumin, human serum albumin, human gamma globulin, keyhole limpet he ocyanin, bovine gamma globulin, and the like. The choice is primarily one of convenience, availability, and intended purpose. The manner of linking is conventional, employing such reagents as p-maleimidobenzoic acid, p-methyldithio- benzoic acid, maleic acid anhydride, phthalic anhydride, succinic anhydride, or glutaric anhydride. 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 otherwise prepared. Any convenient technique may be used for preparing the conjugates, including conventional chemical coupling. The resulting conjugate may comprise a single modified peptide or a plurality of peptides so as to provide multivalent polymeric form. The subject peptides may be prepared as repetitive, conveniently tandem, sequences, with or without inter¬ vening bridges of from 1 to 20 atoms in the chain. The repetitive sequences may be joined by divalent func¬ tionalities, such as cystine or bis-maleimido compounds. Preparation of CD4 derivatives The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively small size, the peptides may be synthesized in solution or on a solid support. 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 Tarn 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); p-toluene s.ulfonyl (R); and benzyl (C) . In many cases, protecting groups such as benzyl, chlorobenzyl, substituted benzyl, benzyla- mide, and chlorobenzamide can be employed for E, C, T and Y. When using a. t-butyloxycarbonyl (t-Boc) procedure, side-chain protection used during synthesis can include tosyl (R,H), 0-benzyl (D, E), benzyl (S, T, in some cases C), Br-Z (Y), Cl-Z (K), 4-MeO-benzyl (C) and formyl (K) . Z represents 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 hydrophobic (e.g., arylalkyl) substituents on the peptide should be avoided. Generally, 50-80% trifluoroacetic acid (TFA) in dichloromethane (DCM) is used, preferably 70%. Other organic acids such as 70% trifluoromethanesulfonic acid (TFMSA), and hydrobromide (HBr) in TFA and a mixture 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 dodecylsulfate (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- pro- tecting groups of the synthetic peptide are deprotected and the peptide cleaved from the resin simultaneously by addition of, for example, hydrofluoric acid (HF) usually at a temperature of from about -20°C to 0°C. The HF may contain scavengers such as anisole, or thioanisole, p- thiocresol, and/or 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 bicarbonate, or DMF and H j O (2:1 v/v) . Alternatively, the ethyl ether step may be omitted, and any residual scavengers, such as anisole and dimethyl sulfide, can be removed by chromatography on Sephadex G-10 or G-25. Peptide compositions having the ability to inhibit HIV-induced cell fusion ("antisynchytial activity") prepared by either the F-moc or t-Boc method are separated from inactive peptides in the post-resin mixture by conventional means, including high pressure liquid chromatography (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 C84 in the core sequence is replaced with S-benzyl-Cys by sub¬ stitution of t-Boc-S-benzyl-cysteine as the precursor amino acid for t-Boc-S-p-methylbenzyl cysteine. Compositions having antisyncytial activity can also be prepared using as a starting material a peptide comprising a purified, underivatized CD4 peptide containing the core sequence CD4(81-87) or a purified derivative in which C84 of the core sequence is derivatized particularly with an aralkyl substituent having from 7-12 carbon atoms and from 0-2 heteroatoms, such as a benzyl or chlorobenzyl group or another substituted benzyl group. 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, for example by 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,,0-enones, such as maleimide, disulfides, or the like. Peptides prepared from these starting materials will generally comprise at least two derivatizing groups, where one derivatizing group is attached to C84. 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 C84 and E85 can be synthesized by the F-moc method using N-F-moc-S-benzyl- L-cysteine and N-F-moc-L-glutamic acid-E-benzyl ester as a precursor. The remainder of the precursor amino acids and the procedure of cleavage, extractions, and purifi- cation of peptides are similar to those described. Use of CD4 Derivatives The subject compounds and compositions have a number of uses and may be used in vitro and jLn vivo. In vitro, the subject compounds and compositions may be employed for determining 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 cytophatic effects, and the like. In vivo, the subject compounds and compositions may be used prophylactically or therapeutically for prevent¬ ing infection or inhibiting propagation of CD4-dependent retroviruses such as HIV and infection of or cytophatic effects on additional T-cells or other CD4-bearing cells by inhibiting HIV envelope glycoprotein-CD4 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, or tissue therefrom, 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 retro¬ viruses. The subject compounds and compositions will generally be administered so as to enter the blood stream, being administered parenterally, for example intramuscularly, intraperitoneally, intravenously, intransasally, rectal/vaginal suppositories, topically, or the like or orally. The subject compounds are protease resistant and may be made more protease resistant by substituting an unnatural D-amino acid for a unnatural L-amino acid, by N-acetylation, etc. Other modifications may also be used to extend the in vivo lifetimes of the subject compounds. Any physiologically acceptable medium may be employed, such as deionized water, saline, phosphate- buffered saline, aqueous ethanol, and the like. The concentration of the active ingredient of the subject composition will vary, depending upon the solubility, use, frequency of administration, and the like. The amount used will depend upon a number of factors, in¬ cluding the route of administration of the composition, the number and frequency of treatments, the formula weight of the active component of the composition, and the relative biological activity (for example, anti¬ syncytial activity or anti-infectivity activity) of the formulation employed. Where the biologically active component has a formula weight of about 1,600-2,000, 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 1-10 g, more usually in the range of about 200 mg to about 600 g, and may be in the range of about 1 mg to about 200 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 μϋ to about 10 μϋ. The subject compositions 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 conserved region. The compositions of the subject application may act by interfering 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 sequence of the CD4 binding domain on gpl20 may be used to design a vaccine to protect against HIV infection. The gpl20 molecule 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 albumin, keyhole limpet hemocyanin 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. Some evidence indicates that there is more than one sequence of amino acids proximal (first half of molecule from N-terminus) to the N-terminus of CD4 involved in interactions between CD4 and gpl20 crucial for HIV infectivity and cytopathic consequences of infection. Peptides and derivatives thereof having any of these sequences and characterized as competitive inhibitors or antagonists of the binding between CD4 and gpl20 can be used as starting fragments for the practice of the present invention. At least one of these sequences on CD4 which interacts with HIV, namely CD4(81-92), appears to be distinct from the MHC site and does not appear to be involved in CD4-dependent immune functions, including responses to alloantigens and soluble antigens. Thus immunization with either this native amino acid sequence or a derivative thereof as described above can protect against infection with HIV without compromising.the physiologic functions of CD . "Active sequence" as used hereinafter includes peptides that are fragments of the total CD4 molecule and which have the native sequence and peptides having substan¬ tially the native sequence, particularly those peptides which have been derivatized in accordance with the subject invention. In addition to use as a vaccine, the compositions can be used to prepare antibodies to the active sequences on C4 and gpl20 involved in the interaction between CD4-bearing cells and HIV. The antibodies can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the active sequence itself, or, as appropriate, bound to a carrier as described above. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the relevant sequence. The gamma globulin fractions or the IgC antibodies can be obtained, for example, by use of saturated ammonium sulfate, DEAE Sephadex, or other techniques known to those skilled in the art. To enhance the specificity of the anti-active- sequence antibody composition, the composition can be purified by adsorbing the preparation with gpl20 fragments or CD4 (as appropriate) lacking the active sequence of interest. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs. 