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WO1990013562A1 - Fragments de peptides de cd4 modifies chimiquement presentant des proprietes anti-retrovirales - Google Patents

Fragments de peptides de cd4 modifies chimiquement presentant des proprietes anti-retrovirales Download PDF

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Publication number
WO1990013562A1
WO1990013562A1 PCT/US1990/002360 US9002360W WO9013562A1 WO 1990013562 A1 WO1990013562 A1 WO 1990013562A1 US 9002360 W US9002360 W US 9002360W WO 9013562 A1 WO9013562 A1 WO 9013562A1
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Prior art keywords
polypeptide
peptide
cells
group
hiv
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English (en)
Inventor
Lee E. Eiden
Blair Fraser
Peter L. Nara
Jeffrey D. Lifson
Kou Hwang
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Genelabs Inc
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Genelabs Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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 major-histocompatibility-c ⁇ mplex (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.
  • MHC major-histocompatibility-c ⁇ mplex
  • CD4 is a 55-58 kD glycoprotein which, while originally 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, including 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).
  • HIV-1 also referred to as HTLV-III, LAV-1 or ARV
  • AIDS Acquired Immuno-deficiency Syndrome
  • 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).
  • CD4+ cells recognize MHC Class II determinants whereas CD8+ cells recognize MHC Class I determinants (Engleman et al. , J. Immunol.
  • CD4 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) 328:351-353; Rosoff et al., Cell
  • 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
  • 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 characteristic 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.
  • CD4 antigen When HIV-1 is bound to CD4+ T-cells, detection of the CD4 antigen by a specific anti-CD4 antibody (OKT4a) is blocked. Although the CD4 antigen cannot be detected on the surface of HIV-1 infected cells, intracytoplasmic complexes of CD4 and the HIV envelope glycoprotein
  • 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- terminus 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).
  • 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.
  • the hydrophobic domain will have at least two aryl substituents.
  • the molecule may be further substituted with substituents on
  • Cellular responses which can be modulated, particularly inhibited, include retrovirus-induced cell fusion and virion infectivity, as well as virus transmission following infection.
  • 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 spectrometry, 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.
  • compositions and methods for their use are provided for
  • 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).
  • 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.
  • Substituents normally will be selected to enhance the hydrophobic character of the hydrophobic domain.
  • the cysteine at position 84 (C84) will be substituted, particularly with a hydrophobic sub- stituent, more particularly an aryl group, e.g.,
  • phenyl 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.
  • 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.
  • 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.
  • 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.
  • polypeptide analog that consists of a backbone and sidechain groups that are otherwise identical to
  • 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
  • the peptide sequences may also be modified by N-terminal methylation, esterification by addition of an alcohol, C-terminal carboxyl reduction to 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
  • the subject polypeptides may be subject to various changes, such as insertions,
  • 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 providing 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.
  • heteroatom is intended any atom excluding carbon, including atoms located in ring structures and non-ring structures.
  • one of the heteroatoms is the sulfur of the cysteine at position 84 of the CD4
  • substitution s 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.
  • the derivatized group is usually the heteroatom of the amino acid C84, although a glutamic acid, usually E85, may alternatively be derivatized.
  • a glutamic acid usually E85
  • the derivatized group is usually the heteroatom of the amino acid C84, although a glutamic acid, usually E85, may alternatively be derivatized.
  • more than one heteroatom is derivatized, conveniently two adjacent chalcogens having an atomic number of 16 or less are derivatized.
  • chalcogen having an atomic number of 16 or less is intended oxygen and sulfur.
  • the chalcogens are
  • T81 threonine
  • Y82 tyrosine
  • C84 cysteine
  • E85 glutamate
  • compositions include those in which at least one
  • T81, C84 and E85 are derivatized.
  • the derivatizing groups generally have from about 1 to about 36 carbon atoms and may be aliphatic, alicyclic, 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.
  • 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 8 or C 18 columns eluted by a gradient consisting of (1) water and 0.1% TFA and (2) acetonitrile 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 derivatizing 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.
