WO1995031212A1 - Human cd7 related compositions and methods of using the same - Google Patents

Abstract

Essentially pure soluble human CD7 proteins are disclosed. Anti-CD7 antibodies are disclosed. Anti-CD7 antisense compounds are disclosed. Pharmaceutical compositions which comprise active ingredients selected from the group consisting of: soluble human CD7 protein, anti-CD7 antibodies and anti-CD7 antisense compounds are disclosed. Transgenic animals that comprise human CD7 are disclosed. Methods of purifying human immunodeficiency virus and methods of diagnosing and treating individuals infected with human immunodeficiency virus are disclosed.

Description

HUMAN CD7 RELATED COMPOSITIONS AND METHODS OF USING THE SAME

Acknowledgement of Government Rights

This invention was made with United States Government support under AI28767 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

Field of the Invention

The present invention relates to human CD7 and fragments thereof; antibodies, antibody fragments and peptides that comprise portions of antibodies which each bind to human CD7; and anti-CD7 antisense compounds. The present invention relates to pharmaceutical compositions to treat individuals infected with human immunodeficiency virus (HIV) which comprise human CD7 and fragments thereof; antibodies, antibody fragments and peptides that comprise portions of antibodies which each bind to human CD7; and anti-CD7 antisense compounds, and to methods of using the same. The present invention also relates to transgenic animals that comprise human CD7.

Background of the Invention

The HIV envelope glycoproteins alone are capable of mediating the cell surface membrane fusion required for infection by cell-free virions and in syncytium formation (Kowalski, M. , et al . 1987 Science 237:1351) . In contrast, the requirements for the target cell membrane in this fusion process appear to include molecules other than the CD4 receptor, such as cell-surface proteases and LFA-1 as well as several as of yet unidentified proteins (Qureshi, N.M. , et al. 1990 AIDS 4:553, Ehenbichler, C.F., et al . 1993 AIDS 7:489, Hildreth, J.E.K., et al. 1989 Science 244:1075, einer, D.B., et al . 1989 Vaccines 1989 Cold Spring Harbor Press:115, Dragic, T., et al . 1992. J. Virol . 66:4794, and Kido, H. , et al . 1990 J. Biol . Chem. 265:21979, each of which is incorporated herein by reference) . Identification of such molecules and characterization of their exact roles in HIV-directed membrane- fusion is important in the development of strategies to limit the spread of infection and to reduce the pathologic consequences of infection.

There is a need to identify the components of the cell membrane required for cell-free infection by HIV virions and in syncytium formation. There is need for anti-HIV therapeutics, for starting materials to make anti-HIV therapeutics, for assays useful to identify anti-HIV therapeutics, for methods of treating individuals infected with HIV and for transgenic animals useful as HIV models and/or models to identify and evaluate anti-HIV therapeutics.

Summary of the Invention

The present invention relates to essentially pure soluble human CD7 and fragments thereof.

The present invention relates to a pharmaceutical composition comprising soluble human CD7 or fragments thereof and a pharmaceutically acceptable carrier.

The present invention relates to antibodies, antibody fragments and peptides comprising portions of antibodies which bind to human CD7.

The present invention relates to a hybridoma cell line which produced monoclonal antibodies that bind to human CD7.

The present invention relates to a monoclonal antibody that binds to human CD7.

The present invention relates to a pharmaceutical composition comprising antibodies, antibody fragments and peptides comprising portions of antibodies which bind to human CD7, and a pharmaceutically acceptable carrier. The present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7. The present invention relates to a pharmaceutical composition comprising an isolated nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 and a pharmaceutically acceptable carrier. The present invention relates to a method of treating an individual suspected of being infected by human immunodeficiency virus comprising the steps of administering a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and an active ingredient selected from the group consisting of: soluble human CD7 protein, fragments thereof, antibodies that bind to human CD7, antibody fragments that bind to human CD7, peptides comprising portions of antibodies which bind to human CD7 and isolated nucleic acid molecules that comprise a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7.

The present invention relates to a non-human transgenic mammal, essentially all of the somatic cells of which comprise a nucleotide sequence that encodes human CD7 operably linked to regulatory sequences required for expression of said nucleotide sequence in said cells, wherein said cells produce human CD7 protein.

Brief Description of the Figures

Figure 1 has two graphs showing the effect of CD7-6B7 on HIV-l mediated syncytium formation. Figure 1 has two panels: panel A and panel B. Representative data of anti-CD7 antibody CD7-6B7 (closed box) inhibition cell-to-cell fusion mediated by HIV-1 isolate III_B (panel A) or MN (panel B) as compared to positive (anti-CD4 antibody Leu3a, closed circle) and negative (mouse serum IgG, open box) controls are shown. The total number of syncytia per well as determined by visual inspection is plotted against the concentration of antibody used.

Figure 2 is a bar graph showing results from experiments performed for epitope mapping by competition binding assay. SupT 1 cells were pretreated with each test antibody prior to binding of 3A1-FITC monoclonal antibody. All anti-CD7 antibodies except CD7-6B7 compete with 3A1 for binding. Control anti-beta-2-macroglobulin antibody BBMI does not affect 3A1-FITC binding. Figure 3 is a bar graph showing FACS Analysis of CD7- cell line. A CD7- variant of SupTl, line F8.E5 (white bars), was selected as described in the Materials and Methods and tested in flow cytometric analysis in comparison to SupTl

(black bars) for cell surface expression of relevant antigen components: CD4 (Leu3a) , CD7 (3A1, CD7-6B7, B-F12) , and LFA-1

(TS1/18, TS1/22) .