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 by using an antibody of the same species as the host animal, for example, by using anti-active-sequence antibodies from human/human hybridomas (see below). Anti-active-sequence antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-active-sequence antibody preparation prepared as described above is used to induce anti-idiotype anti ody 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 produced by the same species as the host animal can be used or the Fc region of the antibodies to be administered 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-active-sequence antibodies or by affinity chromatography using anti-active-sequence antibodies bound to the affinity matrix. When the active site used as an immogen is CD4 active sequence, the anti-idiotype antibodies produced are specific for the region of gpl20 implicated in interactions with CD4. When used as a means of inducing anti-active- sequence antibodies in a patient, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like at an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desir- able. The induction of anti-active-sequence antibodies can alleviate problems which may be caused by passive administration of anti-active-sequence 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-viral agents, antibodies to proteins comprising the active sequence and anti-idiotype antibodies also find use in diagnostics. For both in vivo use and in diagnostics it may be preferable to use monoclonal antibodies. Monoclonal anti-active-sequence antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized 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 CD4-dependent retro- virus (where infection has been shown for example by the presence of viral antibodies in the blood or by 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 subject 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 antibodies of the desired specificity. For monoclonal anti-active- sequence antibodies, the antibodies must bind to gpl20 so as to inhibit binding of gpl20 to CD4. For monoclo- nal anti-idiotype antibodies, the antibodies must bind to the binding site region of anti-active-sequence 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 Example 1 Automated Synthesis and Bioassay of Synthetic CD4(74-92) Peptides and Fragments Thereof Prepared by t-Boc Procedure A. Synthesis (a) Basic Synthesis A series of substituted peptides derived from a portion of the sequence of the extracellular portion of the CD4 molecule were synthesized using automated solid phase peptide synthesis methodology (Applied Biosystems, Inc., Foster City, CA, Model 430A Peptide Synthesizer; Milligen/Biosearch Peptide Synthesizer 9600 Novato, CA) 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), 0-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 from the resin (post-resin mixture) com¬ prises about 65-95% inactive underivatized peptides as well as other peptide species having antisyncytial activity. After removal of HF and other volatile components 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 dimethylsul¬ fide, were removed by Sephadex G-10 chromatography. The lyophilized post-resin mixture 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 further purified by HPLC. The post- resin mixtures were heterogeneous as assessed by HPLC. (b) Synthesis of S-benzylated Analogs Derivatives of CD4(74-92) in which C86 was replaced with S-benzyl-cysteine were synthesized by substituting t-Boc-S-benzyl-cysteinefor 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 comprising a linear gradient of 10-80% acetonitrile in 20% ammonium acetate buffer. Aliquots were taken for both FAB mass spectrometric analysis and assay of antisyncytial 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 TYICbzlEVEDQKEE. This material had no antisyncytial activity at ≤ 500 /_M, the highest concentration tested. B. Bioassay of Synthetic Peptides (a) Effect on HIV-induced, CD4-dependent Cell Fusion The ability of the compositions synthesized as described in Example Al(a) 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) 323:725-728. Briefly 5xlθ4 H9.HIVHXB-2 cells were preincubated with varying concentrations of the heterogeneous peptide composition, obtained as described in Example 1, in the wells of flat-bottomed 6-well icrotiter plates for 30 minutes at 37°C. 5x104 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 2, where titer indicates the nominal peptide concentration required for complete blockade of HIV-envelope-induced CD4-dependent cell fusion after an overnight (•"'12-16 hours) 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 indicated sequence but in which only C-84 is derivatized. Purified nonderivatized CD4(74-92), which Has the sequence LKIEDSDTYICEVEDQKEE, was inactive (at £500 μH, the highest concentration tested) as measured by ability 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(74-92) were deleted had a slightly reduced ability to block HIV-induced cell fusion. The post-resin mixture from synthesis of a derivatized peptide in which the amino terminal 8 residues of CD4(74-92) were deleted had no fusion inhibiting activity at £500 μVL. 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. Table 2 Inhibition of HIV Induced Cell Fusion by CD4 Derived Synthetic Peptide Compositions Peptide Sequence ' Titer2 LKIEDSDTYICbzlEVEDQKEE4 60 μH KIEDSDTYICbzlEVEDQKEE 125 μH EDSDTYICbzlEVEDQKEE4 125 μH SDTYICbzlEVEDQKEE 125 μH TYICbzlEVEDQKEE4 125 μH YICbzlEVEDQKEE Not Active LKIEDSDTYICbzlEVEDQKE 125 μΑ TYICbzlEVEDQKE 125 μH TYICfazlEVEDQK 250 μH. TYICbzlEVE 250 μH TYICbzlEVE 500 μH YICbzlEVE Not Active 1 Preparations tested were post-resin peptide mixtures prepared by the t-Boc method, and were not homogeneous by HPLC. 2 Not active indicates no anti-syncytial activity at the highest concentration tested, 500 μH. 3 Designation Cbzl indicates synthesis conducted using as a side protection group benzyl rather than paramethyl-benzyl. The sequence shown is the nominal peptide sequence expected for the dominant synthetic product. 4 FAB-MS analysis of HPLC fractionated post resin mixtures demonstrated that the indicated mixtures comprised peptides in which C-84 and at least one other amino acid residue were derivatized. The post-resin mixture from synthesis of a derivatized peptide, in which the carboxy terminal 6 amino acids of CD4(74-92) were deleted from the carboxy terminal end of the peptide and then added back to the amino terminal end of the molecule in other than the order found in native CD4, had no activity, further demonstrating the requirement for sequence specificity of the peptide to obtain inhibition of HIV-induced cell fusion. Furthermore, the post-resin mixtures from synthesis of CD4(74-92) in which C-84 was replaced with alanine, serine or phenylalanine were not active; likewise, post-resin mixtures from synthesis of CD4(81- 92) in which C-84 was replaced with alanine, methionine or serine were not active. The ability of the post-resin mixture from synthesis of CD4(81-92) (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. H9.HIVHXB2 cells were pre-incubated for 30 min at 37°C with post-resin peptide mixtures at the nominal concentrations shown. Either VB cells or peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin A (1 μg/ml. Burroughs, Beckenha , England) were then added to the H9.HIVHXB2 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 3 indicate that the peptide blocked CD4-dependent fusion of both primary and transformed CD4-expressing cells. Table 3 Comparison of CD4(81-92) Post Resin Mixture to Inhibit HIV-induced Cell Fusion of VB Indicator Cells Compared to PHA Activated, Fresh Human Peripheral Blood Mononuclear Cells D Doossee P PHHAA PPBBMMCCss VB 500 μH — 250 μH - 125 μH - 4 6 633 μ μHH - 4 32 μH 3 4 16 μH 4 4 8 μH 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 J,- HIV-IDV, HIV-1HXB2 OR SIVucDavis were preincubated for 1 hr at 37 β C with CD4(81-92)BZL in 96-well micro- titer plates. Levels of viral expression in each infected 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 at previously reported. Results shown in Table 4 are for 24 hours after initiation of co-culture: -, no visible syncytia or pre-syncytial aggregates observed in duplicate wells; 4, maximum number of syncytia, comparable to number observed in the absence of treatment with peptide. As shown in Table 4 below, the post-resin mixture from synthesis of CD4(81-92) (t-Boc synthesis, S-benzyl protected cysteine) also completely inhibited fusion induced by SIV and two additional HIV isolates at nominal peptide concentrations for the dominant synthetic product of between 63 and 250 μH, but did not show any inhibition of cell fusion induced by HTLV-I, a non-CD4-dependent human retrovirus (not shown) . Table 4 Ability of Post-Resin Mixture of CD4(81-92) to Inhibit Fusion Induced by Several Isolates of HIV and by SIV SIV Dose HIV-TJ HIV-DV HIV-HXB2 (UC Davis)! 25 μ — — — 63 μ 4 4 - 32 μ 4 4 4 4 16 μ 4 . -_ ■ ' 4 4 4 1 Obtained from Dr. Preston Marx, California Regional Primate Center, Davis, California. ( ) ect on M xe Leukocyte Reaction Post-resin mixtures having antisyncytial activity were analyzed 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 centrifugation 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, L-glutamine, and antibiotics. One-way mixed leukocyte cultures were performed in triplicate in round bottom 96-well 4 icrotiter 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 product) of 125 μH. Cells were cultured for 6 days at 37°C in humidified 5% CO, 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(74-92) which completely blocked HIV-induced cell fusion, had significant inhibitory effects on the mixed leukocyte reaction. Representative results are shown in Table 4a for an experiment using CD4(81-92) derivatives, where the compounds are substituted at the following positions with a benzyl group: Bzl-1: C84; Bzl-2: C84, E85; Bzl-3: T81, C84, E85. The results are reported as a percent of control mixed leukocyte reaction response observed for untreated cultures. Table 4a Lack of Inhibition of Mixed Leukocyte Reaction Responses by CD4 Derived Peptide Peptide (uH) BZL-1 BZL-2 BZL-3 0 100 100 100 5 96 92 100 10 100 88 101 50 100 100 88 100 102 95 101 In an additional study, a peptide was evaluated at different concentrations in comparison to the mAb anti- Leu3a(anti-CD4) . The peptide was CD4(81-92) C84(bzl), E85(bzl) where bzl is benzyl. The compounds were tested against primary cells obtained from blood donors (number indicates donor) where the R indicates the cells were irradiated so as to serve as stimulating cells: 83 x 87R; 87 x 90R; 87 x 83R and 87 x 90R. Table 4b indi¬ cates the results, reported as percentage of central mixed leukocyte reaction response observed for untreated cultures for each stimulator responder pair. Table 4b Treatmen : NONE Anti-leu3a A*250**M A*125αM A*63/_M Concentration NA 25mg/ml 83 x 87R 100 7 96 92 1 87 x 90R 100 5 83 100 87 x 83R . 