WO1990000566A1

WO1990000566A1 - Inhibition of hiv-1 infection with soluble cd4

Info

Publication number: WO1990000566A1
Application number: PCT/US1989/002966
Authority: WO
Inventors: Daniel R. Littman; Nathaniel R. Landau; Richard M. Myers
Original assignee: The Regents Of The University Of California
Priority date: 1988-07-11
Filing date: 1989-07-07
Publication date: 1990-01-25

Abstract

Compositions and methods for the inhibition and prevention of HIV-1 infection are provided. The compositions include polypeptides which are homologous with a region of the human CD4 molecule found at amino acids 37-159, more particularly 37-131, and still more particularly at 37-84, of the natural protein. The polypeptides comprise the effective binding region of CD4 to gp120 of HIV-1, and are useful as a blocking agent in preventing cellular infection. Compositions comprising the polypeptides in a suitable pharmaceutical carrier are also provided.

Description

INHIBITION OF HIV-1 INFECTION WITH SOLUBLE CD4

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compositions and methods for inhibiting the infection of T-lymphocytes by HIV-1. More particularly, the present invention relates to the preparation and use of a soluble polypeptide capable of binding to gp120 on HIV-1 and thus inhibiting binding of the virus to the target T-lymphocytes.

Human immunodeficiency virus (HIV-1), previously designated HTLV-III and

lymphoadenopathy-associated virus (LAV), has now been identified as the etiological agent responsible for acquired immunodeficiency syndrome (AIDS). As the spread of AIDS threatens to become pandemic, methods for treating and preventing HIV-1 infection become increasingly important.

AIDS is characterized by a depletion of

T-lymphocytes bearing the T4 surface glycoprotein (T4⁺ lymphocytes). As a result of this depletion, cellular immunity is severely impaired in AIDS patients,

frequently resulting in opportunistic infections, such as rare forms of pneumonia, and unusual neoplasms, such as Kaposi's sarcoma.

Although the detailed mechanism of HIV-1 infection is not entirely understood, it is known that HIV-1 binds to T4⁺ lymphocytes via binding to the T4 receptor itself. The T4 cell surface glycoprotein is generally referred to as CD4. In its natural function, CD4 on helper T-lymphocytes and macrophage interacts with target B-lymphocytes bearing class II major histocompatibility complex (MHC) molecules, playing an important role in the patient's natural immune

response. At present, the only accepted therapy for the treatment of AIDS relies on the use of drugs that block viral replication, such as AZT. Unfortunately, none of these blocking agents have proved to be sufficiently effective and many have severe side effects. Thus, it would be highly desirable to provide improved methods for the treatment of AIDS and HIV-1 infection,

preferably methods which would inhibit initial

infection of T-lymphocytes by HIV-1 as well as

preventing proliferation of the virus in a

previously-infected individual.

One approach that has been proposed involves the use of a soluble form of CD4 as a blocking factor to prevent binding of HIV-1 gp120 to the CD4 (T4) receptor on T-lymphocytes. Although such blockage has been demonstrated in vitro, very high concentrations of the soluble CD4 have been required. Such high

concentrations would be difficult or impossible to achieve in vivo in patients at risk or suffering from AIDS. Moreover, binding of the soluble CD4 to the class II MHC molecules on target B-lymphocytes will interfere in the patient's natural immune response, even if the damage to the T-lymphocytes population is otherwise reduced.

For these reasons, it would be desirable to provide polypeptides which are capable of binding to gp120 of HIV-1 with high affinity in order to

effectively block binding of HIV-1 to T-lymphocytes bearing the CD4 surface glycoprotein. In particular, it would be desirable if such soluble polypeptides displayed much lower affinity binding, or were

substantially free of binding, to the class II MHC molecules on a patient's B-lymphocytes.

2. Description of the Background Art

Cloning of the human CD4 gene is reported in

Maddon et al. (1985) Cell 42:93-104. Cloning of the mouse CD4 gene is reported in Littman and Getner (1987) Nature 325:453-455. The use of a soluble form of CD4 receptor to block HIV-1 infectivity is suggested in Smith et al. (1987) Science 238:1704-1707; Fisher et al. (1988) Nature 331:76-78; Hussey et al. (198) Nature 331:78-81; Deen et al. (1988) Nature 331:82-84; and

Traunecker et al. (1988) Nature 331:84-86. Except for Traunecker et al., each of these papers teach the use of substantially the entire extracellular domains of CD4 as the blocking composition. Traunecker et al.

teach the use of a CD4 fragment containing the two amino-terminal immunoglobulin-like domains, comprising amino acids 1 through 167.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful for inhibiting the infection of human T-lymphocytes by HIV-1. The compositions

comprise polypeptides of at least about 10 amino acids and having at least about 90% sequence homology with a CD4 fragment comprising amino acids 37-131 or more particularly amino acids 37-81, of a natural CD4 molecule. The polypeptides of the present invention will generally be free from regions homologous to amino acids 1-36 and 132-435 of the CD4 molecule, although they may be joined to other non-homologous sequences, or proteins, such as proteases, glycosidases, and the like, which can disrupt the HIV-1 particle after binding by the CD4 homologous region. Preferably, the polypeptides may be modified to enhance binding with gp120 of HIV-1 and to reduce or eliminate undesired binding with class II MHC molecules present on target