Figure 4 is a graph showing proliferation rates of CD7+ and CD7- cell lines. 5 x 10⁴ SupTl or F8.E5 cells per well of a 24-well plate were grown at 37oC and cell counts taken every 12 hours, up to 48 hours. Total number of cells per well are shown with standard deviations for four experiments. Open circles = SupTl cells, closed circles = F8.E5 cells.

Figure 5 is two graphs showing Anti-I-FA- 1 inhibition of HIV-mediated syncytium formation in CD7- cells. Figure 5 has two panels: panel A and panel B. Syncytium inhibition assays were performed with SupTl (panel A) and F8.E5 cells

(panel B) . Whereas the effects of Leu3a (closed circles) and mlg (open circles) treatment were essentially unaltered, the CD7 epitope-negative cells exhibited sensitivity to inhibition by anti-LFA-1 antibodies, TS1/22 (closed boxes) and TSl/18

(open boxes) that had no effect upon the parental CD7+ SupTl line's fusion capacity.

Figure 6 shows results from experiments performed to study cell-free infection of CD7+ and CD7- cell lines. Culture supernatants taken at day 10 following infection were analyzed for evidence of reverse transcriptase activity. Background activity (Lanes 1 and 2) for SupTl cells (row A) and F8.E5 cells was minimal. Evidence of productive infection in SupTl cells (row A, lanes 3 and 4) but not F8.E5 cells (row B, lanes 3 and 4) exposed to 100 TCID₅₀ of cell-free HIV-1 III_B demonstrates significantly reduced susceptibility of the CD7- cells to infection.

Detailed Description of the Invention and Preferred Embodiments

The CD7 antigen is a T cell surface antigen with a poorly understood immunological function. CD7 is expressed on a wide number of cells in the immune system, including approximately 80% of peripheral T cells (Reiter, C. 1989, Cluster report:CD7. In Leukocyte Typing IV, ed. by W. Kapp et al . , Oxford Univ. Press, Oxford 341) . The level of cell surface CD7 is directly related to the activation state of the T cell. CD7 is rapidly induced in vi tro through PHA or anti- CD3 treatment (Lazrovits, A.I., et al . 1987, Modulation of CD7 is associated with T lymphocyte function. In Leukocyte Typing III, ed. by A. NcMichael et al . , Oxford Univ. Press, Oxford 29, which is also observed with T activation antigens such as IL-2R and LFA-1. Specifically, down modulation of CD7 on T cells by anti-CD7 antibody leads to the inhibition of allogenic and autologous MLR as well as inhibition of antigen- or lectin- driven proliferation (Lazrovits, A.I., et al . 1988 Transpl . Proc. 6:1253, and Lazarovits, A.I., e al. 1989 Transpl . Proc. 21:3325) .

In addition to CD4, it has been observed that the presence of another cell surface molecule is required in order for cells to be infectable by cell-free virions and to be involved in syncytium formation. It has been discovered that human CD7 acts as an accessory molecule in HIV-1 infection. The presence of CD7 in addition to CD4 renders cells infectable by cell free virions. Infected cells which contain both CD7 and CD4 can form syncytium. As an accessory protein, CD7 binds to HIV proteins, or interact with proteins which may bind to HIV proteins. It has been discovered that the human CD7 antigen fulfills several criteria necessary for an accessory molecule for HIV fusion. Antibodies to the CD7 molecule are capable of limiting the scope of infection and in vi tro cytopathic effects 5 of HIV, the infectious agent for acquired immunodeficiency syndrome (AIDS) which selectively impairs the ability of T lymphocytes to respond to antigenic challenge. Furthermore, the dependence of SupTl cells on the presence of CD7 for infection by cell-free virions suggests that the role of CD7 in

10 HIV-mediated fusion is more profound than simply providing additional adhesive contacts as LFA-1 is thought to do in syncytium formation.

Both CD4 and CD7, but not LFA-1 or class I MHC, have previously been reported to be downmodulated on chronically

15. infected CEM cells (Wrightham, M. , et al . 1991 Clin . Exp .

Immunol . 85:75) . That CD7 may be specifically downmodulated by viral infection as CD4 is suggestive of a direct interaction between it and viral components. The size of CD7

(approximately 40 kDa) correlates with that of an unknown

20 protein band previously identified through anti-idiotypic serum preparations to be part of a potential secondary interaction complex for HIV-1 on human T cells (Weiner, D.B., et al . 1989 Vaccines 1989 Cold Spring Harbor Press:115, which is incorporated herein by reference) . The same size range of

25 protein has been identified by two other groups using crosslinking of T cell lysates to gp41-derived peptides (Qureshi, N.M. , et al . 1990 AIDS 4:553, and Chen, Y.H. , et al. 1992 AIDS 6:533) or bacterially produced soluble external gp41 (Ehenbichler, C.F., et al . 1993 AIDS 7:489) .

30 It has been discovered that CD7 serve as a receptor for a component of the virion, possibly as a co-receptor for the viral envelope proteins which in this system is important for virus entry. In contrast, the role of CD7 in syncytia formation may be substituted by another unknown molecule which

35 may be involved in cell adhesion.