100 , 27 107 84 87 x 90R 100 5 83 100 1 A*=T IC bzl E bzl VEDQKEE (c) Effect on Viral Infectivity The ability of the post-resin peptide mixtures to inhibit infection of CD4-pos cells by cell-free HIV virions using the assay of Nara et al. , AIDS Research and Human Retroviruses (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 virual adsorption to the cells. Medium was removed and replaced with RPMI 1640/10% fetal calf serum with or without additional peptide to maintain the desired concentration. Cells were re-fed with fresh medium on day three, and syncytia counted when cell confluency was reached 5-6 days later. These data are shown in Table 5. Table 5 Inhibition of HIV-1 and HIV-2 Infection of CEM-SS Cells by CD4 ( 74- ■92 ) Post-resin Peptide Mixture Peptide . Vn/Vo 2 Vn/Vo Concentration HTLV-IIIB HIV-2 250 μH 0. 003 0 . 10 125 μH 0.003 0.34 63 μH 0.003 0.60 32 μH 0.003 0.71 16 μH 0. 15 0. 79 8 μH 0.33 0. 80 4 μH 0.47 0. 95 2 μH 0.78 >1.00 1 Peptide was present at the indicated concentration 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) . To confirm that the post-resin peptide CD4(74- 92) mixture inhibited infection of CEM-SS cells as well as the formation of syncytia, HIV-1 viral antigen p24 was measured (as described by Arthur et al., Proc. Natl. Acad. Sci. USA (1987) 84_:8583) in culture supernatants on day 6 post-infection, at the time cells were observed and quantitated for syncytia. Duplicate cultures contained an average concentration of ρ24 of 68 ng/ml. Duplicate cultures inoculated with HTLV-IIIB in the presence of 500 μH CD4(74-92) peptide mixture (without further exposure to peptide) contained an average concentration of p24 of 4.6 ng/ml when the supernatants 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(74-92) . A representative chromato- gram of 1.8 mg of CD4(74-92) (t-Boc synthesis; S- menthyl-benzyl protected cysteine) on a Vydac C8 (10x250 mm) bonded-phase semi-preparative column is shown in Figure 1. Material was post-resin CD4(74-92) dissolved in 10 mM ammonium acetate at pH 7.0. Mobile phase was (A) ammonium acetate buffer and (B) 20% ammonium acetate buffer/80% acetonitrile. The percen- tage 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 submitted to bioassay at nominal concentrations of 500 to 30 μH, in the fusion assay described above. Bioactivity (hatched bar) is expressed as doses of antisyncytial activity/reaction. (One dose is the smallest amount of material necessary to completely inhibit fusion between 50,000 H9.HIV HT TV. T IIB cells and 50,000 VB indicator cells over a 24-hr period under standard assay conditions.) (ii) CD4(81-92). Ten mg of CD4(81-92) dis¬ solved in 10 mM ammonium acetate at pH 7.0 and prepared using the t-Boc method using S-benzyl-protected cysteine was chro atographed on a Vydac C8 (10x250 mm) bonded-phase semi-preparative column. A representative chromatogram is shown in Figure 2. The mobile phase was (A) ammonium acetate buffer and (B) 20% ammonium acetate buffer/80% acetonitrile. The percentage of B in the mobile phase was varied as shown (dashed line). Bio¬ activity (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 H.9HIV HT V _ IIIB 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(74-92) . As shown in Figure 1, all of the bioactivity (antisyncytial activity) present in the post-resin mixture eluted in a series of peaks having a retention time of 5 to 8.5 min (hatched area). Aliquots of the major peak (3 to 4.5 min retention time) and the hatched area of the chromatogram in which bioactive material eluted were submitted to fast atom bombardment- mass spectrometric analysis as described by Buky & Fraser, Biomed. Mass Spectrum (1985) 12:577. The major peak 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-O (18 atomic mass units). The major peak had no measurable anti-syncytial activity. The material in the hatched area 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(81-92). 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 Buko & Fraser, supra. The major peak, which had no measurable anti¬ syncytial activity, gave a parent fragment mass (M+H = 1576) consistent with the mass of a peptide having the sequence TYICEVEDQKEE wherein the C residue has a benzyl substituent. 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) E ects o B oactive Fraction on Viral Infectivity of Heterogeneous HIV Isolates The ability of a peptide preparation to inhibit 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(81-92) (t-Boc synthesis; S-benzyl protected cysteine), 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- pretreated CEM-SS cells. To determine the effect of addition of DEAE-dextran on the anti-viral efficacy of CD4(74-92)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(74-92) 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 μH nominal concentrations of peptide, respectively (average of duplicate determinations). In the presence of DEAE-dextran, a 1:2 dilution of the 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 μH 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-dextran-pretreated CEM-SS cells for 1 hr at 37°C. Inocula were removed from the cultures by aspiration and replaced with fresh medium or medium containing the nominal concentrations of CD4(81-92)BZL shown. Results (shown in Table 6) 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 6 Relative Anti-viral Efficacy of CDR(81-92) Peak 7 on Infection of . CEM-SS Cells with Multiple Isolates of HIV-1 X Dose IIIB RF MN CC 125 μH 0.005 0.003 0.003 0.003 63 μH 0.005 0.43 0.54 0.05 32 μH 0.005 0.69 1.03 1.01 16 μH 0.04 0.84 ±1.00 ±1.00 8 μH 0.31 .±1.00 ±1.00 ±1.00 4 μH 1.06 1.00 ±1.00 ±1.00 See Footnote 1, Table 5. Example 2 Comparison of Bioactivity of S-benzylated Analogs of CD(74-92) and Fragments Thereof Prepared by Solid Phase Synthesis and by Post-synthesis Modification A. Preparation of Benzylated Derivatives A series of benzylated derivatives and other analogs of CD4(74-92) were synthesized, using benzyl bromide and other reaction compounds as indicated in Table 7 and 9 (below) and the following methods as described below. Method 1. One and one-half mg of HPLC-purified CD4(74-92) was dissolved in 120 μl acetonitrile plus 150 μl deionized water and 60 μl sodium bicarbonate (0.5 N) . Acetonitrile 200 μl , was added followed by a 40- olar excess of benzyl bromide or other reaction compound (see Table 7) . 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 chloroform. The mixture was vortexed, then the water layer and interface were collected and used for bioassay. Method 3. One mg of HPLC-purified CD4(74-92) 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 7 and 9) was then added to the CD4(74-92) solution and reacted at room temperature for 6 hrs. After completion of the reaction, the product was dried by centrifugal 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. Method 4. Purified non-derivatized CD4(74-92), prepared as described in Example l.A, was benzylated by incubation with α-bromotoluene or -bromoxylene according to the method of Erickson et al. , J. Amer. Chem. Soc. (1973) 9_5:11. Briefly, 5 mg (2.2 /unol) of CD4(81-92) was dissolved in 1.4 ml triethylamine (11 mmol) and 1.22 mg 4-methylbenzyl bromide (7.1 mol) 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 reconstituted in phosphate-buffered saline and tested for fusion-inhibiting activity. B. Preparation of S-Benzyl Derivatives S-benzyl derivatives of CD4(81-92) were prepared by one of the following methods: Method 1. Seven and one-tenth μmole of CD4(81-92) (peak 4, see Figure 2) prepared as described about in Example 2A(b) 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 aqueous phase lyophilized repeatedly. Method 2. Five mg of CD4(81-92) (peak 4, see Figure 2) purified by HPLC as described in Example l.C was submitted to chemical derivatization as described in Example 2, Method 1. The resultant peptide derivative was evaporated to dryness, reconstituted in water, extracted with one volume of chloroform, and the aqueous phase lyophilized and tested for anti-syncytial activity 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„ χ _ 2 -induced cell fusion. C. Comparison of Effect on Cell Fusion of S-benzylated Analogs (a) CD4(74-92) The antisyncytial activity of derivatives of CD4(74-92) 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 7). Table 7 Comparison of Anti-syncytial Activity of CD4(74-92) as Benzyl or Xylyl Derivatives Preparation Activity CD4(74-92) 2 Not Active Xylyl CD4(74-92) 125 μH Benzyl CD4(74-92) 63 μH Not Active = No anti-syncytial activity at £500 μH. 2 HPLC purified authentic CD4(74-92) 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(74-92) was not active. (b) CD4(81-92) The anti-syncytial activity of three preparations of derivatized CD4(81-92) were also compared. The three preparations were: (A) TYIC ,EVEDQKEE, the peptide mixture obtained as described in Example l.C by solid-phase synthesis of TYICEVEDQKEE, using the t-Boc method with S-benzyl protected cysteine; (B) the purified peptide S-benzyl- CD4(81-92) obtained by HPLC fractionation of the peptide mixture described in (A) (peak 4, see Figure 2); and (C) the peptide mixture obtained by post-synthesis derivatization of (B), using Method 2 in Example 2B above. The results are shown in Table 8 below. Table 8 Comparison of Anti-syncytial Activity of Solid-Phase and Post-Synthesis Analogs of CD4 ( 81-92 ) Method of Nominal Material Assayed Preparation Potency A. CD4(81-92) Solid phase, 250 μH post-resin B. Peak 4 CD4(81-92) Solid phase, Not Active post-HPLC [Peak 4] BZL CD4(81-92) Post-synthesis 250 μH modification of peak 4 post-HPLC As shown, only the post-resin peptide mixture and CD4(81-92) benzylated post-synthesis were biologically active. HPLC-purified S-benzyl CD4(81- 92) prepared by the t-Boc method using S-benzyl protected cysteine (peak 4) was inactive at ≤500 μH. - Table 9 Derivatives of CD4(74-92)1 Method of Reaction Compound Preparation Titer3 1. Benzyl bromide 1,2,3 60-120 μH 2. Xylyl bromide 1 cytotoxic 3. 