  • aryl-containing substituents 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 containing 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;
  • aryl group may have from 0 to 3 substituents, usually not more than 2 substituents, which substituents may be the same or different.
  • the derivatizing group on each heteroatom may be the same or different.
  • Preferred derivatizing groups include benzyl and chlorobenzyl.
  • the olefin will usually be conjugated with a second site of unsaturation; e.g., a carbonyl group, acyl group, maleimido group, conjugated polyolefin, or the like.
  • a second site of unsaturation e.g., a carbonyl group, acyl group, maleimido group, conjugated polyolefin, or the 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 carbodiimide, carbonyl diimidazole, or the like such as anhydride for reaction with an amine or alcohol, for example, in a protein.
  • 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:
  • n 0-20 and X is selected from H, OH, OR, SH, SR, CO 2 H, CO 2 R, NH 2 , NHR, N(R) 2 , SO 3 H, SO 2 CH 3 , or halogen, being other than H only when n is other than 0, in which R is lower alkyl (especially C 1 -C 4 , most preferably CH 3) .
  • n 1-10 and a hydrogen on any of the ring carbons is replaced by X as described in (a) above.
  • heterocyclic or substituted heterocyclic compounds such as (i) substituted pyridyl, (ii)
  • n 0-3 and R' is a pair of electrons, H, or O.
  • maleimide adducts such as m-maleimidobenzoate, N-hydroxysuccinimide ester; m-maleimidobenzoylsulfo- succinimide ester; N-succinimidyl-4-(p-maleimidophenyl) butyrate; N-succinimidyl-4-(N-maleimidomethyl)cyclo- hexane-1-carboxylate; sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate; bis-maleimidohexane; bis-maleimidomethyl ether; or N-maleimidobutyryloxysuccinimide.
  • maleimide adducts such as m-maleimidobenzoate, N-hydroxysuccinimide ester; m-maleimidobenzoylsulfo- succinimide ester; N-succinimid
  • m is 1-3 and X is N 3 ; OH; OR; NH 2 ; NHR, NO 2 ; SH; SR; halogen; CO 2 H; or aryl of from 6 to 12 carbon atoms; or
  • cytotoxic agents such as alkylating agents, for example pipobroman; thio-TEPA; chlorambucil; cyclo- phosphamide; nitrogen mustard; mephalan; or uracil mustard.
  • membrane-perturbating agents for example adriamycin; ionophores, such as valinomycin; or surface active agents, such as detergents.
  • anti-retroviral agents such as 5-azidothy- midine (AZT); dideoxycytidine (DDC); dideoxyadeno ⁇ ine (DDA); or dideoxyinosine (DDI).
  • toxins such as ricin A chain, diphtheria toxin A chain, abrin, trichosanthin, etc.
  • carbon atoms in the basic derivatizing group 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.
  • the hydrophobic domain extends to V86, and the hydrophilic domain begins with D88.
  • 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.
  • the various substituents may have different effects upon the different roles the subject compounds may play in modulating viral activity.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • an amide bond can be formed between the amino group of the amino terminus and a side-chain or
  • a side-chain carboxy group in the amino-terminus amino acid in an aspartate or glutamate
  • an amino group in a side chain in the carboxy-proxxmal half of the molecule 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.
  • 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.
  • particular derivatives may be found to be cytotoxic for in vivo use. It will then be necessary to modify the derivatives to reduce cytotoxicity or substantially eliminate toxicity at
  • the subject peptides may be employed for some applications linked to a soluble macromolecular carrier.
  • the carrier may be a polypeptide, a polysaccharide, or a combination thereof, either
  • 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
  • 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 intervening bridges of from 1 to 20 atoms in the chain.
  • the repetitive sequences may be joined by divalent functionalities, such as cystine or bis-maleimido compounds.
  • 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.
  • side-chain protecting groups may be as follows: t-butyl (D, E, S, T, Y); t-Boc (K); trityl (H); p-toluene sulfonyl (R); and benzyl (C).