As used herein, the term "soluble human CD7 protein" is meant to refer to human CD7 free from cells and fragments of human CD7 which retain there ability to bind to HIV proteins and/or inhibit HIV infection. Soluble CD7 protein is preferably a truncated form of the naturally occurring CD7 in which all or part of the transmembrane region has been deleted. As used herein, the term "HIV-binding fragment" is meant to refer to a fragment of human CD7 which includes the region of CD7 which binds to HIV proteins and which retains its ability to bind to HIV proteins and/or inhibit HIV infection. Fragments of CD7 comprise a portion having at least about 5 amino acids derived from CD7 and may further comprise non-CD7 amino acid sequences. The portion of an HIV-binding fragment that is derived from CD7, which is referred to herein as a "CD7 portion" directly interacts with HIV proteins and/or inhibits HIV infection. Truncated versions of CD7 may be prepared and tested using routine methods and readily available starting material. One having ordinary skill in the art can readily determine whether a protein or peptide is an HIV-binding fragment of CD7 by: 1) comparing the sequence of the peptide or protein with the sequence of CD7 to identify a CD7 portion, 2) testing the peptide or protein to determine whether it has the ability to bind to HIV proteins and/or inhibit HIV infection and 3) determining whether the CD7 portion is involved in binding of the peptide or protein to HIV proteins and/or inhibiting HIV infection. One having ordinary skill in the art can readily determine whether a protein or peptide is an HIV-binding fragment of CD7 without undue experimentation. Sequence analysis can be performed routinely. Similarly, binding studies to determine affinity of a peptide or protein to an HIV protein and infection assays to determine whether a peptide or protein inhibits HIV infection can be performed routinely. Using antibody mapping techniques, the portion of a peptide or protein that binds to another peptide or protein can be determined without undue experimentation. Contemplated equivalents of soluble human CD7 protein including HIV-binding fragments thereof include peptides, polypeptides, molecules comprising amino acids linked by non- peptidal bonds, or proteins which comprise an amino acid sequence that is identical or substantially homologous to at least a portion of the CD7 protein amino acid sequence and which are capable of binding to HIV proteins and/or inhibit HIV infection. The term "substantially homologous" refers to an amino acid sequence that has conservative substitutions.

One aspect of the present invention provides essentially pure, isolated soluble human CD7 protein. The isolated soluble human CD7 protein is provided essentially free from other cellular proteins and materials.

Soluble human CD7 protein may be used in diagnostic assays and kits to identify HIV in a sample. For example in some embodiments, an assay is performed wherein soluble human CD7 protein is combined with a sample and complexes that are formed which include soluble human CD7 protein bound to HIV particles which can then be detected. In some embodiments, the presence of the complex is detected by migrating the assay products through an electrophoresis gel and comparing the distance travelled by proteins in the assay product with a control which include uncomplexed soluble human CD7 protein and/or a control that includes complexed soluble human CD7 protein/HIV particle or a molecule that migrates with the same apparent molecule weight of the complex. In another embodiment, the soluble human CD7 protein may be fixed to a solid phase and contacted with a sample. Complexes formed between the immobilized soluble human CD7 protein and HIV particles can be detected indicating the presence of HIV particles in the sample. Kits are designed to provide each of the various reagents in containers. Those having ordinary skill in the art can readily use soluble human CD7 protein and well known techniques and starting materials to practice these or other assays to identify the presence of HIV particles in a sample. By obtaining samples from individuals and performing such assays, individuals can be identified as being infected with HIV.

The soluble human CD7 protein may also be used in HIV purification assays and kits. For example, columns can be loaded or charged with soluble human CD7 protein to produce columns with immobilized soluble human CD7 protein and samples which contain HIV are passed through the column. HIV in the sample binds to the immobilized soluble human CD7 protein and the column is washed of other components form the sample. The conditions in the column are then changed to bring about the release of the HIV from the immobilized soluble human CD7 protein thereby allowing the pure HIV to be collected. Those having ordinary skill in the art can readily apply standard techniques to purify HIV using soluble human CD7 protein.

The pure, isolated soluble human CD7 protein is useful as an anti-HIV agent. Anti-HIV therapeutics which comprise soluble human CD7 protein bind to HIV proteins when contacted with cell-free HIV virions or cells that are infected with HIV and which display HIV proteins on their cell surfaces. By binding to the HIV protein, the soluble human CD7 protein prevents the HIV protein from interacting with CD7 on the cell surface and thus interferes with HIV infection and cell to cell spread of HIV. Accordingly, one aspect of the present invention is a pharmaceutical composition comprising soluble human CD7 protein in combination with a pharmaceutically acceptable carrier. Soluble human CD7 protein is combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical compositions which can be administered in therapeutically effective amounts to persons diagnosed as or suspected of being infected with HIV in a method of treating such persons. Such persons are readily identifiable by those having ordinary skill in the art. Another aspect of the present invention is a method of treating an individual suspected of being infected with HIV which comprises the step of administering to said individual a therapeutically effective amount of a soluble human CD7 protein. As used herein, the term "effective amount of a soluble human CD7 protein" is meant to refer to the amount of protein necessary to inhibit HIV infection of cells in the individual and/or the amount of anti-viral material necessary to inhibit cell-cell spread of viral infection by inhibiting syncytium formation between infected and uninfected cells. Effective amounts include both the amount effective to eliminate the progress of infection as well as the amount effective to slow the progress of infection relative to the rate of progress that would be observed in the absence of the soluble human CD7 protein.