2-chlorobenzyl bromide 3 60-120 μH bromide 4. 4-(N-Maleimidomethyl) cyclohexane-1- carboxylic acid N-Hydroxysuccinimide ester 1,2,3 <8 μH 5. 3-(2-Pyridyldithio) propionic acid N-Hydroxy-succinimide ester 2,3 32 μH 6. None not active D. 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 1 Preparations tested were not homogeneous by HPLC. 2 500 μH was the highest concentration tested. All concentrations 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. 3 Example 2A Example l.B(a). The following were the results obtained. As shown in Table 9 (above), derivatives of CD4(74-92) which inhibited HIV-induced cell fusion included those prepared using benzyl bromide, 2- chlorobenzyl bromide, 4-(N-maleimidomethyl)-cyclo- hexane-1-carboxylic acid N-hydroxysuccinimide ester or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester. Two derivatives prepared using naphthyl reaction compounds were ineffective at the con¬ centrations tested. Benzyl cysteine, including "N- terπT-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 μH) . Example 3 Solid Phase Synthesis of CD4 Derivatives by F-moc Procedure A series of CD4-derived synthetic peptide derivatives 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 (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% trifluoro- acetic acid containing 30% dimethylsulfide 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 sequences as shown in Table 10 were prepared. Table 10 Inhibition of HIV-induced Cell Fusion by CD4-derived Synthetic Peptide Composition No. Compound 1 Potency ( μH) Purity IV. T-Y-I-C(Cl-Bzl)- E(Cl-Bzl)-V-E-D-Q-K-E-E 125 ND V. T-Y-I-C(Cl-Bzl)-E(Bzl)- V-E-D-Q-K-E-E 125 ND VI. T-Y-I-C(Bzl)-E(Cl-Bzl)- 125 ND V-E-D-Q-K-E-E VII. S-D-T-Y-I-C(Bzl)- E(Benzamide)-V-E-D-Q-K-E-E 500 ND VIII. T(Bzl)-Y(Bzl)-I-C(Bzl)-E V-E-D-Q-K-E-E 63 75 X. T(Bzl)-Y-I-C(Bzl)-E(Bzl)- V-E-D-Q-K-E-E 63 80 T(Bzl)-Y-I-C(Bzl)-E(Bzl)- V-E-D-Q-K-E-E 40 >99 XI. T(Bzl)-Y-I-C(Bzl)-E- V-E-D-Q-K-E-E> 500 ND XII. T-Y-I-C(ACM)-E(Bzl)- V-E-D-Q-K-E-E> 500 ND XIII. T-Y-I-C(ACM)-E(Cl-Bzl)- V-E-D-Q-K-E-E> 500 ND XIV. T-Y-I-C(Bzl)-E(p-N0,-Bzl)- V-E-D-Q-K-E-E 250 >99 XVII. T-Y-I-C(Bzl)-E(p-methyl-Bzl) V-E-D-Q-K-E-E 125 ND XVIII, T-Y-I-C(Bzl)-E(Bzl)- V-E-D-Q-K-E-E ~200 ~100 XIX. T-Y(methyl-Bzl)-I-C(Bzl)-E- V-E-D-Q-K-E-E 250 ND XXIV. T-Y-I-S(Bzl)-E-(Bzl)- V-E-D-Q-K-E-E 500 ~65 XXIX. T(Bzl)-E-V-E(Bzl)-I-K- C(Bzl)-Q-E-D-V-E >500 >80 Table 10 Inhibition of HIV-induced Cell Fusion by CD4-derived Synthetic Peptide Composition No . Compound Potency ( μH) Purity XXX . K-E-E-I-C(Bzl)-E-(Bzl)- V-E-D-Q-T(Bzl)-Y >500 ~83 XXXI . T(Bzl)-Y(Bzl)-I-C-E- V-E-D-Q-K-E-E 500 -57 XXXIII Y-I-C(Bzl)-E(Bzl)- V-E-D-Q-K-E-E >500 -89 XXXVI . T(Bzl)Y(Bzl)IC(Bzl)EVEDQKEE- NH 2 125 ~75 " 18 " T(Bzl)YIC(Bzl)E(Bzl)VEDQKAc EE 16 >98 Abbreviations are as follows: Bzl = benzyl; Cl-Bzl - chlorobenzyl; ACM = acetamide, (-CH-CONH,) E. Analysis of Analogs bY FAB-MS The post-resin mixtures obtained from automated 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. The samples were ionized by means of 6 Kev Xenon using different matrices. 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 peptide were modified by benzyl or chlorobenzyl as shown. These data are summarized in Table 11 below. Table 11 FAB-MS Analysis of Compositions Having Anti-syncytial Activity Derivatized Compos. Subst. Amino Acids 81 TYICE di(chloro¬ Cys p .(Cl-Bzl)+ VEDQKEE 92 benzyl) GlUggCl-BZl) di(benzyl) Cys 8 .(Bzl)+ Glu°*(Bzl) 81 TYICE dibenzyl Cys 84 (Bzl)+ VEDNKEE 92 GlujjgfBzl) Cys a .(Bzl)+ Aspjjjj(Bzl) 74 LKIED benzyl & Cys 84 (Bzl)+ SDTYICE chlorobenzyl Glu°*(Bzl) VEDQKEE 92 Cys g4 (Bzl)+ Glu 85 (Cl-Bzl) Cys 84 (Bzl)+ Glu ?7 (Cl-Bzl) Example 4 ' Anti-infective, Anticytopathic and Virustatic Effects of CD4-Derived Peptides The standard CEM-SS based assay system of Nara et al. was modified to examine the anti-HIV activity of derivatized peptide preparations during various stages of the viral life cycle. In the standard assay, peptide preparations were mixed with the viral inoculum, then the virus/peptide mixture was used to inoculate CEM-SS cell monolayers for 1 hr at 37°C. After this 1-hr incubation, the virus/peptide mixture was aspirated and replaced with fresh medium, with or without peptide added at the desired concentration for the remaining 6 days of culture. In order to distinguish the various antiviral effects of the peptides, a modified assay was developed. Division of the assay into distinct kinetic phases allows examination of the effects of peptides at distinct phases of the viral life cycle. For instance, having peptide present only during the initial 1-hr virus adsorption and absent during the remainder of the culture allows specific assessment of the effects of peptides on the virus binding/adsorption process alone. Similarly, if peptides are not present during the adsorption phase but are added after removal of the virus inoculum (at the conclusion of the 1-hr bind¬ ing/adsorption phase), effects of the peptides on post- adsorption processes may be studied. Effects of peptides on early aspects of virus infection and replication may be studied by having peptides present only from the inoculation phase through the first 48 hrs of culture, and compared to effects of peptides on later stages of infection/viral replication by examining the effects of peptides present from 48 to 144 hrs of culture. Finally, effects of peptides on reducing the actual number of infected cells obtained following inoculation of a given culture may be assessed by quan- titating infectious centers obtained in the secondary infectious cell center assay (ICC) cultures. Modified As8ay Procedure Peptides were prepared as described in this application and peptide mixtures prepared and purified as described in Lifson et. al. Science (1988) 241:712- 716. The CEM-SS syncytium-forming microassay [CEM- SS/SFA/HIV] for HIV infection was performed essentially as described by Nara et. al. AIDS Res. Hum. Retroviruses (1987) 3_:283-302 with modifications as described below. Briefly, frozen viral stocks of or HIV-1- K-.—_.lJ.. obtained from supernatants of chronically infected H(9 human T-lymphoblastoid cells were thawed at room temperature, diluted into RPMI-1640/10% FBS con¬ taining antibiotics, and added 1:1 (v:v) to solutions of either RPMI-1640, PBS, or RPMI-1640/10% FBS contain- ing 100 to 140 mM sodium bicarbonate, or to otherwise identical solutions containing in addition various concentrations of peptides. These mixtures were incu¬ bated at 25°C for 60 min. 50 μ of the viral inocula, with or without peptides present, were added to 96-well- plate, poly-L-lysine-coated wells containing 50,000 CEM- SS cells from which medium had been aspirated by pipet. After 1 hr at 37°C to allow viral adsorption to the cells, media containing the viral inoculum were removed and replaced with 200 μl of RPMI-1640/10% FBS with or without peptides. Cells were incubated 37°C in a humidified atmosphere in 5% C0 2 /95% air. 48 hrs later, all media were replaced with fresh medium with or without peptides added. 72 to 96 hrs after this (upon growth to confluency of the cell monolayer) wells were examined under an inverted-phase contrast microscope and the number of syncytia counted. Vn/Vo was scored as the number syncytia counted in a treatment well divided by the average number of syncytia counted in multiple control (virus only during entire incubation) wells. When statistical comparisons are made, they are generally between the means of 3 or more treatment wells in an experiment, and 3 or more control wells, in which the standard error of the mean of treatment group is obtained by calculating individual 'Vn/Vo' values in which the denominator is the average number of syncytia in control wells and the numerator is the number of syncytia in an individual control well. After syncytia were scored, medium was removed from each well, centrifuged to remove any CEM-SS cells, and placed in a solubilization buffer for determination of viral p24 antigen levels by radioimmunoassay. The CEM- SS infectious-cell center assay (ICC) for HIV cell- associated infectivity [CEM-SS/ICC/HIV] was performed as follows: on day 5-6 after virus inoculation in the SFA media containing about 1,000,000 cells inoculated with HIV-1 and incubated in the presence or absence of peptides for varying periods of time during the previous 5 to 6 day period as described above was harvested. Medium was removed from individual wells for p24 RIA, and the cells in the well were resuspended in RPMI-1640, centrifuged to remove residual peptide, and resuspended again in RPMI-1640/10% FBS at 500 to 50,000 cells per 50 μl . These cell suspensions were added to 50,000 fresh CEM-SS cells in 50 μl medium plated onto poly-L- lysine-coated 96-well plate wells as described above. The cells were co-incubated for 48 hrs at 37°C in humidified CO-/air, and syncytia counted in each well as an index of the cell associated infectivity of the cell population derived from the previous SFA. Table 12 Effects of CD4 Peptideε ; oni Distinct Kinetic Phases of CEM-SS HIV Infectivity Assay: HIV- —HTLV-IIIB- Isolate Vn/Vo CEM/SS/ICC RIA /HIV HTLV _ IIIB Syncytia 750,000 cells p24, ng/ Peptide ! Present During Hours Compound 0-48 49-144 0-48 49-144 0-24 49-144 T4DTEbzl 167 μH .004 .004 1.3 J L2.3 3.5 4 83 μH .212 .025 n.d. n.d. 34.1 45.3 40 μH .373 .O99 n.d. n.d. 82.7 ° 93.3 Peak 7 167 μH .004 .004 0 7 30.4 0 83 μH .004 .004 n.d. n.d. 39.0 0 40 /*M .004 .005 n.d. n.d. 2.6 .23 T,C,E tribenzyl 167 μH .004 .187 0 275 0 83.6 83 μH .011 .442 n.d. n.d. 0 101 40 μH .25 .660 n.d. n.d. 13.5 71.6 Control Vo is 121.3+ 16.9 (s .e.nt. , n *•= 10) ; control ICC is 283 + 4.7 (s.e.m.. n = 6); contr is 175.8 + 56 ng/ml~(s.e.m. , n ■ 10). Table 13 Effects of CD4 Peptides on Distinct Kinetic Phases of CEM-SS HIV Infectivity Assay: HIV-1 Isolate