  • protecting groups such as benzyl, chlorobenzyl, substituted benzyl, benzyla- mide, and chlorobenzamide can be employed for E, C, T and Y.
  • t-butyloxycarbonyl t-Boc
  • side-chain protection used during synthesis can include tosyl (R,H), O-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.
  • TFA trifluoroacetic acid
  • DCM dichloromethane
  • 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,
  • 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.
  • detergents such as sodium dodecylsulfate (SDS).
  • 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.
  • HF hydrofluoric acid
  • the HF may contain scavengers such as anisole, or thioanisole, p- thiocresol, and/or dimethylsulfide.
  • 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, preferably ammonium carbonate or bicarbonate, or DMF and
  • a carbonate-containing buffer preferably ammonium carbonate or bicarbonate, or DMF
  • 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 prepared by either the F-moc or t-Boc method are
  • HPLC high pressure liquid chromatography
  • 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 substitution 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
  • the source of the starting materials, both underivatized and derivatized, may be the post-resin mixture obtained from solid phase
  • derivatized starting materials may additionally be prepared by reaction of purified underivatized or derivatized peptide under mild conditions with reagents known to react with nucleophilic groups such as mercaptans, active halides, pseudohalides, active olefins, e.g., ⁇ , ⁇ -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 sidechain protecting groups.
  • a peptide comprising 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 purification of peptides are similar to those described.
  • the subject compounds and compositions have a number of uses and may be used in vitro and in vivo. In vitro, the subject compounds and compositions may be employed for 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.
  • the subject compounds and compositions may be used prophylactically or therapeutically for preventing infection or inhibiting propagation of CD4-dependent retroviruses such as HIV and infection of or 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 compounds additionally may be used, for example, during pregnancy or at the time of delivery to prevent
  • the subject compounds are protease resistant and may be made more protease
  • any physiologically acceptable medium may be employed, such as deionized water, saline, phosphate- buffered saline, aqueous ethanol, and the like.
  • 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, including 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, antisyncytial activity or anti-infectivity activity) of the formulation employed.
  • 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 mg, and may be in the range of about 1 mg to about 200 mg for compounds having a high relative biological
  • composition which is capable of blocking HIV-induced cell fusion in vitro at a
  • compositions also find use as
  • compositions of the subject application may act by interfering with binding between CD4 and gp120.
  • the subject derivatized peptides may also find use in binding studies with purified gp120 followed by cross-linking and biochemical analysis to localize the CD4 binding domain on the gp120.
  • 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 gp120 may be used to design a vaccine to protect against HIV infection.
  • the gp120 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.
  • 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.
  • active sequence includes peptides that are fragments of the total CD4 molecule and which have the native sequence and peptides having substantially the native sequence, particularly those peptides which have been derivatized in accordance with the subject invention.
  • compositions can be used to prepare antibodies to the active
  • the antibodies can be used directly as antiviral agents.
  • 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 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.
  • the composition can be purified by adsorbing the preparation with gp120 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.
  • 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.
  • 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.
  • serum or plasma is removed to provide an antibody composition.
  • the composition can be purified as
  • anti-active-sequence antibodies or by affinity chromatography using anti-active-sequence antibodies bound to the affinity matrix.
  • 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.
  • 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.
  • antibodies to proteins comprising the active sequence and anti-idiotype antibodies also find use in anti-viral agents.
  • antibodies to proteins comprising the active sequence and anti-idiotype antibodies also find use in anti-viral agents.
  • Monoclonal anti-active-sequence antibodies or anti-idiotype are preferred for both in vivo use and in diagnostics.
  • Monoclonal anti-active-sequence antibodies or anti-idiotype are preferred for both in vivo use and in diagnostics.
  • 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.
  • 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.
  • the antibodies For monoclonal anti-active- sequence antibodies, the antibodies must bind to gpl20 so as to inhibit binding of gpl20 to CD4.