Soluble CD7 may be produced by routine means using readily available starting materials as described above. The nucleic acid sequence encoding soluble CD7 as well as the amino acid sequence of the protein are well known. Complete nucleotide and amino acid sequences are reported in Schanberg, L.E. et al . (1991) Proc . Na tl . Acad. Sci . USA 88:603-607 and Aruffo, A and B. Seed (1987) EMBO ι7.6 (11) .3313-3316, each of which is incorporated herein by reference.

DNA molecules that encode CD7 may be isolated by those having ordinary skill in the art from readily available starting material routine techniques. Isolation of a DNA sequence encoding human CD7 permits the production of soluble human CD7 protein using recombinant techniques now known in the art as well as the design and production of truncated forms of human CD7 which are also soluble human CD7 proteins.

Nucleic acid molecules that comprise nucleotide sequences that encode human CD7 can be obtained from human genetic material or can be prepared chemically using nucleotide sequence synthesizer or other standard techniques. When the coding DNA is prepared synthetically, advantage can be taken of known codon preferences of the intended host where the DNA is to be expressed.

One having ordinary skill in the art can, using well known techniques, obtain a DNA molecule that comprises a nucleotide sequence that encodes a soluble human CD7 protein and insert that DNA molecule into a commercially available expression vector for use in well known expression systems. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, CA) may be used for production in E. coli . The commercially available plasmid pYES2 (Invitrogen, San Diego, CA) may be used for production in S. cerevisiae strains of yeast. The commercially available MaxBac™ (Invitrogen, San Diego, CA) complete baculovirus expression system may be used for production in insect cells. The commercially available plasmid pcDNA I (Invitrogen, San Diego, CA) may be used for production in mammalian cells such as Chinese Hamster Ovary cells.

One having ordinary skill in the art can use these commercial expression vectors systems or others to produce soluble human CD7 protein using routine techniques and readily available starting materials.

One having ordinary skill in the art may use other commercially available expression vectors and systems or produce vectors using well known methods and readily available starting materials. Expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art for a variety of hosts. See e . g. , Sambrook et al. , Molecular Cloning a Laboratory Manual , Second Ed. Cold Spring Harbor Press (1989) . Thus, the desired proteins can be prepared in both prokaryotic and eukaryotic systems, resulting in a spectrum of processed forms of the protein. The most commonly used prokaryotic system remains E. coli , although other systems such as B. subtiliε and Pseudomonas are also useful. Suitable control sequences for prokaryotic systems include both constitutive and inducible promoters including the lac promoter, the trp promoter, hybrid promoters such as tac promoter, the lambda phage PI promoter. In general, foreign proteins may be produced in these hosts either as fusion or mature proteins. When the desired sequences are produced as mature proteins, the sequence produced may be preceded by a methionine which is not necessarily efficiently removed. Accordingly, the peptides and proteins claimed herein may be preceded by an N-terminal Met when produced in bacteria. Moreover, constructs may be made wherein the coding sequence for the peptide is preceded by an operable signal peptide which results in the secretion of the protein. When produced in prokaryotic hosts in this matter, the signal sequence is removed upon secretion. A wide variety of eukaryotic hosts are also now available for production of recombinant foreign proteins. As in bacteria, eukaryotic hosts may be transformed with expression systems which produce the desired protein directly, but more commonly signal sequences are provided to effect the secretion of the protein. Eukaryotic systems have the additional advantage that they are able to process introns which may occur in the genomic sequences encoding proteins of higher organisms. Eukaryotic systems also provide a variety of processing mechanisms which result in, for example, glycosylation, carboxy-terminal amidation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth.

Commonly used eukaryotic systems include, but is not limited to, yeast, fungal cells, insect cells, mammalian cells, avian cells, and cells of higher plants. Suitable promoters are available which are compatible and operable for use in each of these host types as well as are termination sequences and enhancers, as e.g. the baculovirus polyhedron promoter. As above, promoters can be either constitutive or inducible. For example, in mammalian systems, the mouse metallothionene promoter can be induced by the addition of heavy metal ions.

The particulars for the construction of expression systems suitable for desired hosts are known to those in the art. For recombinant production of the protein, the DNA encoding it is suitably ligated into the expression vector of choice and then used to transform the compatible host which is then cultured and maintained under conditions wherein expression of the foreign gene takes place. The protein of the present invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art. One having ordinary skill in the art can, using well known techniques, isolate and purify the soluble human CD7 protein produced using such expression systems.

In addition to producing these proteins by recombinant techniques, automated amino acid synthesizers may also be employed to produce soluble human CD7 protein. If soluble human CD7 proteins are made synthetically, substitution by amino acids which are not encoded by a gene may also be made. Alternative residues include, for example, the ω amino acids of the formula H₂N(CH₂)_nC00H wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine (Sar) , t-butylalanine

(t-BuAla) , t-butylglycine (t-BuGly) , N-methyl isoleucine (N-

Melle) , and norleucine (Nleu) . Phenylglycine, for example, can be substituted for Trp, Tyr or Phe, an aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic. The conformation conferring properties of the proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp) .