RF-II- Vn/Vo CEM/SS/ICC RIA Svncytia/50,000 cells p24, ng HIV HTLV-] EIIB Peptide Present During Hours Compound 0-48 49-144 0-48 49-144 0-24 49-144 T4DTEbzl 167 μH .392 .343 31 29 0.9 ^ .87 83 μH .503 . ' 739 n.d. n.d. 2.0 2.2 40 μH .912 .915 n.d. n.d. 1.1 3.4 Peak 7 167 μH .004 .004 0 37 .3 .6 83 μH .110 .138 n.d. n.d. 0 2.3 40 μH .385 .745 n.d. n.d. 1.1 2.9 T,C,E tribenzyl 167 μH .004 1.00 0 274 0 3.5 83 μH .017 1.00 n.d. n.d. 0 2.4 40 μH .242 1.00 n.d. n.d. 0.5 2.9 Control Vo is 220.5 + 84 (s.c.m., n ■ 8); control ICC is 102 + 26 (s.e. ., n = 6); control is 2.3 + 0.35 ng/ml (s.e.m., n = 8). As shown in Tables 12 and 13, studies of peptides using these assay procedures, broken down into distinct kinetic phases, demonstrate that peptides including (1) T4DTE-Bzl; (2) T4DTE-S-Bzl ("Peak 7"); and (3) T(Bzl)YIC(Bzl)E(Bzl)VEDQKEE show activity during multiple distinct phases of the viral life cycle. LKIEDSDTYICEEDQKEE(SBzl) AND TYIC(Bzl)e(Bzl)-VEDQKEE also showed activity during multiple distinct phases of the viral life cycle (data not shown). Thus peptides present only during the viral adsorption phase resulted in concentration-depended inhibition of Vn/Vo values of readout. When peptides were present for the entire duration of culture, greater inhibition was observed. Interestingly, addition of peptides at 48 hrs of culture, well after the viral adsorption phase was completed, still resulted in substantial concentration- dependent inhibition of Vn/Vo values at readout, indicating a virustatic effect of the peptides on post binding/adsorption phases of the viral life cycle. A decrease in the number of infected calls obtained in such treat cultures was also documented based on diminished numbers of syncytial centers seen in the secondary ICC assays. A series of experiments analogous to the immediately previous experiments were performed using substantially the same conditions and material, except as specifically indicated below. Example 5 Anti-infective, Anticytopathic and Virustatic Effects of CD4-Derived Peptides A. Measurement of inhibition of HIV-induced cell fusion by CDA peptides Peptides at nominal concentration (concentration based on actual weight of material and molecular weight of parent or major peptide species) of l-2mM were dissolved in phosphate-buffered saline or 100-140 mM NaHCO, and diluted to appropriate working concentrations with RPMI 1640/10% FBS. 50 μl of peptide solution was added to 26 μl of RPMI 1640/10% FBS containing 50,000 HIV-infected H9 cells (^.HlV-l^g^) in individual wells of 96-well microtiter plates. After incubation of this mixture at 37°C for 30 min, 25 μl of RPMI 1640/10% FBS containing 50,000 CD4+ VB indicator cells were added and plates incubated for 24 h at 37°C in a humidified air/5% CO- atmosphere. At the end of incubation, cultures were scored for the presence or absence of fused, multinucleated cells, as described above. Nominal potency of each peptide preparation is given as the lowest concentration of peptide within a 20- fold- dilution series giving complete inhibition of fusion at 24 h. B. Quantitative Syncytial-Forming Microassay (SFA) Peptides were dissolved in PBS or 100-140 mM NaHCO, as 1 mM solutions, and diluted with RPMI to give working stocks. Peptide solutions, or medium alone, were incubated with cell-free virus in RPMI/10% fetal bovine serum with penicillin, streptomycin and antimycotic for 60 min at 25°C, and the mixtures in a total volume of 50 μl placed onto 50,000 CEM-SS cells in 96-well microtiter plates coated with poly-L-lysine which had been plated in RPMI 1640/fetal bovine serum, allowed to settle for 1 h, and medium removed just prior to addition of viral inoculum. After 1 h of viral inoculation at 37°C, supernatants containing virus were carefully pipetted from the cells, and replaced with 100 μl of fresh RPMI 1640/10% fetal bovine serum, or RPMI containing peptide. Forty-eight hours later, medium was again carefully removed by pipet, and replaced with either fresh medium, or fresh medium containing peptide. All incubations were carried out at 37°C in a humidified, 95% air/5% CO- atmosphere. Approximately 120 h later, cultures were scored for syncytia as previously described. C. p24 Assay At the end of the quantitative microassay (B above), supernatants were removed, centrifuged at 12,000 rpm for 8 min, and the clarified supernatant diluted with lysis buffer, and stored at 4°C prior to radioimmunoassay for p24 as previously described. D. Infectious Cell Center Assay (ICC) To assess potential infectious activity associated with cells inoculated with virus, with or without peptide treatment, cells were collected from duplicate microtiter wells after being scored for syncytia in the quantitative syncytial-forming micro-assay (B above), pooled, washed once in 1 ml RPMI, suspended in 2 ml RPMI, and serially diluted in the same medium. 100 μl of suspended cells, corresponding to 20-50,000, 2-5,000 and 200-500 cells, was then added to wells containing 50,000 fresh CEM-SS indicator cells on poly-L-lysine coated wells prepared as described in B above. 48 h later, plates were scored for the presence of syncytia, exactly as for scoring of the SFA. Since the incubation period of the ICC is only 48 h, syncytia formed during this time period arise exclusively from fusion of infected input cells and fresh indicator cells, and not from infection and subsequent fusion of indicator cells. Thus, quantitation of secondary syncytia formed in the ICC assay gives in index of the number of infectious cells that were present at the end of the preceding syncytial-forming assay (SFA). E. Preparation of Virus Stocks HIV ~ 1 HTLV-IIIB and HIV " 1 RF wβrβ Propagated from chronically infected H9 or CEM-SS cells and kept as frozen tittered stocks. The following data were obtained using the specific procedures described below. 50,000 CEM-SS cells/well were plated into 96 well microtiter plates coated with polylysine. 1 h later medium was removed and either fresh medium (C), or thawed, tittered virus stocks diluted into RPMI 1640/10% fetal bovine serum and previously pre-incubated at 37°C for 1 h with (V+P) or without (V) 167 μH CD4(81-92)BZL were added to each well. After 60 min at 37°C, viral inoculum (V, V+P) or medium (C) were removed and replaced with fresh medium (V, C) or medium containing 167 μH CD4(81-92)BZL (V+P). 48 h later, mediums were removed and again replaced with fresh medium (V, C) or medium containing 167 μH CD4(81- 92)BZL (V+P). 72 h after this medium change, medium was removed, clarified by centrifugation, and prepared for p24 radioimmunoassay. Cells were washed with RPMI 1640/10% fetal bovine serum and 50,000 cells plated onto 50,00 fresh uninfected CEM-SS cells in polylysine-coated microtiter plate wells. Syncytia in an entire well were counted for both SFA and ICC using an inverted phase- contract microscope. Table 14 Continuous Peptide Treatment Inhibits Syncytium Formation, p24 Elaboration and Subsequent Formation of Infectious Cell Centers After HIV-1 HTL V-IHB or HIV ~ 1 RF-II Inoculation of CEM Cells IIIB SFA (syncytia p24 ICC Compound /well) (nq/ml) (syncytia/wel CD4(83-94)BZL 31 μH 0 2 0 63 μH 0 3 0 125 μH 0 3 n.d. C,E-dibenzyl- TYICEVEDQKEE 63 μH 27 51 n.d. 125 μH 2 18 22 250 μH 0 2 n.d. Control (no peptide) 120 664 >300 B. HIV-l^ j SFA (syncytia p24 ICC Compound /well) (ng/ral) (syncytia/well CD4(83-94)BZL 7.8 μH 111 11 n.d. 31 μH 56 5 n.d. 125 μH 0 0 0 C,E-dibenzyl TYICEVEDQKEE 63 μH 160 64 n.d. 125 μH 66 16 n.d. 250 μH 0 0 0 Control (no peptide) 168 64 >300 Values are averages of duplicate (experimental) or octuplicate (control) wells from a single experiment which was repeated fou times for CD4(81-92)BZL and twice for C,E-dibenzyl-TYICEVEDQKEE with similar results. ' - Nld. ,' not determined. ' Similar values to the non-peptide control were obtained for syncytia in SFA, syncytia in ICC and p24 by treatment with 100-500 μH of the control peptide GGa-26 (Lifson et al. r Science (1988) 241:712-7 which has an acidic amino acid composition similar to the CD4(8 92) based peptides. A number of observations were made in addition to the results reported in Table 14. In this study, the period from viral inoculation to the first medium change 48 h later is referred to as the infection phase, since during this period virus binding to the surface of the cell, virus internalization and intra-cellular viral replication (in aggregate, infection) has occurred, and production of viral protein products begun, but cells have not yet produced viral progeny or expressed viral envelope proteins in significant amounts, as evidenced by lack of measurable p24 antigen in culture medium and lack of syncytia induced by production of viral envelope glycoprotein on the surface of infected cells. Incuba¬ tion continued to a total of 120 h post-inoculation during which time the cells reach confluence. This period is referred to as the transmission phase, since during this time cells infected with virus become competent to infect other CD4-positive cells by release of viral progeny or by cell fusion. Cells inoculated with HΪV' HTVL-IIIB exhibit multiple syncytia composed of 10-150 fused cells at the end of the transmission phase. Cells treated with virus plus CD4(81-92)BZL are indistinguishable in appearance from control cells not treated with virus. Results are obtained with dibenzyl and tribenzyl congeners of TYICEVEDQKEE. When present continuously during the infection and transmission phase of the syncytia-forming microassay, the di- and tri¬ benzyl compounds block syncytium formation completely in the SFA at 250 and 167 μH, respectively, with HTLV-IIIB as the input virus at a multiplicity of infection of 100-150 infectious particles per 50,000 CEM-SS cells. The above assay alone does not distinguish between the ability of CD4(81-92) derived peptides to block cell fusion and their ability to inhibit viral infection or inhibit the infectiousness of already viral infected cells. In a first study, cells were monitored for their potential to transmit virus through release of viral progeny by measurement of viral antigen p24 in culture supernatants at the end of the syncytial-forming microassay. Cells were monitored for their potential to transmit virus and induce cytopathicity via syncytium formation by measurement of infectious cell centers (ICCs) after extensive washing of cells to remove peptide and released virus particles, followed by coincubation of 5 x 10 4 infected cells with 5 x 104 uninfected indicator cells. Incubation of virally infected, otherwise untreated cells in the ICC assay results in formation of multiple infectious cell centers. Cells inoculated with virus and treated with CD4(81-92)BZL showed no infectious cell centers and were otherwise indistinguishable from control cells not exposed to virus and transferred onto fresh CEM-SS cells in a mock ICC assay. Levels of the core viral antigen p24 were likewise greatly decreased in cultures infected with HTLV-IIIB and treated with CD4(81-92)BZL. The data support the conclusion that CD4(81-92)BZL acts to inhibit primary infection of CEM-SS cells inoculated with HIV-1 in vitro, since if the peptide were only suppressing syncytium formation and not primary infection, p24 levels would be expected to be unaltered and the unused, still infectious, cells would be expected to fuse with fresh indicator cells upon removal of peptide. Analogous data was obtained for the struc¬ turally defined peptides C^E.