B-lymphocytes. In a particularly preferred embodiment, polypeptides may be joined in a multivalent construct, typically including from two to five substantially identical CD4 polypeptides, to increase binding avidity of the polypeptide to gp120. Therapeutic compositions may be prepared by combining the polypeptides with a pharmaceutically-acceptable carrier. The polypeptides of the present invention corresponding to truncated analogs of CD4 provide several benefits. First, it is expected that the shortened polypeptides will display reduced interaction with class II MHC molecules present on target

B-lymphocytes, even without further specific

modification of the amino acid sequence. Second, the shorter polypeptides will be generally easier to

produce and to combine with other amino acid sequences and proteins. Finally, the reduced size of the

polypeptide will increase the concentration at which they can be administered in therapy, providing more effective blocking activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates a series of mouse-human chimeric CD4 molecules utilized in the Experimental section hereinafter.

Fig. 2 illustrates the results of FACS analysis of the binding of human CD4 (CD4-h) and mouse CD4 (CD4-m) expressed on the cell surface of Chinese hampster ovary (CHO) cells to gp120 of HIV-1 in panel A, and the binding of eight of the chimeric CD4 as illustrated in Fig. 1 expressed on the surface of CHO cells to gp120 in panel B. The dotted lines represent binding of gp120, while the solid lines indicate binding of anti-CD4 antibody. The broken lines are controls representing the binding of anti-gp120

antibody.

Fig. 3 summarizes the essential binding regions of human CD4 identified by gp120 and various monoclonal antibodies specific for CD4 and other receptors.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides novel

compositions and methods for inhibiting HIV-1 infection of susceptible T-lymphocytes, in particular by

interfering with binding between gp120 of HIV-1 and the CD4 (T4) surface receptor present on the target

T-lymphocytes. Polypeptides of the present invention mimic the essential binding region (s) of CD4 which are found on amino acid nos. 37-159. In addition to acting as blocking agents, the polypeptides of the present invention will be useful in diagnostic assays for the detection of HIV-1 in biological samples.

Human CD4 is a 55 kilodalton (kD) cell surface glycoprotein (435 amino acids in length) found on T helper cells and macrophage. The protein is composed of four extracellular domains, a transmembrane domain, and a carboxy-terminal cytoplasmic domain. A complete amino acid sequence of CD4 with the domains shown is provided in Appendix A. The amino-terminal domain has a structure which is strongly analogous to the Ig light chain variable region and is therefore referred to as the Ig-like domain. Cysteine residues are found in the remaining three extracellular domains, providing disulphide bonding. In its natural function, CD4 binds to class II MHC molecules on target

B-lymphocytes as part of the host cellular immune response. In a less useful function, CD4 in human T-lymphocytes acts as a receptor for HIV-1, allowing HIV-1 infection of the cells.

In a broad aspect of the present invention, polypeptides comprise at least about 10 amino acids, usually being from about 30 to 150 amino acids, more usually from about 44 to 122 amino acids, and

frequently being from about 50 to 94 amino acids, and are homologous to a natural sequence within a human CD4 molecule. The natural sequence of the human CD4 molecule comprises at least amino acids 37-81, usually comprising amino acids 37-131, and more usually

comprising amino acids 37-159. Thus, the polypeptides of the present invention will be homologous to at least about 10 contiguous amino acids within the sequence of amino acids 37-81 of human CD4 and will be substantially free from homology with amino acid

sequences 1-36 and 160-435 of CD4. The polypeptides may include two or more contiguous sequences within the region 37-159, so long as at least one of the sequences comprises 10 amino acids from the amino acid sequence 37-81. Other contiguous sequences may be provided which are homologous within the region of amino acids 37-159, and as discussed in more detail hereinbelow, particular sequences may be repeated within the same polypeptide construct.

The polypeptides of the present invention will generally have at least about 90% sequence

homology with a natural human CD4 sequence, such as that provided in Appendix A. Mere sequence homology, however, will not necessarily be sufficient to provide a polypeptide according to the present invention. It is necessary also that the polypeptides have the ability to bind with most or all polymorphic forms of gp120 on HIV-1 with a high affinity of at least about 10⁶ liters/mole, usually being at least about 10⁷ liters/mole, preferably being at least 10⁸ liters/mole and more preferably being at least about 10⁹

liters/mole, or above, where binding affinity is defined as the reciprocal of the molar concentration of CD4 polypeptide required to produce one-half maximal binding to gp120.

The sequence of amino acids 37-159 of a natural CD4 sequence is as follows.

37

KILGN QGSFL

47

TKGPS KLNPR

57

ADSRR SLWDQ

67

GNFPL IIKNL KIEDS DTYIC

87

EVEDQ KEEVQ

97

LLVFG LTANS

107

DTHLL QGQSL

117

TLTLE SPPGS

127

SPSVQ CRSPA

137

GKNIQ GGKTL

147

SVSQL ELQDS

157

GTW.

The amino acid sequences of the polypeptides of the present invention need not correspond precisely to the sequences set forth above and in Appendix A. Rather, it is essential only that the peptides are capable of binding with a desired affinity to gp120 from a wide variety of HIV-1 strains. Thus, the polypeptides may embody substitutions of particular amino acids, although the substitutions will generally be within the 90% homology characteristic of the polypeptides. In particular, substitutions and random mutations may be provided in order to enhance binding affinity of the polypeptides to gp120.