The soluble human CD7 proteins can be formulated as pharmaceutical compositions which can then be administered to individuals infected with or suspected of being infected with HIV. Another aspect of the present invention provides antibodies, antibody fragments which specifically bind to human CD7 and peptides that specifically bind to human CD7 which include portions of antibodies that specifically bind to human CD7. As used herein, the term "fragments of antibodies" is meant to refer to Fab fragments, (Fab)₂ fragments, and any other truncated form of an antibody molecule which retains affinity to CD7 including peptides and proteins which comprise a portion of such antibodies. As used herein, the term "anti-CD7 antibodies" is meant to refer to: rodent-derived antibodies which specifically bind to human CD7 including murine antibodies which specifically bind to human CD7 and rat antibodies which specifically bind to human CD7; fragments of rodent-derived antibodies which specifically bind to human CD7 including fragments of murine antibodies which specifically bind to human CD7 and fragments of rat antibodies which.specifically bind to human CD7; human antibodies that specifically bind to human CD7; fragments of human antibodies which specifically bind to human CD7; humanized antibodies that specifically bind to human CD7; fragments of humanized antibodies which specifically bind to human CD7; chimeric antibodies that specifically bind to human CD7 and fragments of chimeric antibodies which specifically bind to human CD7.

One aspect of the invention is anti-CD7 antibodies. A preferred embodiments is monoclonal antibodies that specifically bind to human CD7. One aspect of the invention is hybridomas which generate monoclonal antibodies that specifically bind to human CD7.

Anti-CD7 antibodies may be used in soluble human CD7 protein purification assays and kits. For example, columns can be loaded or charged with anti-CD7 antibodies to produce column with immobilized anti-CD7 antibodies and samples which contain human CD7 protein such as cell extracts are passed through the column. Soluble human CD7 protein in the sample binds to the immobilized anti-CD7 antibodies and the column is washed of other components from the sample. The conditions in the column are then changed to bring about the release of the soluble human CD7 protein from the immobilized anti-CD7 antibody thereby allowing the pure soluble human CD7 protein to be collected. Those having ordinary skill in the art can readily apply standard techniques to purify soluble human CD7 protein using anti-CD7 antibodies.

When contacted cells that have CD7, anti-CD7 antibodies are useful as anti-HIV agents. Anti-HIV therapeutics which comprise anti-CD7 antibodies bind to membrane-bound CD7 on human cells which display CD7 and thereby prevent HIV particles from binding to the CD7. By preventing HIV from interacting with CD7 on the cell surface, HIV infection and cell to cell spread of HIV by HIV infected cells is inhibited.

Accordingly, one aspect of the present invention is a pharmaceutical composition comprising anti-CD7 antibodies in combination with a pharmaceutically acceptable carrier. Anti- CD7 antibodies are combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical compositions which can be administered in therapeutically effective amounts to persons diagnosed as or suspected of being infected with HIV in a method of treating such persons. Such persons are readily identifiable by those having ordinary skill in the art.

Another aspect of the present invention is a method of treating an individual suspected of being infected with HIV which comprises the step of administering to said individual a therapeutically effective amount of anti-CD7 antibodies.

As used herein, the term "effective amount of anti-CD7 antibodies" is meant to refer to the amount of anti-CD7 antibody necessary to inhibit HIV infection of cells in the individual and/or the amount of anti-viral material necessary to inhibit cell-cell spread of viral infection by inhibiting syncytium formation between infected and uninfected cells. Effective amounts include both the amount effective to eliminate the progress of infection as well as the amount effective to slow the progress of infection relative to the rate of progress that would be observed in the absence of the anti-CD7 antibodies.

Those having ordinary skill in the art can produce monoclonal antibodies which specifically bind to CD7 and are useful as anti-HIV therapeutics using standard techniques and readily available starting materials. The techniques for producing monoclonal antibodies are outlined in Harlow, E. and D. Lane, (1988) ANTIBODIES: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor NY, which is incorporated herein by reference, provide detailed guidance for the production of hybridomas which produce monoclonal antibodies which specifically bind to target proteins and the production of the monoclonal antibodies themselves. Briefly for example, human CD7 is injected into mice. The spleen of the mouse is removed, the spleen cells are isolated and fused with immortalized mouse cells. The hybrid cells, or hybridomas, are cultured and those cells which secrete antibodies are selected. The antibodies are analyzed and, if found to specifically bind to the protein of interest, the hybridoma which produces them is cultured to produce a continuous supply of antigen specific antibodies.

Another aspect of the present invention provides an isolated nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 and pharmaceutical compositions which contain such nucleic acid molecules. Nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 may be used in methods for modulating the activity of RNA that encodes human CD7. Accordingly, nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 relate to the field of "antisense" compounds, compounds which are capable of specific hybridization with a nucleotide sequence of an RNA molecule.

As used herein, the term "anti-CD7 antisense compound" is meant to refer to nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7. Anti-CD7 antisense are capable of specific hybridization with a nucleotide sequence of an RNA molecule and thereby block translation of mRNA that encodes CD7, thus inhibiting production of the protein.

The anti-CD7 antisense compounds of the present invention are useful to inhibit production of human CD7 in cells that would otherwise produce the protein. Anti-CD7 antisense compounds are useful in methods of inhibiting production of human CD7 and thereby making cells less infectable by HIV and less capable of forming syncytia with HIV infected cells. Anti-CD7 antisense compounds are useful to regulate gene expression, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA. Anti-CD7 antisense compounds of the present invention inhibit the production of human CD7 by interactions with molecules that direct their synthesis, intracellular RNA. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to CD7 production. One method for inhibiting specific gene expression which has been adopted to some degree is the "antisense" approach, where oligonucleotide analogues complimentary to a specific, target, messenger RNA, mRNA sequence are used. A number of workers have reported such attempts. Pertinent reviews include CA. Stein & J.S. Cohen, Cancer Research, vol. 48, pp. 2659-2668 (1988); J. Walder, Genes & Development, vol. 2, pp. 502-504 (1988); CJ. Marcus-Sekura, Anal. Biochemistry, vol. 172, 289-295 (1988); G. Zon, Journal of Protein Chemistry, vol. 6, pp-131-145 (1987); G. Zon, Pharmaceutical Research, vol . 5, pp. 539-549 (1988); A. R. Van der Krol, J.N. Mol, & A.R. Stuitje, BioTechniques, vol. 6, pp. 958-973 (1988) and D.S. Loose-Mitchell, TIPS, vol. 9, pp. 45-47 (1988) . Each of the foregoing provide background concerning general antisense theory and prior techniques.