-dibenzyl and T.C 4 E-- tribenzyl-TYICENVEDQKEE. Levels of p24 in culture supernatants and number of syncytia ICC were reduced more than 90% compared to virally-infected untreated cultures at 250 and 167 μH of dibenzyl- and tribenzyl- CD4(81-92), respectively. The data shown for a single dose of CD4(81-92)BZL with HIV* "1 H T J - T HB as t * ιe • :Ln P ut virus, were extended by measuring syncytium formation in SFA, p24 levels, and syncytium formation in ICC assay after continuous treatment with either CD4(81-92)BZL or its structurally defined conjugate, Cl, E4-dibenzyl-CD4(81-92)BZL. Upon inoculation of CEM-SS cultures with either HIV-1 HTLV _ IIIB or a second HIV-I isolate, RF-II, both compounds exhibited a dose dependent inhibition of syncytium formation, p24 production and infectious cell centers, for both viral isolates tested. CD4(81-92)BZL was much more potent that the dibenzyl compound, inhibiting syncytium formation completely for HIV-1 RTLV _ IIIB AT 31 μH compared to 125 μH for the dibenzyl compound. Complete inhibition of RF-II infection required two-four times more peptide than corresponding inhibition of infection by HIV_1 H T LV-IIIB (see Table 14) . In a second set of experiments analogous to experiments described in Example 4, where peptide derivatives were added only during either the infection (0-48 h) or transmission (38-120 h) phases of the syncytium-forming microassay, CD4(81-92)BZL showed a complete or nearly complete inhibition of syncytium formation when added during either phase of the assay. The tribenzyl compound likewise was a relatively potent inhibitor of syncytium formation when added during either the infection or transmission phases of the assay. Good correlation was observed between inhibition of syncytia in the SFA, p24 production during the last 72 h of the assay, and the number of secondary syncytia generated during the infectious cell center assay. p24 production as well as secondary syncytia were signifi¬ cantly decreased even when peptide was present only during the final 72 h of the assay, 48 h after viral inoculation. Example 6 S-Cysteine Derivatives Derivatives of T 1 E 5 ~dibenzyl-CD4(81-92) are prepared by joining to the sulfur of the cysteine a single amino acid or oligopeptide comprising cysteine. The peptide segment (17mg, lOμmole) was dissolved in 0.1 M phosphate buffer, pH 7.4, and 5 mg of DTNB (Ellman's reagent, dissolved in 1 ml of acetonitrile) added. After stirring at room temperature for 3 h, the solution was passed through a G-25 column equilibrated with deiόnized water and peak one was collected and lyophilized. After the lyophilizaon, the portion of the resulting power was screened on HPLC (0.1% TFA in water) to demonstrate reaction. The dry power prepared above was dissolved in 0.1 M phosphate buffer and 20/κnoles of the appropriate amino acid or oligopeptide added. There was an immediate change of color from yellowish white to intense yellow indicating the progress of the reaction, which was continued for 3 h. The solution was passed through a G25 column and the first major peak was isolated and lyophilized to a powdered product. The activity of the compounds was determined by the SFA assay described previously. Table 15 indicates the result. Table 15 Amino acid or oligopeptide S-(T.,E.)-dibenzyl CD4(81-92) derivatized peptides Substituent Potency, μH C 50 T-C-T-A 250 - 500 E-L-T-C-T-A > 500 C* 500 * The peptide lacked the two benzyl groups Example 7 Amine Derivatives of T 1 ,C 4 ,E 5 -Tribenzyl (T 1 C 4 E 5 TriBzl CD4(81-92) or Analogs Thereof The~derϊvatizing reagent, dissolved in a 1:1 acetonitrile: 0.1 N aqueous sodium bicarbonate solution, was added to T.C 4 E_ CD4(81-92) in aliquots with vortex mixing to provide a molar ratio of T-C 4 E-TriBzl CD4(81- 92) to derivating agent of about 1:3-5. The reactions were allowed to proceed at room temperature for 1 h and the at 37°C for 30 min. The reaction mixtures were dried by Speed-Vac for several hours. The dried pellets were then washed twice with acetone and once with anhydrous ether. The washed pellet or powder was used for HIV infection and cytopathicity in the SFA assay. The results are reported as IC. QQ , the concentration necessary for complete inhibition of syncytium formation. The formulation reaction was carried out by a somewhat different procedure, where T-C 4 E 5 TriBzl CD4(81- 92) was first suspended in dichloromethane. Formic acid was then slowly added to the T.^E-TriBzl CD4(81-92) suspension, in 0.33 volume of glacial acetic anhydride and after completion of the slow addition the mixture allowed to sit for 30 min at room temperature. The reaction mixture was dried by Speed-Vac, the dried pellet washed with anhydrous ether 2X, and the power used for bioassays. The following table indicates the results. Table 16 Inhibition of HIV Fusion Assays By T 1 C 4 E 5 TriBzl CD4(81-92) Derivatives Derivatizing Compound IC 1Q0 ,/*M Al succinimidyl 4-(N-maleimidomethyl) . cyclohexane 1-carboxylate (SMCC) 4-8 (c) A2 sulfo SMCC 4-8 (c) A3 m-Maleimidobenzoyl NHS ester 4-8 A4 methyl £-hydroxybenzimidate 32-64 AS 2 * ~ n droχ yp hen yi*aF 1 y° χal 32-64 A6 N-succinimidyl3-(4-hydroxy phenylpropionate) 32-64 A7 E-nitrophenyl chloroformate 32-64 A8 phthalic anhydride 13 A9 glutaric anhydride 13 A10 chloroacetyl chloride 32-64 All isobutyl chloride 32-64 A12 HC0 2 H/AC 2 0 < 16 1. (c) - cytotoxic 2. NHS - N-hydroxy succinimidyl In the next study a number of maleimido derivatives were made of various derivatives of the CD4-(81-92) peptide. The procedure was described previously. The assay is the SFA with continuous peptide presence, using the strain and counting the SFA on day 5. The following table indicates the results. Table 16a t** Peptide* 12.5 μH 1.25 μH 0.125 μH VI TCEtri -MBS 0.004 0.004 0.730 (0,0) (0,0) (38,38) V4 TYCtri -MBS 0.004 0.067 0.336 (0,0) (4,3) (21,14) V5 CEdi -MBS 0.004 0.004 0.404 (0,0) (0,0) (19,23) V7 CE ,Cl -MBS 0.004 0.125 0.279 (0,0) (10,3) (18,11) V8 19mer(H)-MBS 0.004 0.154 0.529 (0,0) (9,7) (47,8) V9 TCdi -MBS 0.004 0.087 0.221 (0,0) (7,2) (13,10) VI 0 TCEdi^, Cl -MBS 0.004 ' 0.076 0.365 (0,0) (4,4) (22,12) VI 1 YCdi -MBS 0.010 0.004 0.404 (0,1) (0,0) (19,23) V12 YC ,MefS-MBS 0.004 0.019 0.231 (0,0) (1,1) (3,21) 125 MM 12.5 MM 1.25 M TCEtri 0.004 0.192 0.490 (0,0) (6,14) (23,28) * The peptides indicate the amino acids which are modified and the modifying group, where φ is benzyl, Clφ is chlorobenzyl and Me^ is £-methylbenzyl. ** The ntimbers in parenthesis indicate the observed number of syncytia and the fractional results indicate the Vn/vo for average, Another series of experiment is modification of T BZL YIC BZL E BZ L VED Q KEE b Y reductive alkylation (Borch et al. J. Am Chem. Soc. (1971) 9.1*2896-2904. A 4-8 molar excess of the carboxy1 compound was used with a 4-8 molar excess of NaBH-CN based on peptide in 0.1-1% acetic acid in methanol and the reaction continued to completion. Product was separated on Sephadex G-25 followed by purification by preparative HPLC on a Vydac C8 silicon column. In the next series of experiments, T.C 4 E-TriBzl CD4(81-92) was modified by alkylating agents as described previously. The resulting products were then tested in the SFA assay as previously described. The following are the results. Table 17 Inhibition of HIV fusion assays by CD4(81-92) Derivatives T j C 4 E 5 TriBzl CD4(81-92) Derivatives IC 10 o (μH) Bl o-Br-butyrolactone < 16 B2 Benzyl bromide 50 B3 3-chlorobenzyl bromide 50 T 1 C 4 E 5 TriBzl CD4(81-92) Derivatives IC 10Q (μH) B4 2-chlorobenzyl bromide 50 B5 p-toluenesulfonyl isocyanate 50 B6 p-toluenesulfonyl chloride 50 B7 7-methylcoumarin 50 B8 4-(Bromomethyl)-6,7- dimethoxycoumarin 13 B9 2-picolyl chloride 50 B10 3-picolyl chloride 50 Bll 2-Bromopropane 50 B12 1-Bromopropane 50 B13 cyclohexyl bromide 50 B14 cyclohexyl bromide 50 B152-(bromomethyl)-tetrahydro-(2H)- pyrazine 50 B16 Marfey's reagent 32 B17 Dabsyl chloride 32 B18 Fluoroaldehyde 16 B19 Dansyl chloride 16 B20 Clytaraldehyde < 18(c) In the next study, a series of oligopep ides, for the ' most part substituted with benzyl or substituted benzyl groups were employed and reacted with MBS as described previously. The following table indicates the results. Table 18 Oli g o p e p tide derivative IC 100' ■ * uM D l T lBzl' C 4Bzl' E 5Bzl CD4(81-92) 4 D 2 T lBzl' C 4Bzl' E 5Bzl CD4(81-92) amide 6 D 3 T lBzl EYEIKC Bzl QE Bzl DVE 4 D 4 T lBzl' Y 2Bzl' C 4Bzl CD4(81-92) 6 D 5 C 4Bzl' E 5Bzl CD4(81-92) 4 D 6 TE Bzl EVE Bzl IKC Bzl QEDVE 10 D 7 C 4B Z 1' E 4(C1-B Z 1) CD4(81-92> 4 Dg LKIEDSDTYICCNEDQKCT >500 D 9 T lBzl' C 4Bzl CD4(81-92) 8 D 10 T lBzl' C 4Bzl' 4 D ll Y 2Bzl' C 4Bzl 4 D 12 Y 2(MeBzl)' C 4Bzl' CD4(81-92) 4 Where a letter and subscript appear, e.g., T., this intends the first amino acid threonine substituted with benzyl. Where a sequence appears, the sub¬ script intends a substituent, e.g., Bzl intends benzyl. Where the subscript Cl-Bzl or Me-Bzl appears this intends the substituent, chlorobenzyl and p_-methylbenzyl, respectively. In the next study, derivatives were made, where a D-amino acid was used in place of the natural L-amino acid. Again, the base oligopeptide was T.C 4 E g TriBzl CD4(81-92). The following table indicates the various sequences and their activity in the SFA assay. Table 19 Oligopeptide Derivatives 1 IC. 00 ,/*M2 El D-T χ (Bzl) 72 E2 D-Y 2 128 E3 D-I 3 32 E4 D-C 4 (Bzl) 32 E5 D-E 5 (Bzl) 128 E6 D-K 10 (Bzl) 32 E7 D-Y 2 , D-C 4 , D-E 5 , D-E ? , D-K 10 250 1. The parent oligopeptide has T., C and E5 benzyl substituted. The derivatives indicate which amino acids have been substituted and the substitution. 