Additionally, such substitutions and

mutations may also be provided in order to further diminish binding capability of the polypeptides to the class II MHC molecules of the target B-lymphocytes. Generally, the truncated polypeptides of the present invention will display an affinity below about 10 7 liters/mole toward class II MHC molecules, preferably below about 10 liters/mole, and more preferably below about 10 5 liters/mole. Ideally, the polypeptides of the present invention will be substantially free from binding toward such class II MHC molecules.

The polypeptides of the present invention may also include additional amino acid sequences not corresponding to those enumerated above and in

Appendix A. Such additional sequences may be present at either the C-terminus, N-terminuε, or both and can either be synthesized at the same time as the

homologous CD4 sequences, or may be conjugated or attached to the CD4 sequences by well known techniques.

Usually, such additional sequences will be useful in separation, conjugation, or other manipulation of the polypeptides which facilitate their use or purification in some known manner. In particular, polypeptides may include additional amino acids or be otherwise modified at the C-terminus or N-terminus to provide for binding or conjugation of the peptide to a solid phase or other protein. For example, a gly-gly-cys sequence may be added to either terminus to facilitate coupling to a carrier. Hydrophobic residues or lipid-containing moieties may be added to enhance liposome or membrane binding. When additional polypeptides not

corresponding to the CD4 sequences of the present invention are added to the polypeptides, the length will often exceed 150 amino acids.

In a particular embodiment of the present invention, the polypeptides will be joined to active substances, such as proteolytic enzymes or

glycosidaseε, in order to enhance interference with

HIV-1. The polypeptides will bind to the free viral particles as they are released from T-lymphocytes and before they bind to target T-lymphocytes, and the active substances will act to destroy the viral particles. The combination of inhibiting binding to target T-lymphocytes as well as destroying the viral particle themselves will enhance the protective effect of the compositions of the present invention. The proteolytic enzymes will break down the envelope glycoproteins, while the glycosidases will cleave sugar residues on the glycoproteins. Without the sugar residues, the viral particles cannot bind to the target cells.

In a second preferred embodiment of the present invention, multi-valent polypeptides comprising two or more repeating sequences of the present

invention may be formed.

The polypeptides of the present invention may be natural, i.e., fragments of CD4 isolated from suitable T-lymphocytes or macrophage, but will more usually be synthetic. Natural polypeptides may be isolated by conventional techniques, such as affinity chromatography. Conveniently, polyclonal or monoclonal antibodies raised against the peptide of interest may be used to prepare a suitable affinity column by well known techniques. Such techniques are taught, for example, in Hudson and Hay, Practical Immunology,

Blackwell Scientific Publications, Oxford, United

Kingdom, 1980, Chapter 8. The particular polypeptides of interest may be obtained by chemical or enzymatic cleavage of the intact CD4 molecule. Because of the difficulty in isolating intact CD4 from natural

sources, however, it is preferred to produce synthetic polypeptides based on the sequence of human CD4, as set forth above and in Appendix A. Synthetic polypeptides which are capable of binding gp120 may be produced by either of two general approaches. First, polypeptides having up to 150 amino acids, usually having fewer than about 100 amino acids, and more usually having fewer than 75 amino acids may be synthesized by the

well-known Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain. See, Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156. Apparatus for automatically synthesizing polypeptides using such solid-phase methodology is now commercially available.

The second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of

recombinant DNA molecules encoding the desired portion of the human CD4 gene. The CD4 gene may itself be natural or synthetic, with the natural gene obtainable from cDNA or genomic libraries using degenerate probes based upon the known amino acid sequence set forth above and in Appendix A. The use of a specific human cDNA clone is described in detail in the Experimental section hereinafter. Alternatively, polynucleotides may be synthesized by well-known techniques. For example, single-stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett. Letters 22:1859-1862. A double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

The natural or synthetic DNA fragments coding for the desired CD4 fragment will then be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture. Usually, the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria.

Alternatively, it may be desirable to introduce and integrate the DNA constructs into the genome of

cultured mammalian or other eukaryotic cell lines. DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the CD4 DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the CD4 DNA sequence, and transcriptional and

translational termination regulatory sequences joined to the 3'-end of the CD4 sequence. The transcriptional regulatory sequences will include a heterologous promoter which is recognized by the host.

Conveniently, available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the CD4 DNA sequences may be

employed. For transformation of mammalian and other eukaryotic cell lines, co-transfection of cell lines in the presence of a suitable marker, such as the DHFR gene, may be employed. Transfection may be

accomplished using chemical or electroporation

techniques.

To be useful in the methods of the present invention, the peptides are generally obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants. Preferably, the CD4

peptides are isolated or synthesized in a purity of at least about 80% w/w and, more preferably in at least about 95% w/w purity. Using conventional protein purification techniques, homogeneous peptides of at least 99% w/w can be obtained. For example, the proteins may be purified by use of antibodies specific for the polypeptides using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking the antibodies to a solid phase support and then contacting the linked antibodies with the source of the CD4 polypeptides, e.g., lyεates of lymphocytes or other cells which naturally or recombinantly produce CD4, or of supernatants which contain secreted CD4 polypeptides.