Antisense compositions according to the present invention comprise oligonucleotide molecules which are complementary to the nucleotide sequence of the DNA molecule that encodes human CD7. The oligonucleotides in accordance with this aspect of the invention preferably comprise from about 5 to about 200 nucleotides. The oligonucleotides in accordance with this aspect of the invention more preferably comprise from about 5 to about 50 nucleotides. It is more preferred that such oligonucleotides comprise from about 8 to 25 nucleotides, and still more preferred to have from about 12 to 25 nucleotides.

The oligonucleotides used in accordance with this aspect of the invention may be conveniently and routinely made through the well-known technique of solid phase synthesis using the information provided in Aruffo, A and B. Seed (1987) EMBO J. 6 (11) :3313-3316, which has been incorporated herein by reference, between nucleotide 50 and nucleotide 1480. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed, however the actual synthesis of the oligonucleotides are well within the talents of the one having ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. In accordance with this aspect of the present invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5' -untranslated region, the 3' -untranslated region, the 5' cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. In preferred embodiments, the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation site, an intron/exon junction or sequences in the 5'- or 3'- untranslated region or 5' cap region. Oligonucleotides useful in the invention are complementary to the DNA or to the corresponding messenger RNA (mRNA) or pre-messenger RNA. Thus, the oligonucleotides in accordance with the invention preferably have one of the foregoing sequences or an effective portion thereof. Thus, it is preferred to employ any of these oligonucleotides as set forth above or any of the similar nucleotides which persons of ordinary skill in the art can prepare from knowledge of the preferred antisense targets for the modulation of CD7 production. Pharmaceutical compositions according to the invention comprise a pharmaceutically acceptable carrier in combination with an active ingredient selected from the group consisting of soluble human CD7 protein, anti-CD7 antibodies and anti-CD7 antisense molecules. The pharmaceutical compositions of the invention may be formulated by one having ordinary skill in the art with compositions selected depending upon the chosen mode of administration. Suitable pharmaceutical carriers are described in the most recent edition of Remington 's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

The active ingredient is formulated based upon the nature of the ingredient and how it is to be administered. For parenteral administration, the active ingredient can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) . The formulation is sterilized by commonly used techniques. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution. The pharmaceutical compositions according to the present invention may be administered as a single doses or in multiple doses. The pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. For example, soluble human CD7 protein may be administered in conjunction with soluble CD4 and/or AZT. The treatments of the present invention may be combined with conventional therapies, which may be administered sequentially or simultaneously.

The pharmaceutical compositions may be administered by any means that enables the active agent to reach the agent's site of action in the body. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption. Intravenous is the preferred route of administration.

The dosage administered varies depending upon factors such as: pharmacodynamic characteristics; its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment; and frequency of treatment. Usually, a daily dosage of soluble human CD7 protein can be about 1 μg to 100 milligrams per kilogram of body weight. Ordinarily 0.5 mg to 50 mg, and preferably 1 mg to 10 mg per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results. Similarly, a daily dosage of anti-CD7 antibody can be about 5 μg to 5000 mg of antibody. In some preferred embodiments, 50 μg to 500 mg of antibody may be administered. In other preferred embodiments, 500 μg to 50 mg of antibody may be administered. In a preferred embodiment, 5 mg of antibody is administered. The pharmaceutical compositions may be administered in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.

According to another aspect of the invention, transgenic animals, particularly transgenic mice, are generated. The transgenic animals according to the invention contain a nucleic acid molecule which encodes human CD7. Such transgenic mice may be used as animal models for studying HIV infection and for use in drug evaluation and discovery efforts to find compounds effective to prevent or impede HIV infection mediated by CD7. Transgenic animals according to the present invention may further comprise a nucleic acid molecule that encodes human CD4. One having ordinary skill in the art using standard techniques, such as those taught in U.S. Patent No. 4,873,191 issued October 10, 1989 Wagner and U.S. Patent No. 4,736,866 issued April 12, 1988 to Leder, both of which are incorporated herein by reference, can produce transgenic animals which produce the human CD7 and use the animals in drug evaluation and discovery projects. Example

A major cytopathic effect seen upon in vi tro infection of CD4+ human T cells by the human immunodeficiency virus (HIV) is cell-to-cell fusion which results in giant cell (or syncytium) formation. Membrane fusion is required for infection by cell-free virions and in syncytium formation. In vi tro syncytium formation is considered to be a model for cell-cell transmission of infection. The human T cell surface molecule, CD7, has been discovered to be important for fusion process. CD7 is a roughly 40 kDa glycoprotein member of the immunoglobulin supergene family that is expressed early in the ontogeny of thymocytes and on the majority of peripheral blood T cells, as well as on natural killer (NK) cells and a small subpopulation of B cells. Anti-CD7 monoclonal antibodies inhibited HIV-1 induced cell-cell fusion in several CD4+ T cell lines tested, (SupT 1, HuT-7 8, and CEM-SS) . The anti-syncytial activity of the CD7 antibodies is not due to crossreactivity with CD4 or with viral proteins. Epitope mapping revealed at least two regions of the molecule which are important for this effect. Cells rendered CD7- are poorly infectable by cell-free virus. Additionally, cells rendered CD7- are more easily inhibited from fusing in syncytium formation assays. The collective results support a central role for human CD7 in the process of HIV infection.