  • the antibodies 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 material obtained following cleavage of the peptide from the resin comprises about 65-95% inactive underivatized peptides as well as other peptide species having antisyncytial activity.
  • Example Al(a) 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 5x10 4 H9.HIV HXB-2 cells were preincubated with varying concentrations of the heterogeneous peptide composition, obtained as described in Example 1, in the wells of flat-bottomed 96-well microtiter plates for 30 minutes at 37°C. 5x10 4 CD4+ VB cells were then added. The total volume was 100 ⁇ l .
  • syncytia (defined as at least four nuclei within a common cell membrane) were scored by inverted phase contrast microscope (x100) 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.
  • 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 ⁇ M .
  • 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.
  • H9 cells (50,000) infected with the viral isolates HIV- 1 TJ , HIV-I DV , HIV-1 HXB2 OR SIV UCDavis were preincubated for 1 hr at 37oC with CD4(81-92)BZL in 96-well micro- titer plates.
  • 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
  • microtiter plates using 5x10 4 responder cells and an equal number of irradiated (3000R) stimulator cells/well in a total volume of 200 ⁇ l .
  • Assays were performed using post-resin mixtures at a nominal concentration
  • Bzl-3 T81, C84, E85. The results are reported as a percent of control mixed leukocyte reaction response observed for untreated cultures.
  • 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 indicates the results, reported as percentage of central mixed leukocyte reaction response observed for untreated cultures for each stimulator responder pair.
  • 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.
  • Vn/Vo the number of syncytia/well at confluence
  • 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 ⁇ 24 of 68 ng/ml.
  • 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
  • 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 HTLV-IIIB cells and 50,000 VB indicator cells over a 24-hr period under standard assay conditions.)
  • CD4(81-92) Ten mg of CD4(81-92) dissolved in 10 mM ammonium acetate at pH 7.0 and prepared using the t-Boc method using S-benzyl-protected
  • cysteine was chromatographed on a Vydac C8 (10x250 mm) bonded-phase semi-preparative column. A representative 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). Bioactivity (hatched bar) is expressed as doses of antisyncytial activity per fraction. (One dose is the smallest amount of material necessary to completely inhibit fusion between 50,000 H.9HIV HTLV-IIIB cells and 50,000 VB indicator cells over a 24-hr period under standard assay conditions.)
  • 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.
  • cysteine with demonstrated antisyncytial activity, was tested for its effect on infectivity of multiple HIV isolates.
  • 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
  • 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.
  • ammonium acetate, pH 7.0 extracted with one volume of chloroform, and the resultant aqueous phase lyophilized repeatedly.
  • Figure 2 purified by HPLC as described in Example 1.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 HXB2 -induced cell fusion.
  • the anti-syncytial activity of three preparations of derivatized CD4(81-92) were also compared.
  • the three preparations were: (A)
  • TYIC bzl EVEDQKEE the peptide mixture obtained as described in Example 1.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
  • Example 2A Example 1.B(a). The following were the results obtained.
  • 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 concentrations tested.
  • Benzyl cysteine including "N- term”-t-Boc-blocked and CBZ-blocked "N-term”-blocked benzyl cysteine, had no effect on HIV-induced cell fusion at all concentrations tested (up to 500 ⁇ M) .
  • the peptide was side-chain deprotected and cleaved from the resin by addition of 70% trifluoroacetic 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 1.A for the t-Boc procedure.
  • 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.
  • 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.
  • 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.
  • 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 binding/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 quantitating infectious centers obtained in the secondary infectious cell center assay (ICC) cultures.
  • ICC secondary infectious cell center assay
  • 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.
  • HIV-1 RF-11 obtained from supernatants of chronically infected H(9 human T-lymphoblastoid cells were thawed at room temperature, diluted into RPMI-1640/10% FBS containing antibiotics, and added 1:1 (v:v) to solutions of either RPMI-1640, PBS, or RPMI-1640/10% FBS containing 100 to 140 mM sodium bicarbonate, or to otherwise identical solutions containing in addition various concentrations of peptides. These mixtures were incubated at 25°C for 60 min.