Antibodies to the CD4 polypeptides of the present invention may be obtained by injecting the purified polypeptide into a wide variety of vertebrates in accordance with conventional techniques. Suitable vertebrates include mice, rats, sheep, goats, and in particular mice. Usually, the animals are bled

periodically with successive bleeds having improved titer and specificity. The antigens may be injected intramuscularly, intraperitoneally, subcutaneously, or the like. Usually, a vehicle is employed, such as a complete or incomplete Freund's adjuvant. If desired, monoclonal antibodies can be prepared.

To obtain monoclonal antibodies, spleen cells from immunized vertebrates are immortalized. The manner of immortalization is not critical. Presently, the most common method is fusion with a myeloma fusion partner. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes,

retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies. Human monoclonal antibodies may be obtained by fusion of the spleen cells with an appropriate human fusion partner. A detailed technique for producing mouse x mouse monoclonal antibodies is taught by Oi and Herzenberg in: "Selected Methods in Cellular Immunology," Mishell and Shiigi (eds.), W.H. Freeman and Company, San Francisco, (1980) , pp.

351-372. The antibodies of the present invention may be of any immunoglobulin class, i.e., IgG, including IgGl, IgG2A, and IgG2B, IgA, IgD, IgE, and IgM, usually being IgG or IgM.

Polypeptides and antibodies according to the present invention may be utilized in a wide variety of assay methods suitable for detecting HIV-1 in a biological sample. In particular, the polypeptides will be reactive with substantially all strains of HIV-1, making them ideal for use for capturing HIV-1 from the sample. The polypeptides may be immobilized on a solid phase, and the solid phase exposed to the biological sample suspected of containing HIV-1, typically blood or serum. Binding of HIV-1 to the solid phase can then be detected by exposure to a labelled reagent capable of binding to the immobilized HIV-1. Labelled CD4 polypeptides according to the present invention or anti-HIV-1 antibodies would be suitable for the detection step.

The polypeptides of the present invention can be incorporated as components of pharmaceutical

compositions useful to attenuate, inhibit, or prevent HIV-1 infection of T-lymphocytes. The composition should contain a therapeutic or prophylactic amount of at least one polypeptide according to the present invention present in a pharmaceutically-acceptable carrier. The pharmaceutical carrier can be any

compatible, non-toxic substance suitable to deliver the polypeptides to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents, and the like, may also be incorporated into the pharmaceutical

compositions. Such compositions can contain a single polypeptide or may contain two or more polypeptides according to the present invention to form a

"cocktail."

The pharmaceutical compositions just described are useful for oral or parenteral

administration. Preferably, the compositions will be administered parenterally, i.e., subcutaneously, intramuscularly, or intravenously. Thus, the invention provides compositions for parenteral administration to a patient, where the compositions comprise a solution of the polypeptides in an acceptable carrier, as described above.

The concentration of polypeptide in the pharmaceutical compositions can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight, to as much as 20% by weight. A typical pharmaceutical composition for intramuscular injection would be made up to contain, for example, 1 ml of sterile buffered water and 20-100 μg of the polypeptide of the present invention. A typical

composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100-250 mg of polypeptide. Actual methods for

preparing parenterally administrable compositions are well known in the art and are described in more detail in various sources, including, for example. Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, PA (1980), which is incorporated herein by reference.

The pharmaceutical polypeptide compositions of the present invention can be administered for prophylactic and/or therapeutic treatment of HIV-1 infection. In therapeutic application, the

pharmaceutical compositions are administered to a patient already infected with HIV-1 in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Such effective dosage will depend on the severity of the infection and on the general state of the patient's own immune system, but will generally range from about 1 to 200 mg of polypeptide per

kilogram of body weight, with dosages of from about 5 to 25 mg per kilogram being more commonly employed. In life-threatening situations, it may be desirable to administer dosages substantially exceeding those set forth above.

In prophylactic applications, the pharmaceutical compositions of the present invention are administered to a patient not already infected by HIV-1, but perhaps recently exposed to or thought to have been exposed to, or at risk of being exposed to the virus. The polypeptides will then be able to block initial infection of the patient's T-lymphocytes with

HIV-1. The amount of effective dosages for this purpose, referred to as "prophylactically effective dosages," will generally be in the range from about 0.1 mg to 25 mg per kilogram of body weight, usually in the range from about 0.5 mg to 2.5 mg per kilogram of body weight.

The following examples are offered by way of illustration, not by way of limitation.

EXPERIMENTAL

Materials and Methods

1. CD4 cDNA and Preparation of Chimeric

Expression Vectors

Murine CD4 and cDNA (Littman and Gettner (1987) Nature 325:453-455) and human CD4 cDNA (Maddon et al. (1985) Cell 42:93-104) were employed. Chimeric mouse-human (mh) and human-mouse (hm) CD4 cDNA

molecules (Fig. 1) were prepared as follows.

Restriction endonuclease sites were created at

analogous positions on the mouse cDNA and human cDNA using oligonucleotide directed mutagenesis (Kunkel (1973) Proc. Natl. Acad. Sci. USA 82:488-492). The resulting mutations did not alter the encoded amino acid sequence. The new restriction sites and their positions were as follows: Avrll, 37; Xbal, 57; Ndel, 83; Kpnl, 105; Bglll, 131; ApaLI, 159; Seal, 177.