As in cell-free infection, the major lymphocyte receptor for gpl20 utilized in cell-to-cell infection resulting in syncytium formation is CD4. Binding of gp 120 depends primarily upon a short (approximately 20 amino acid) region of the first domain of CD4 which is structurally similar to an immunoglobulin kappa light chain CDR2 loop. A second region of CD4 is also suggested to play a role in gpl20 binding CD4 not only serves as a receptor but appears to induce a conformational shift in gp 120 which increases the antibody accessibility of the V3 loop of gp 120 and of certain gp4l epitopes. Furthermore, the N-terminal region of the transmembrane gp4l includes a hydrophobic 'spike' . Following gpl20-CD4 binding, this 'spike' is believed to protrude into the target membrane and facilitate membrane fusion by bringing the two membranes into close apposition. A separate receptor molecule for gp41 has been hypothesized to be important for fusion to occur.

In contrast to CD4, the leukocyte adhesion molecule, LFA-1, has been shown to be important in mediating syncytium formation but not infection by cell-free virions. Syncytium formation can be prevented by treatment of target PHA blasts with several anti-LFA-1 antibodies. Anti-LFA-1 antibody inhibition of syncytium formation in T cell lines CEM and Jurkat has been reported, but inhibition of fusion by the same antibodies has not been observed in other T cell lines, including SupTl. The inability of anti-LFA-1 antibodies to inhibit syncytium formation of certain T cell lines may relate to the function of LFA-1 as an adhesion molecule. It is likely that cell to cell adhesion is a prerequisite for syncytia formation. Accordingly, different T cell lines or even different subclones of the same line may demonstrate differences in their ability to form cell-cell conjugates and that only certain cell lines can prevented frog forming fusion permissive contact through antibodies directed against LFA-1 alone. The failure of metabolic inhibitors such as cycloheximide and aphidicolin to block cell-cell fusion following viral infection suggests that cell surface adhesion itself and not an associated signaling function is necessary for syncytium formation.

Antiserum directed against the T cell surface and recognizing several membrane associated proteins has been reported to be able inhibit cell fusion driven by the HIV envelope glycoproteins. This antiserum did not react with the human CD4 antigen. A multimolecular complex upon immunoprecipitation of human T cell lysates with a soluble form of the external domain of HIV-1 gp41 or HIV-2 gp36 has been identified. The present invention is based upon the discovery that CD7, a 40 kDa immunoglobulin supergene family member expressed early in the ontogeny of thymocytes and on the majority of peripheral blood T cells is an accessory molecule for HIV entry. Anti-CD7 but not anti-LFA-1 monoclonal antibodies blocked cell-cell fusion in three CD4+ T cell lines tested, (SupTl, HuT-78, and CEM-SS) . At least two distinct epitopes on the cell surface marker CD7 have been observed to be important for HIV-1 mediated syncytium formation. SupT 1 cells made negative for the syncytia-related CD7 epitopes were able to fuse upon exposure to HIV infected cells. However, in contrast to the parental SupTl cells, the CD7 epitope-negative cell lines were now sensitive to inhibition by anti-LFA-1 antibodies. Furthermore, the CD7-cells were resistant to cell- free HIV-1 infection by both the III_B and MN isolates. These data support a central role for CD7 in the HIV infective process. MATERIALS AND METHODS:

Cells and Viral Isolates. H9/III_B and H9/MN were obtained from the AIDS Reference Reagent Repository (Frederick MD) . Sup Tl, CEM-SS, SP2/0 and H9 cells were obtained from the American Type Culture Collection (ATCC; Rockville MD) . Cell lines were maintained at 37oc and 5% C0₂ in RPMI 1640 or Kennett's HY media (JRH Biosciences; Lenexa KS) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan UT) and L- glutamine. Transfected cells including the SP2-CD4 cell line

(30) were maintained in the above media with added Geneticin

(G418; Gibco, Grand Island MI) for selection of drug-resistant clones. Virus stocks were prepared and TCID50 values were determined by titration upon MT-2 target cells as described in Wang, B., et al . (1993) Proc . Natl . Acad. Sci . USA 90:4156, which is incorporated herein by reference.

Antibodies. Leu3a (Engleman, E.G., et al. J. Exp. Med. 154:193, which is incorporated herein by reference) , an anti-CD4 antibody with established anti-syncytial activity, was obtained from AIDS Reference Reagent Repository (Frederick MD) . Monoclonal anti-CD7 ascites were provided by other investigators as follows: B-F12 by Dr. John Wijdenes (Centre Regional de Transfusion Sanguine) ; CD7-6B7 by Dr. 0. Majdie (Institut Fur Immunologie der Universitat Wein) ; 124-1D1, 142- 9, and 142-24 by Dr. R. Villela (Servei d' Immunologia, Hospital 5 Clinic) ; 63.7 by Dr. Malek Kamoun (University of Pennsylvania) ; and N-170A by Dr. M. Agadganyan (Institute of Viral Preparation) . Ascites antibody to beta-2-macroglobulin (BBMI) as well as concentrated supernatants of anti-LFA-1 antibodies TSl/18 and TSl/22 were provided by Dr. B. Perussia (Thomas

10 Jefferson University) . Anti-LFA-1 antibodies H52 and MHM.24 were the kind gift of Dr. J. Hildreth (The Johns Hopkins Medical Institute) . Purified anti-CD7 antibody 3A1, 3A1-FITC conjugated antibody, and control mouse IgG were purchased from Sigma Chemicals.