2. T 1 C 4 E 5 TriBzl CD4(81-92) had an activity of 40 μH. A number of the derivatives described above were vested in the syncytial-forming microassay and viru¬ static assay as described previously, where the compound was introduced initially and maintained or introduced a ter 3 days and maintained for an additional 3 day period. The observed control values were the same for both the 1 to 3 days and the 3 to 6 days, so that the 8 SFA values obtained and the 6 ICC values obtained were pooled. All other values given are duplicates. "1-3" means that peptide or antibody were present during pre- incubation with virus, 1 h virus inoculation and 48 h subsequent, with removal at 48 h. "3-6" means the agent was not present during viral pre-incubation, inoculation or 48 h subsequently, but the agent was added at 48 h and removed at 120 h, at which time syncytia were counted, cells washed once, and 50,000 cells (less for anti-Leu3a) plated for secondary ICC. The procedures are described above, the virus being HIV-1 HTLV _ IIIB . Three separate studies were carried out, with some overlap as to compositions tested. The following table indicates the result. , Table 20 Dose SFA ICC SFA ICC Compound μH (i- • 3) (1- 3) (3-6 ) (3-6) I. Peak 7 prep 100 0, 0 o. 0 0, 0 3, 4 50 0, 0 o. 0 0, 0 5 25 0, 0 - 0, 0 - - 6 11, 0 - - 0, 0 - Bl 125 0, 0 o. 0 0, 0 5, 10 62.5 0, 0 - 0, 0 — — 31.25 0, 0 - - 0, 0 - A3 126 0, 0 o. 0 0, 0 1, 2 62.5 0, 0 o. 0 31.25 0, 0 o. 0 - - A12 127 0, 0 o. 0 o. 0 4, 7 62.5 0, 0 - o. 0 31.25 0, 0 - - o. 0 - Anti- Leu3a 10 tg/ml 1, 0 2, 6 o. 0 35, 35 5 μg/ml 1, 0 2, 2 o. 0 34, 35 2.5 μg/ml 0, 0 - - o. 0 - - 1.25 μg/ml 0, 0 - — o. 0 - - Control Medium only 71 160 (50-87) (120-184) II. Peak 7 prep 100 1, 0 0, 0 o, 0 0, 4 50 0, 0 o. 0 o, 0 0, 2 25 0, 0 o, 0 6 3, 5 o. 0 T 1 C 4 E 5 TriBzl 125 0, 1 o. 0 o. 34 59, 54 CD4( 81-92) 62.5 2, 2 - 56, 51 - 31.25 29, 20 - 52, 48 A3 100 - - - - 0, 0 3, 4 6 13, 7 12, 31 Table 20 ' (Con * - t) Dose SFA ICC SFA ICC Compound μH (1-3) (1-3) (3-6) (3-6) Anti- Leu3a 5 /_g/ml - - - 0, 0* 41, 29 5 g/rnl 0, 0* 69, 61 2.5 μg/ml 0, 0 0, 0 1.25 μg/ml 0, 0, - - 0, 0 - D2 125 6, 4 9, 3 43, 68 94, 105 62.5 5, 3 78, 41 31.25 24, 18 55, 73 D3 125 7, 20 6, 2 6, 2 20, 40 62.5 29, 41 36, 29 31.25 20, 57 68, 66 Control 3.5 mM bicarbonate 68, 56 150,106 40, 39 50, 52 1.8 mM bicarbonate 75, 59 - - 79, 59 - - Medium alone 66(49-84) 83(48-123) III. T.C.E-TriBzl 1 4 5 100 0, 0 0, 0 0, 0 17, 16 50 0, 0 1, 1 0, 3 39, 60 K. 0 (AC) 25 19, 24 22, 28 32, 16 112, 44 CD«(81-92) 12 35, 31 72, 88 59, 44 132, 156 T.C.E-TriBzl 100 0, 0 0, 0 17, 49 71, 69 CD4(81-92) 50 0, 0 0, 0 75, 94 78, 98 25 16, 31 57, 62 106, 88 91, 116 12 77, 52 71, 87 108, 66 78, 101 Control Bicar bonate (nM) 1.5 81, 82 70, 61 57, 87 56, 90 0.77 97, 99 57, 72 93, 91 69, 87 0.39 139, 118 85, 110 102, 83 115, 104 0.19 113, 84 72, 104 85, 61 139, 98 Some clumping of cells. Anti-Leu3a probably underestimated since cell recovery less than control or peptide treated. The above results were further substantiated by work at a second laboratory from the one in which the above results were obtained. Where the lysine is acetylated, the acetylated lysine was used in the synthesis, rather than by post modification. The peptides were synthesized by FMOC chemistry, as benzyl (b) or acetyl (ac) derivatives. Does are given for 100% inhibition of HIV-induced cell fusion (Lifson et al. Science (1986) 232_:1123), >50% inhibition of HIV ~ 1 HTLV-IIIB inIec tion of CEM-SS cells quantitated by a syncytial-forming microassay (Nara et al. (1987) AIDS Res. Hum. Retrovir. 3_:283) and >50% inhibition of cell- mediated transmission measured by formation of syncytia between HIV-l H _-. v ____ B _infected cells incubated 120 h post-inoculation (peptide present during the final 72 h of incubation) , and .fresh uninfected CEM-SS cells (Nara and Fischinger Nature (1988) 332:469). Table 21 Fusion Infection Transmission Peptide (tfM) (tfM) (tfM) TbYICbEbVEDQKEE 63 63 125 TbYICbEbVEDQKacEE 16 10 n.d. TbYICbEVEBDQKEE >500 >250 >250 KEEICbEbVEDQTbY >500 >250 >250 Prior results have been reported indicating that soluble CD4, like anti-Leu3a, failed to block cell-mediate infection when added post-infection. As evident from the above results, a number of the subject compound, particularly the peak 7 prep, Bl, A3, and A12 are more potent that the tribenzyl derivatives T j C 4 E 5 TriBzl CD4(81-92) in inhibiting infection (1-3) and much more potent than tribenzyl or even the CD4 antibody anti-Leu3a in inhibiting cell-mediated infectivity. Interestingly, the core benzylated compound D2, while about as active as the tribenzyl CD4(81-92) to inhibit infection, is much less efficacious to inhibit cell-mediated fusion and virtually inactive as a virostatic agent. Additional modified peptides were synthesized based on the findings that for the parent peptide T(Bzl)YIC(Bzl)E(Bzl)VEDQKEE, substitution of the amino terminal group and the epsilon amino group of K10 was either tolerated with no decrease in HIV activity, or associated with an increased potency of anti-HIV activity. Thus, solution phase derivatization was employed utilizing T(Bzl)YIC(Bzl)E(Bzl)VEDQKEE produced by solid phase automated F-moc synthesis and purified to >95% purity by reverse phase HPLC as the peptide starting material and 1,2,4,5-benzenetetracarboxylic acid dianhydride and 3,3',4,4'-benzyphenone- tetracarboxylic dianhydride as the derivatizing reagents. The anti-syncytial potency of the resulting derivatized compounds, designated "XI" and "W3" respectively, is compared to the starting material in Table 22. Table 22 Effect of modification of psilon amino group of K- Q on antisyncytial potency of CD4(81-92) peptides ana evidence for cyclic nature of modified CD4 peptide W3 Antisyncytial Compound Designation Potency T(φ)YIC(φ)E(φ) VEDQKEE GLH328" 63/iM T( )YIC( )E( )VEDQK(Ac)EE "#18" 16/iM T( )YIC( )E( )VEDQK(Nicotinyl)EE - 8-16/iM 1-4 Cyclic AC 2 0 H 2 NT(^)YIC(^)E(^)VEDQK(AC)EE Non-Cyc Ac 2 0 (AC)H 2 NT(^)YIC(^)E(^)VEDQK(AC)EE-C0NH 2 16 Non-Cyc Ac 20 H 2 NT( )YIC( )E( )VEDQK(AC)EE-CONH 2 16 Non-Cyc (φ) represents benzyl (BZL) . FAB-MS analysis of an HPLC purified sample of W3 with potent antisyncytial activity indicated that the likely structure of the compound was a cyclic peptide in which the bifunctional derivatizing reagent bridged the amino groups at the amino terminus and the epsilon position of K. Q (Table 22). The relatively weak fragment ion series spectrum detected in the analysis of W3, despite the presence of a strong signal from the parent molecular ion, is relatively typical for cyclic peptides, particularly those which do not have a weak bond in the ring. Additional evidence for the probable cyclic nature of the compound is the presence of a carboxyl terminal fragment ion series corresponding to the parent molecular ion (MW 2078), and ions at 1930 and 1802, corresponding to successive losses of the two carboxyl terminal Glu residues, which would be removed before coming to K. Q involved in the putative cyclic bond with the amino terminal amino group. The absence of a clear T-Bzl signal in the spectrum from W3, despite consistent evidence of this residue in all other T(Bzl)YIC(Bzl)E(Bzl)BEDQKEE based peptides we have analyzed, provides additional evidence consistent with cyclic structure, and correlates with inability to detect the modified threonine residue by amino acid sequencing of the product, using Edman degradation chemistry. Finally, the fact that derivatization using the same reagent and reaction conditions, but using two peptides with additional substitutions to block either only the epsilon amino group of the lysine residue at position 10 in the peptide, H 2 N-T(Bzl)YIC(Bzl)E(Bzl)VEDQK(Ac)EECONH 2 , or both the amino terminal amino group and the epsilon amino group of the lysine residue at position 10 in the peptide (Ac)-HN-T(Bzl)YIC(Bzl)E(Bzl)VEDQK(Ac)EE, yielded FAB-MS spectra consistent with further derivatization and consistent with no further derivatization, respectively, as would be expected based on sites available. The increased potency of W3 may be explained by the cyclic structure functioning to lock in a conformation which is preferred for binding interactions with HIV envelope glycoproteins. This "conformation locking" has been comfirmed by preparing a cyclic analogue of TYICbzlEVEDQKEE. The analogue was synthesized with an aspartic acid residue (D) at the amino terminus of the indicated sequence. The resulting molecule was cyclized by forming an amide bond between the aspartic acid side- chain carboxylate group and the e-amino group of the lysine (K) residue in the fragment. The resulting molecule, DTYICbzlEVEDQKEE, was more potent in inhibiting syncytia formation than the non-cyclized version. Effects of compound W3 and XI on HIV-induced cell fusion were assayed using the standardized HIV-induced cell fusion assay (described above), utilizing H9 cells chronically infected with the HIV-1 isolate designated HXB-2, co-cultivated with the strongly CD4 expressing cell line VB. Representative prototype compounds included for comparison were T(Bzl)YIC(Bzl)E(Bzl)VEDQKEE (also designated "T,C,E-tri- Bzl(CD4 gl _ g2 )" or alternatively, "GLH328"); T(Bzl)YIC(Bzl)E(Bzl)VEDQK(Ac)EE (also designated "#18"); and (Ac)HN-T(Bzl)YIC(Bzl)E(Bzl)VEDQK(ac)EE-CONH 2 (also designated "#30"). Representative results are presented in Table 23. Results are expressed as the nominal molar concentration required for complete blockade of HIV- induced cell fusion in this assay. Due to the extremely high valencies of interaction which obtain in the assay, which utilizes a strongly CD4 expressing indicator cell and a chronically infected cell line which expressed high levels of viral envelope proteins, the assay con¬ stitutes an extremely robust challenge for an inhibiting agent, particularly when results are reported for complete blockade of fusion, I .