(Amino acid numbering differs by 2 amino acids from that previously reported by Maddon et al. (1985), supra . , where the actual N-terminal residue of the mature protein is incorrectly shown at position 2. A segment of each mutated CD4 cDNA was removed by

restriction endonuclease cleavage at the new

restriction site and a second site within the plasmid sequence and the reciprocal mouse or human fragment was substituted. The structure of each chimeric cDNA

(Fig. 1) was confirmed by restriction mapping with enzymes diagnostic of human or mouse CD4 cDNA, and in several cases, by DNA sequence analysis of the junction region. The chimeric cDNA was excised from the plasmid vector by cleavage with EcoRI and Sail and ligated to similarly cleaved pSV7d expression vector DNA derived from pHS210 from which the gB gene has been removed

(Stuve et al. (1987) J. Virol. 61:326-335). pSV7d contains the simian virus-40 origin of replication, early region promoter and polyadenylation sequence.

In Fig. 1, the positions of the four extracellular domains of CD4 are schematically

illustrated, with S-S denoting intra-chain disulphide bonds; V denoting the region homologous to an

immunoglobulin variable region; J denoting the region homologous to an immunoglobulin J region; T denoting the transmembrane domain; and C denoting the

cytoplasmic domain. Black bars indicate mouse CD4 sequences while white bars indicate human CD4

sequences.

2. Preparation of Cell Lines Expressing Human, Mouse, a^nd Chimeric CD4 cDNA.

The CHO DHFR- cell line DXB11 was co-transfected by the calcium-phosphate method (Graham and Van der Eb (1973) Virology 52:456-467) with CD4 expression vector DNA plus the DHFR expression plasmid, pMGlP (Mitchell et al. (1986) Molec. Cell Biol.

6:425-440). Surviving colonies were then selected for amplification of the transfected genes by growth in increasing concentrations of methotrexate (Numberg et al (1978) Proc. Natl. Acad. Sci. USA 75:5553-5556).

3. Screening of Cell Lines with gp120 and anti-CD4 Monoclonal Antibodies.

Cells were tested for ability to bind gp120 and various anti-CD4 monoclonal antibodies as follows: Recombinant gp120 (1 microgram) was incubated with 0.5 × 10⁶ transfected CHO DHFR- cells for 30 minutes at 0ºC in 100 microliters of phosphate buffered saline containing 1% fetal calf serum (PBS-FCS). The cells were washed and resuspended in 100 microliters PBS-FCS containing 200 nanograms anti-gp120 monoclonal antibody 110-3 (Genetic Systems, Seattle, Washington), and then incubated for 30 minutes at 0ºC. The cells were washed in 2.0 ,; PBS-FCS and resuspended in 100 microliters of PBS-FCS containing 4 microliters fluorescein-conjugated goat anti-mouse immunoglobulin (Becton Dickinson,

Sunnyvale, CA) and incubated for 30 minutes at 0ºC.

The cells were washed again and resuspended in 0..5 ml PBS-FCS containing 0.2 microgram/ml propidium iodide. Anti-mouse CD4 monoclonal antibody YTA-312 was used on CD4-m and CD4-hm cell lines. Anti-human CD4 monoclonal antibody IKT4 was used on CD4-h, CD4-mh and CD4-hmh cell lines. Both antibodies recognize regions of CD4 that lie carboxy-terminal to amino acid 198 (data not shown) and were therefore used to determine expression levels of chimeric CD4 cell surface protein. A number of other monoclonal antibodies specific for the

external domain of human CD4 were also tested. Cells treated with gp120 or antibody were stained with

FITC-GAMIg. Cell surface fluorescence was analyzed on a FACSCAN (Becton-Dickinson, Sunnyvale, CA) and

nonviable cells were removed from the analysis by virtue of high propidium iodide fluorescence.

RESULTS

The cell line transfected with the human CD4 expression vector (CD4-h) showed a high level of fluorescence with OKT4 (solid line; Fig. 2A) and a similar high level with gp120 plus anti-gp120 (dotted line). In contrast, the cell line transfected with the mouse CD4 expression vector (CD4-m) showed a high level of fluorescence with YTA-312 (solid line)

(corresponding to a level of CD4 expression

approximately 10-fold greater than that of mouse thymomas), but showed no significant increase over background of fluorescence with gp120 plus anti-gp120 (dotted line). Based on this result, and on additional results with CHO cells expressing highly amplified mouse CD4 genes, the minimum difference in

gp120-binding capacity between mouse and human CD4 is approximately 1000-fold. The broken line used in both Figs. 2A and 2B is a control based on binding of

anti-gp120 in the absence of gp120. No binding was observed to occur with the anti-gp120 alone. The first two domains of CD4 were examined to identify sequences that contribute to gp120 binding activity. Chimeric CD4 cDNA's in which sequence encoding the first two domains of the protein were derived from both mouse and human cDNA's were prepared by altering specific

nucleotides to create identical restriction sites at homologous positions in the mouse and human cDNA's, cleaving the cDNA's with the appropriate restriction enzyme, and ligating the reciprocal mouse and human restriction fragments. Fig. 1 shows the three types of chimeric CD4 molecules that were prepared. Those containing murine sequence followed by human sequence are designated CD4-mh, while those containing human sequence followed by murine sequence care designated CD4-hm. The third type containing human sequence followed by murine sequence followed by human sequence are designated CD4-hmh. The chimeric cDNA's were joined with no insertion or deletion of amino acid residues.