  • 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.
  • CEM-SS infectious-cell center assay 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.
  • 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).
  • peptides present only during the viral adsorption phase resulted in concentration-depended inhibition of Vn/Vo values of readout.
  • concentration-depended inhibition of Vn/Vo values of readout When peptides were present for the entire duration of culture, greater inhibition was observed.
  • 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.
  • Peptides at nominal concentration (concentration based on actual weight of material and molecular weight of parent or major peptide species) of 1-2mM were dissolved in phosphate-buffered saline or 100-140 mM NaHCO 3 and diluted to appropriate working concentrations with RPMI 1640/10% FBS. 50 ⁇ l of peptide solution was added to 26 ⁇ l of RPMI 1640/10% FBS containing 50,000 HIV-infected H9 cells (H9.HlV-1 HXB-2 ) in individual wells of 96-well microtiter plates.
  • 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.
  • Peptides were dissolved in PBS or 100-140 mM NaHCO 3 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 ⁇ l 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 ⁇ l of fresh RPMI 1640/10% fetal bovine serum, or RPMI containing
  • 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
  • HIV-1 HTLV-IIIB and HIV-1 RF were Propagated from chronically infected H9 or CEM-SS cells and kept as frozen tittered stocks.
  • the infection phase 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. Incubation 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 HIV-1 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.
  • the di- and tribenzyl compounds block syncytium formation completely in the SFA at 250 and 167 ⁇ M, respectively, with HTLV-IIIB as the input virus at a multiplicity of infection of 100-150 infectious particles per 50,000 CEM-SS cells.
  • CD4(81-92)BZL with HIV-1 H TLV-IIIB as the input 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, C1, E4-dibenzyl-CD4(81-92)BZL.
  • HIV-1 RTLV-IIIB AT 31 ⁇ H compared to 125 ⁇ M for the dibenzyl compound.
  • 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 significantly decreased even when peptide was present only during the final 72 h of the assay, 48 h after viral inoculation.
  • peptide segment (17mg, 10 ⁇ mole) 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.
  • DTNB Ellman's reagent
  • the dry power prepared above was dissolved in 0. 1 M phosphate buffer and 20 ⁇ moles 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.
  • T 1 ,C 4 ,E 5 -Tribenzyl T 1 C 4 E 5 TriBzl CD4(81-92) or Analogs Thereof
  • the der ⁇ vatizing reagent dissolved in a 1:1 acetonitrile: 0.1 N aqueous sodium bicarbonate solution, was added to T 1 C 4 E 5 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 100 , the concentration necessary for complete inhibition of syncytium
  • 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 1 C 4 E 5 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.
  • T 1 C 4 E 5 TriBzl CD4(81-92) was modified by alkylating agents as
  • HIV-1 HTLV-IIIB infection 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 HTLV-IIIB- 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).
  • 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,
  • 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
  • T(Bzl)YIC(Bzl)E(Bzl)VEDQKEE also designated “T,C,E-tri- Bzl(CD4 81-92 )" or alternatively, "GLH328";
  • the 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 constitutes an extremely robust challenge for an inhibiting agent, particularly when results are reported for complete blockade of fusion, IC 100 . Measured potencies for inhibition of HIV infection using cell free virions as the inoculum are typically much greater for a given compound.
  • 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.
  • 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.