- Q . Measured potencies for inhibition of HIV infection using cell free virions as the inoculum are typically much greater for a given compound . Table 23 Effect of prototupe modified CD4 peptides on CD4 dependent Immune responses - MLR Concentration Response (mean cpm x 10 »-"3,) Peptide /Ant ibody (Mg/ml ) D1S1* DΪS2 D23T D232 " N7A " 45.1 57.6 38.5 45.5 #18 3.12 43.9 54.8 37.5 32.5 6.25 46.6 57.2 32.4 41.6 12.4 53.8 54.9 35.4 47.4 25 56.4 60.6 41.5 48.2 50 45.9 57 44.1 47.3 100 41.7 53.6 28.7 35.8 #30 3.12 48.3 58.9 35.4 45.2 6.25 47.5 46.2 44.4 46.3 12.5 41.9 52.3 35.7 40 25 42.6 51.3 38.7 39.8 50 51.2 59.1 26.5 47.9 100 43.1 55.3 28.8 36.3 W3 3.12 45.7 54.6 50 46.8 6.25 45.2 50.2 48.1 52.2 12.5 48.3 55.7 41.1 46.3 25 42.3 53.7 37.8 38.5 50 43.8 61 35.3 39.6 100 43.3 56.8 38.9 22.9 XI 3.12 43.5 57.9 40.1 56 6.25 46.8 50.1 37.1 44.9 12.5 48.2 58.2 33.4 45.9 25 42.3 52 41.8 35.4 50 43.8 58.4 48.3 45.7 100 43.3 58.1 41.2 40.2 KY 3.12 48.6 52.6 45.3 41.9 6.25 38.4 51.4 39.1 47 12.5 47.4 54.6 39.2 44.9 25 40.9 55.2 46.7 52 50 52.2 55.4 31.8 41.2 100 8.3 53 21 28.4 Anti-Leu3a 3.12 16.4 39.3 2.5 22.4 6.25 14.2 37.1 4.7 23.9 12..5 7.4 40.2 4.9 13 25 7.8 40.4 3.5 15.2 50 7.1 27.7 3.4 16.1 100 6.2 29.4 2.4 9.8 * D1S1 represents Donor 1 X stimulator 1; other pairs are numbered the same manner. A number of peptides were tested for their effects on mixed leukocyte responses, including (Ax)-HN- T(Bzl)YIC(Bzl)E(Bzl)VEDQK(ax)EE-CONH 2 ("#18"), (Ac)-HN- T-(Bzl)YIC(Bzl)E(Bzl)VEDQK(Ac)EE-CONH 2 ("#30"), and the solution phase derivatization products "XI" and "W3". Table 23 demonstrates that these compounds did not significantly inhibit MLR responses at concentrations up to 100 /_g/ml. In contrast, the CD4-reactive monoclonal antibody anti-Leu 3a, included as a positive control, show significant inhibition of responses for all stimu¬ lator stimulator-responder pairs. The same prototype modified peptide compounds were tested for their effects on MHC Class II restricted cytotoxic T cell activity. None of these compounds inhibited this CD4 dependent process at concentrations up to 100 μg/ml. In contrast, the CD4-reactive monoclonal antibody anti-Leu 3a, included as a positive control, showed significant concentration dependent inhibition of the cytotoxic activity of the CD4+MHC Class II restricted T cell clone used in the assay. The prototype compounds described above therefore appear to inhibit HIV-induced cell fusion and HIV infectivity, both CD4 dependent processes, but do not appear to affect CD4 dependent immune responses. Example 8 In the next study, a different CD4 locus was employed, namely amino acids 1-25, termed the "E" locus in contrast to the "H" locus peptides previously discussed (e.g., CD481-92)* Synthesis of Peptide E The peptide of sequence corresponding to CD4(l-25) and including an N-terminal tyrosine residue (YQGNKWLGKKGDTVELTCTASQKKS) 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-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 are measured in the SFA microtiter assay, using four isolates of HIV-1. The data are for peptide incubation with virus 30 minutes before inoculation, peptide present during the 60 minute inoculation, and no peptide present during the following 5-6 days prior to coating syncytia in each cell culture well. 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 [CD4(l-25)] were tested for their ability to inhibit HIV-induced cell fusion or infection 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 μH H 63 μH 31 μH 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 50%. A hybrid molecule of two separate epitopes could also be combined for example with a disulfide bond or a flexible polyethylene linker to give a more potent inhibitor if more than one receptor epitope is bound 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. Thus a peptide E/H heterodimer is produced by disulfide bond formation between the two purified peptides, CD4(l-25) and CD4(76- 94) (peptide H) . Accordingly, these peptides are synthesized by solid-phase methodology as described herein and the desired peptides YQGNKWLGKKGDTVELTCTASQKKS and LKIEDSDTYICEVEDQKEE combined in water and allowed to stand overnight at room temperature with slow oxygen bubbling, to effect dimerizatio . Dimers are purified by high pressure molecular sieving chromatography and tested for biological activity. 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 isolates of HIV-1 as well as HIV-2. Thus, the subject compositions may provide for protection against infection and progression of symptoms associated with AIDS-related complex and from AIDS. All publications and patent applications mentioned in this specification 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 at the location where cited. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may 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 (1) at least one heteroatom on a side chain of at least one amino acid in said sequence is joined to a hydrophobic derivatizing group; (2) wherein said polypeptide is characterized as capable of modulating at least one CD4-dependent virus-induced cellular response; and (3) said polypeptide further is characterized by (a) at least one amide linkage in said polypeptide is replaced by an isosteric group or (b) said polypeptide is a cyclic polypeptide in which an amino acid in the amino-proxi al half of said polypeptide is covalently linked to an amino acid in the carboxy-proximal half of said polypeptide. 2. The polypeptide of Claim 1, wherein said at least one heteroatom is a chalcogen. 3. The polypeptide of Claim 2, wherein said chalcogen is on at least one of: the cysteine at position 84 (C84), the glutamic acid at position 85 (E85), the threonine at position 81 (T81), or the tyrosine at position 82 (Y82) of human CD4. 4. The polypeptide of Claim 2, wherein said polypeptide comprises a hydrophobic domain and a hydrophilic domain and two chalcogens of adjacent amino acids are joined to a hydrophobic derivatizing group. 5. The polypeptide of Claim 4, wherein said adjacent chalcogens are on the cysteine at position 84 (C84) and the glutamic acid at position 85 (E85) or on the threonine at position 81 (T81) and the tyrosine at position 82 (Y82) of human CD4. 6. The polypeptide of Claim 4, wherein at least one chalcogen of a non-adjacent amino acid is joined to a derivatizing group. 7. The polypeptide of Claim 6, wherein (1) said adjacent chalcogens are on the cysteine at position 84 (C84) and the glutamic acid at position 85 (E85) and said non-adjacent chalcogen is on threonine at position 81 (T81) or (2) said adjacent chalcogens are the threonine at position 81 (T81) and the tyrosine at position 82 (Y82) and said non-adjacent chalcogen is on the cysteine at position 84 (C84) of human CD4. 8. The polypeptide of 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. 9. The compound of Claim 1, wherein at least one derivatizing group is derived from 1,2,4,5-benzenetetracarboxylic acid dianhydride or 3,3',4, '-benzophenonetetracarboxylic dianhydride which has reacted with said polypeptide. 10. The polypeptide of Claim 8, wherein said aralkyl group is a benzyl or a chlorobenzyl group. 11. The polypeptide of Claim 1, wherein said sequence includes a core comprising: 84 85 T—Y—I—C—E--V--E wherein said 84 and 85 refer to amino acid positions in said human CD4. 12. The polypeptide of Claim 1, wherein said isosteric group is selected from the group consisting of -CH2-C0-, -NHNH-, -N=N-, -NHCH2-, -CH2CH2-, and -O-CO-. 13. The polypeptide of Claim 1, wherein a covalent link joins the amino-terminus amino acid of said polypeptide to said carboxy-proximal-half amino acid. 14. The polypeptide of Claim 12, wherein said covalent link is to a lysine-side-chain amino group in said carboxy-proximal half. 15. The polypeptide of Claim 13, wherein said covalent link comprises a molecule containing activated carboxyl groups that have reacted with said amino acids. 16. The polypeptide of Claim 1, wherein a side chain chalcogen on each of three amino acids in said sequence is derivatized with a benzyl or a chlorobenzyl group. 17. The polypeptide of Claim 16, wherein two of said amino acids are adjacent amino acids. 18. The polypeptide of Claim 17, wherein said three amino acids are T, Y, and C; or T, C and E. 19. The compound of Claim 1, wherein said compound inhibits virus-induced syncytium formation. 20. A pharmaceutical composition useful for interfering with a virus-induced cellular response comprising a polypeptide of Claim 1 and a pharmacologically acceptable carrier. 21. The pharmaceutical composition of Claim 20, wherein said virus induced cellular response is caused by HIV-1 or HIV-2. 22. A polypeptide of the formula TbYICbEbVEDQKEE, wherein X represents b represents a side-chain benzyl group.

Inventors: EIDEN LEE E, FRASER BLAIR, HWANG KOU, LIFSON JEFFREY D, NARA PETER L, EIDEN, LEE, E., FRASER, BLAIR, HWANG, KOU, LIFSON, JEFFREY, D., NARA, PETER, L.
Applicants: EIDEN LEE E, FRASER BLAIR, GENELABS INC, NARA PETER L, EIDEN, LEE, E., FRASER, BLAIR, GENELABS INCORPORATED, NARA, PETER, L.
Application year: 1990
Main classification: C07K7
IPC codes: A61K38, C07K7, C07K14