To compare the relative gp120 binding

abilities of the CD4 molecules encoded by the chimeric cDNA's, CHO cell lines expressing the chimeric proteins were established. Staining of the CD4-hm and

CD4-hmh-expressing cell lines with OKT4 showed a broad profile of CD4 expression (Fig. 2B) . CD4-mh-expressing cells were stained with YTA-312 and showed similar results. As both OKT4 and YTA-312 antibodies recognize membrane-proximal regions of CD4, positive staining confirms CD4 expression. The heterogeneity of CD4 expression level is probably due to the oligo-clonality of the cell lines and to variability in the extent of amplification of the transfected CD4 genes. The majority of the cell lines, however, expressed CD4 at levels higher than those of T-cell tumor lines. It is likely that the chimeric CD4 proteins are not misfolded because they are expressed at the cell surface and are recognized by monoclonal anti-CD4 antibodies.

CHO cells expressing the chimeric CD4

proteins were tested for their ability to bind gp120 (Fig. 2B and Table 1) . Of the cell lines expressing chimeric CD4 containing amino-terminal murine sequence, only CD4-mh-37 binds gp120 (Table 1). This result indicates that amino acids specific to human CD4 in the region from amino acid 1-37 are not necessary for gp120 binding while amino acids from 37-57 are indispensable. Chimeras containing human amino-terminal sequence display a more complex pattern of gp120 binding

(Fig. 2B). Cells expressing CD4-hm-131, CD4-hm-159 and CD4-hm-177 show full or near-full binding to gp120, while cells expressing CD4-hm-83 show low level

binding. Cells expressing CD4-hm-37 and CD4-hm-57 do not bind detectable amounts of gp120. These results suggest that amino acid residues 1-83 of human CD4 are sufficient to bind gp120. Consistent with these results, cells expressing CD4-hmh-57-105 and

CD4-hmh-57-131 fail to bind gp120, indicating a

requirement for human sequence 57-105. Taken together, these data suggest that the minimum region of human CD4 necessary for binding of gp120 lies in the region from amino acid 37-83, and, in addition, that amino acid residues 83-131 contribute to full gp120 binding activity. It is possible that amino acids 37-83 contain the entire gp120 binding site but that the presence of murine-derived amino acid sequence

C-terminal to amino acid 83 in CD4-hm-83 results in a protein whose conformation interferes with gp120 binding.

TABLE 1

Cell Surface Binding

13B

Chimera gp120 Ieu3a OKT4a OKT4b OKT4c OKT4e OKT4f MT151 MT321 VIT4 BU264 G19-2 66. 1 8.2 MAB1 G17.2

CD4-m - - - - - - - - - - - - - - - ND

CD4-h ++ + +++ +++ +++ ++ + + + + +++ +++ + + + +++ + + + + ++ +++ + ++ +++ + + +

CD4-mh-37 ++ + +++ +++ +++ +++ + + + + + + +++ - + + + + + + + + + - +++ + + +

CD4-mh-57 - - +++ +++ - - - - - ++ + + - - - -

CD4-mh-83 - - - +++ - - - - - +++ + + - _- - -

CD4-mh-105 - - - +++ - - - - - - - - - - - ND

CD4-mh131 - - - - - - - - - - - - - - - ND

CD4-mh159 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

CD4-mh-177 - - - - - - - - - - - - - - - ND

CD4-mh-37 - - - - - - - - - - - - - - - ND

CD4-hm-57 - - - - - - - - - - - - - - - ND

CD4-hm-83 + ++ ++ - - - + - - - - - + + +

CD4-hm-105 - + ++ - - - - - - + + + - + + + ++ +++ +

CD4-hm-131 ++ +++ +++ +++ +++ ++ + + + + + ++ ++ + + + ++ + + +++ +++ + + +

CD4-hm-159 +++ +++ ++ + +++ +++ + + + +++ ++ ++ + +++ +++ ++ + +++ +++ +++ ND

CD4-hm-l77 + + + +++ + + + ++ + ++ + +++ + + + +++ ++ + + + + + + + + + + ++ + + + + + + + ND

CD4-hmh- - - - + ++ - - _ _ _ _ _ _ - _ - ND

57-105

CD4-hmh- - - - - - _ _ _ _ _ _ - - _ - ND

57-131

In Table 1, gp120 and antibody binding levels were determined by comparing to binding levels of 0KT4 or YTA-312 for each cell line. (+++) indicates 75-100% of the fluorescence intensity of OKT4 or YTA-312, (++) indicates 10-<75% fluorescence intensity, (+) indicates 2-<10% fluorescence intensity, and (-) indicates <2% fluorescence intensity. MAb1 is a group of antibodies which showed an identical pattern of binding to each of the cell lines. It includes MT310, BW264/123, Fl.01-5, F101-69(ST4), T4/18T3AG, T419THY5D7, 91D6. Cells were assayed for ability to bind monoclonal antibodies as described above for gp120 binding except that anti-CD4 monoclonal antibody (1 microliter of ascites fluid) was used instead of gp120, and anti-gp120 was omitted.