  • 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(1-25) and CD4(76- 94) (peptide H). Accordingly, these peptides are synthesized by solid-phase methodology as described herein and the desired peptides
  • Dimers are purified by high pressure molecular sieving chromatography and tested for
  • compositions 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
  • compositions may provide for protection against

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Abstract

L'invention concerne un procédé général de dérivation et de sélection de fragments de peptides de CD4, présentant une capacité supérieure (par rapport au fragment original) de modulation d'une réponse cellulaire induite par virus, dépendante de la CD4. Un aspect de l'invention décrit des compositions de peptides comprenant une séquence de CD4 humaine, comportant au moins une séquence de noyau de sept acides aminés consécutifs, de la séquence proximale à terminaison L, ou une séquence comprenant la cystéine en position 84 (C84). Au moins une chaîne latérale d'acides aminés est dérivée, telle que la cystéine en position 84 (C84) ou l'acide glutamique en position 85 (E85). On peut utiliser plusieurs groupes en tant que groupes de dérivation, parmi lesquels des substituants contenant de l'aryl, ou dans le cas de la cystéine, un tioéther provenant de la réaction entre le groupe thio de la cystéine et un maléimide. On peut utiliser les compositions ainsi obtenues prophylactiquement ou thérapeutiquement, afin d'inhiber des interactions entre la CD4 et des protéines rétrovirales.
PCT/US1990/002360 1989-05-02 1990-04-27 Fragments de peptides de cd4 modifies chimiquement presentant des proprietes anti-retrovirales Ceased WO1990013562A1 (fr)

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WO1991012271A1 (fr) * 1990-02-09 1991-08-22 Instituto Cientifico Y Tecnologico De Navarra, S.A. Peptides synthetiques pour neutraliser l'infectivite du virus du sida (vih)
WO1991017179A1 (fr) * 1990-04-30 1991-11-14 Temple University Peptides analogues a des proteines de la superfamille de l'immunogobuline
WO1992020704A1 (fr) * 1991-05-21 1992-11-26 Board Of Trustees Of The University Of Illinois Inhibiteurs peptidomimetiques de la liaison du gp 120 du vih au cd4
WO1993024518A1 (fr) * 1992-05-28 1993-12-09 The Board Of Trustees Of The University Of Illinois Conception et synthese d'une substance mimetique a virage beta de cd4 inhibant la liaison gp120 du vih
FR2694560A1 (fr) * 1992-08-10 1994-02-11 Zagury Jean Francois Nouveaux peptides viraux, anticorps dirigés contre ces peptides, procédé de préparation, application à titre de médicaments, compositions pharmaceutiques et kits de diagnostic les renfermant.
US5696229A (en) * 1995-03-16 1997-12-09 The University Of Virginia Patent Foundation Polypeptide with laminin cell adhesion and morphogenesis activity
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WO1991012271A1 (fr) * 1990-02-09 1991-08-22 Instituto Cientifico Y Tecnologico De Navarra, S.A. Peptides synthetiques pour neutraliser l'infectivite du virus du sida (vih)
WO1991017179A1 (fr) * 1990-04-30 1991-11-14 Temple University Peptides analogues a des proteines de la superfamille de l'immunogobuline
WO1992020704A1 (fr) * 1991-05-21 1992-11-26 Board Of Trustees Of The University Of Illinois Inhibiteurs peptidomimetiques de la liaison du gp 120 du vih au cd4
WO1993024518A1 (fr) * 1992-05-28 1993-12-09 The Board Of Trustees Of The University Of Illinois Conception et synthese d'une substance mimetique a virage beta de cd4 inhibant la liaison gp120 du vih
FR2694560A1 (fr) * 1992-08-10 1994-02-11 Zagury Jean Francois Nouveaux peptides viraux, anticorps dirigés contre ces peptides, procédé de préparation, application à titre de médicaments, compositions pharmaceutiques et kits de diagnostic les renfermant.
US5696229A (en) * 1995-03-16 1997-12-09 The University Of Virginia Patent Foundation Polypeptide with laminin cell adhesion and morphogenesis activity
US8440806B2 (en) 2003-03-21 2013-05-14 Biotest Ag Humanized anti-CD4 antibody with immunosuppressive properties
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US8685651B2 (en) 2003-03-21 2014-04-01 Biotest Ag Method for screening for an anti-CD4 antibody suitable for use in immunotherapy
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Also Published As

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EP0462229A1 (fr) 1991-12-27
CA2022523A1 (fr) 1991-10-04
EP0462229A4 (en) 1992-03-11
AU5656890A (en) 1990-11-29

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