In contrast to cells expressing CD4-hm-83, which bind a small amount of gp120, those expressing CD4-hm-105 do not bind a detectable amount of gp120. This result was unexpected because CD4-hm-105 contains more human-derived amino acid sequence than does

CD4-hm-83, It is unlikely that the protein encoded by CD4-hm-105 is misfolded, however, because eight monoclonal antibodies specific for the first external domain of CD4 (leu3a, 0KT3a, VIT4, G19-2, 60.1, 138.2, MABl, and G17.2) bind fully to this chimeric CD4 molecule (Table 1) . The region of CD4 from amino acid residue 83-105 links the first and second domains of the protein and might determine the relative

orientation of the two domains. In CD4-hm-105, human sequence 83-105 followed by mouse sequence 105-131 may result in a protein whose second domain hinders gp120 binding to the first CD4 domain. This hypothesis is supported by the antibody binding data (discussed below) which suggests close proximity of the two domains. Due to this conformational effect it could not be determined whether the two regions (83-105 and 105-131) form part of the gp120 binding site. The majority of anti-CD4 monoclonal

antibodies interfere with HIV infectability (only OKT4 and G19.2 do not; Sattentau et al. (1986) Science

234:1120-1123). A panel of such antibodies was tested for ability to bind to the chimeric CD4 proteins (Table 1 and summarized in Fig. 3) . Results of these studies can be summarized as follows: (1) All of the

antibodies shown in Table 1 require human sequence in the first domain of CD4 (OKT4, which is not shown, is the only exception of 22 examined). The first domain of CD4 therefore appears to be highly antigenic

compared to the rest of the protein. The high

antigenicity of the first domain of CD4 cannot be attributed to clustering of amino acid sequence

differences between mouse and human CD4 in this region as amino acid differences between mouse and human are spread throughout the external domains of the protein. (2) Nearly all of the antibodies require regions of CD4 that overlap with the region involved in gp120 binding (amino acids 37-131). (3) The pattern of binding of many of the antibodies (e.g., MAb1, Leu3a, 0KT4a, OKT4f, G17.2, 66.1) to the chimeric CDE4

proteins is similar to that of gp120; i.e., requiring amino acids 37-105, demonstrating low level binding to the chimera containing human sequence 1-83 (CD4-hm-83), and not requiring human sequence 1-37. (4) Unlike gp120, several antibodies (e.g., MAb1) bind CD4-hm-105. These antibodies do not require human sequence 105-131 and do not show the putative conformational effect previously observed in the lack of gp120 binding to this chimera. (5) Several antibodies require human amino acid residues that lie in both the first and second domains of CD4 (e.g., BU264, MT151, MT321), suggesting that the two domains interact and may be folded close to one another.

The antibodies whose pattern of binding to the CD4 chimeras most resembles that of gp120 are Leu3a, OKT4f and G17.2. Leu3a binding has been shown to be strongly conserved in primate species whose cells are infectable with HIV (OKT4f and G17.2) were not studied; McClure et al. (1987) Nature 330:487-489. In addition, Leu3a and OKT4f bind more effectively to

CD4-hm-83 than to CD4-hm-105, indicating a

conformational effect similar to that shown by gp120. These antibodies may, therefore, recognize CD4 amino acid residues identical to those recognized by gp-120 and might possess binding sites with similarity to the gp120 binding site for CD4.

The binding pattern of antibodies OKT4b and VIT4 supports localization of the minimum gp120 binding site to amino acid residues 37-83. It has been

previously shown that VIT4 blocks HIV infection

completely while OKT4b blocks infection (Sattentau et al. (1986) supra.) and gp120 binding incompletely

(McDougal et al. (1986) Science 231:382-385). As shown in Fig. 3, VIT4 appears to bind CD4 adjacent to the region of CD4 required for low level gp120 binding while OKT4b appears to bind a region of CD4 removed from that required by gp120 for low level binding but within the region required for full binding.

The anti-CD4 antibodies OKT4, G19.2, and 66.1 have been previously reported not to block HIV

infection (Sattentau (1986) supra . ) . OKT4 binds to amino acid residues C-terminal to amino acid 196 (data not shown), consistent with localization of the gp120 binding site within the amino-terminal region of CD4. G19-2 is unusual in that it requires human sequence close to that required by gp120 (Fig. 3) but does not inhibit infection. This antibody may therefore contact amino acids near but not within the region of CD4 required for gp120 binding. 66.1 appears to bind close to the region for gp120 binding. Our results show, however, that it competes effectively with gp120 for CD4 binding (data not shown). Fig. 3 illustrates the human CD4 regions required for gp120 and anti-CD4 monoclonal antibody binding. Results are derived from data in Table 1.

Solid bars indicate minimal human CD4-derived region required for significant binding; shaded bars indicate minimal human CD4-derived region required for full binding. MAb 1 represents the 7 anti-CD4 monoclonal antibodies described in Table 1.

In summary, the first Ig-like domain of CD4 appears to be the region of the protein most available for interaction with other proteins and anti-CD4 antibodies and HIV both recognize this region. The second domain appears to interact with the first domain and is likely to be positioned near it. gp120 appears to bind a portion of the first domain of CD4 and possibly part of the second domain as well, making it less likely that a peptide derived from a single region of CD4 would bind gp120 with high affinity. However, peptides derived from amino acids 37-83 would be the best candidates to exhibit gp120 binding activity.

Several anti-CD4 antibodies (Leu3a, G19.2, and OKT4f in particular) bind to CD4 with sequence requirements similar to the gp120 binding site for CD4 and therefore could be candidates for anti-idiotype mimicry studies.

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 obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide having about 90% or

greater homology with at least about 10 contiguous amino acids within sequence 37-159 of a natural human CD4, and being substantially free from sequences homologous to amino acids 1-36 and 160-435.

2. A polypeptide as in claim l, having about 90% or greater homology with at least about 44 amino acids of the sequence 37-131.

3. A polypeptide as in claim 2 , wherein the 44 amino acids are 37-81.

4. A polypeptide as in claim 1, having about 90% homology with the entire 37-131 amino acid sequence.

5. A polypeptide as in claim 1, present in a pharmaceutically-acceptable carrier.

6. A polypeptide having about 90% or greater homology with at least about 10 contiguous amino acids of a natural sequence of human CD4, said polypeptide being soluble in aqueous solution and being free from sequences homologous to amino acids 1-36 of the natural CD4 sequence.

7. A polypeptide as in claim 6, having about 90% of greater homology with at least about 44 contiguous amino acids of the natural sequence.

8. A polypeptide as in claim 6, present in a pharmaceutically acceptable carrier.

9. A polypeptide consisting essentially of 100 or fewer amino acids, said polypeptide having about 90% of greater homology with at least about 10

contiguous amino acids of the following sequence:

37

KILGN QGSFL

47

TKGPS KLNPR

57

ADSRR SLWDQ

67

GNFPL IIKNL

77

KIEDS DTYIC

87

EVEDQ KEEVQ

97

LLVFG LTANS

107

DTHLL QGQSL

117

TLTLE SPPGS

127

SPSVQ CRSPA

137

GKNIQ GGKTL

147

SVSQL ELQDS

157

GTW.

10. A polypeptide as in claim 9, having about 90% or greater homology with at least about 44 contiguous amino acids of the sequence.

11. A polypeptide as in claim 9, having about 90% or greater homology with at least the entire 94 contiguous amino acids of the sequence.

12. A polypeptide as in claim 9, present in a pharmaceutically-acceptable carrier.

13. A polypeptide consisting essentially of 100 or fewer amino acids, said polypeptide having about 90% or greater homology with amino acids 37-81 of a natural human CD4 sequence.

14. A polypeptide as in claim 13 , wherein said polypeptide has about 90% homology with amino acids 37-131 of the natural human CD4 sequence.

15. A polypeptide as in claim 13 , present in a pharmaceutically-acceptable carrier.

16. A polypeptide capable of specifically forming a complex with gp120 of HIV-1 and soluble in an aqueous solution, said polypeptide being substantially incapable of binding to class II antigens of the human major histocompatibility complex.

17. A polypeptide as in claim 16, having about 90% homology with amino acids 37-131, of human CD4 and being substantially free from sequences

homologous to amino acids 1-36 and 132-435.

18. A polypeptide as in claim 16, consisting essentially of 100 or fewer amino acids and having at least about 90% homology with amino acids 37-131 of a natural human CD4 sequence.

19. A polypeptide as in claim 16, consisting essentially of 100 or fewer amino acids and having about 90% or greater homology with at least about 30 contiguous amino acids of the following sequence:

20. A polypeptide as in claim 16, present in a pharmaceutically-acceptable carrier.

21. A single-stranded polynucleotide

encoding a polypeptide having 90% or greater homology with at least about 30 amino acids of a CD4 molecule, but not coding for the amino-terminal 36 amino acids.

22. A single-stranded polynucleotide encoding for a polypeptide having 90% or greater homology with the following amino acid sequence:

37

KILGN QGSFL

47

TKGPS KLNPR

57

ADSRR SLWDQ

67

GNFPL IIKNL

77

KIEDS DTYIC

87

EVEDQ KEEVQ.

23. A single-stranded polynucleotide encoding for a polypeptide having 90% or greater homology with the following amino acid sequence:

37

KILGN QGSFL

47

TKGPS KLNPR

57

ADSRR SLWDQ 67

GNFPL IIKNL

77

KIEDS DTYIC

87

EVEDQ KEEVQ

97

LLVFG LTANS

107

DTHLL QGQSL

117

TLTLE SPPGS

127

SPSVQ CRSPA

137

GKNIQ GGKTL

147

SVSQL ELQDS

157

GTW.

24. A cDNA molecule comprising the single-stranded polynucleotide of claim 21.

25. A vector capable of expression in cells grown in vitro , said vector comprising the

single-stranded polynucleotide of claim 20.

26. A method for inhibiting HIV-1 infection of T-lymphocytes, said method comprising exposing HIV-1 in the presence of the T-lymphocyte to a polypeptide having about 90% or greater homology with at least about 10 contiguous amino acids within sequence 37-131 of a natural human CD4 molecule, said polypeptide being substantially free from sequences homologous to amino acids 1-36 and 132-435.