15. Syncytia Inhibition Assay. To analyze the effect of the various antibodies on HIV-1 fusion, CD4+ T cell lines SupTl, Hut-78, or CEM-SS were used as target cells for infection. Two-fold dilutions of antibodies were made in 96 well plates in RPMI 1640 media containing 10% fetal calf serum

20 at final concentrations of typically 500 to 16 μg/ml. For certain antibodies, higher two-fold dilutions were tested. HIV-1 infected cells (MN or III_B isolate) were then plated at a density of approximately 10⁴ cells per well. SupTl target cells were added (5 X 10⁴ well) and syncytium formation was

25 qualitatively and quantitatively determined after a 2-day incubation period as described in Weiner, D.B., et al . (1991) Pathobiology 4:1, which is incorporated herein by reference.

Flow Cytometric Analysis. 2-4 x 10⁵ cells per sample were analyzed exactly as described in Osther, K., et al . (1991)

30 Hybridoma 10:673, which is incorporated herein by reference. Briefly, for surface marker analysis, cells were washed with FACS buffer (1% bovine serum albumin, 0.1% sodium azide in phosphate buffered saline) and resuspended in 100 μl total volume. Primary antibody was added to each sample and allowed

35 to bind for 30 minutes on ice. After washing, FITC-conjugated goat anti-mouse whole immunoglobulin antiserum (Fisher Scientific; Pittsburgh PA) was added at 1:200 dilution in FACS buffer and incubated again for 30 minutes on ice. Cells were then washed twice with FACS buffer and then resuspended in PBS containing 1% paraformaldehyde and fixed. The cells were washed and resuspended in FACS buffer prior to flow cytometric analysis.

Epitope Mapping. To determine if the anti-CD7 antibodies recognized overlapping epitopes, two approaches were taken. 2 x 10⁵ SupTl cells were preincubated in 100 μl of 1:50 diluted antibody in FACS buffer on ice for 30 minutes. Cells were washed twice with FACS buffer and then incubated another 30 minutes on ice with 100 μl 1:200 diluted 3A1-FITC in FACS buffer or 1:10 diluted ¹⁵I-B-F12 antibody. (¹²⁵1 labeling of antibody was performed by the lactoperoxidase method) . After washing 3 times with FACS buffer, cells were analyzed by flow cytometry (3A1 binding) . Alternatively, the wells containing treated cells were dried and counted separately in a Beckman gamma counter.

Soluble CD4 ELISA Assay. Dynatech ELISA 96 well plates were precoated with 50 μl of a 1 μg/ml solution of either BSA or soluble CD4 (Repligen, Cambridge MA) in carbonate-bicarbonate buffer (pH 9.6) at 4©c overnight. Plates were washed 5 times with 0.05% Tween 20 in PBS to remove free protein and blocked against non-specific binding by incubation of 200 μl FACS buffer without azide for 1 hour at 37oc Ten- fold dilutions of primary antibody in PBS were allowed to bind for 1 hour at 37oc before washing and addition of 100 μl 1:1000 diluted goat anti-mouse IgG-HRPO (Fisher Scientific; Pittsburgh PA) . Color development was performed using o-phenylenediamine dihydrochloride (OPD) substrate and optical densities of the wells at 492 nm were determined on a Dynatech MR5000 ELISA plate reader.

Construction of CD7 Expressing Cells. The plasmid pCDM7-CD7-28 (Aruffo, A., and B. Seed (1987) EMBO J. 6:3313, which is incorporated herein by reference) which contains a cDNA clone of CD7, and pCDM8 (a vector similar to the pCDM7 vector except that it lacks the polyoma ori) , were kindly provided by Dr. B. Seed (Massachusetts General Hospital) . One μg of column purified plasmid DNA (Qiagen Inc., Chatsworth CA) was used to transfect C0S7 cells by standard DEAE dextran/chloroquine methods (Kriegler, M. (1990) Gene transfer and expression : a laboratory manual . Stockton Press, New York, which is incorporated herein by reference. Cells were washed extensively following transfection and grown for 48 hours at 37oC before analysis.

Selection of CD7-Cells. Cells were rendered cell surface antigen-negative by the method of Hillman et al . (1990) J. Immun . 6:2131, which is incorporated herein by reference. Briefly, SupT 1 cells at a density of 2 x 10-cells/mi were grown in 25 ml of a 1:5000 dilution of ethyl-methanesulfonate (EMS) in RPMI/10% FBS medium for 18 hours. After washing three times in DMEM/2% FBS, cells were resuspended in RPMI/10% FBS and allowed to recover for 48 hours prior to selection. Cells expressing CD7 on their surface were removed by binding to M450-magnetic beads (Dynal) coated with anti-CD7 antibody 3A1 as per manufacturer's instructions. Negative cells were cloned by limiting dilution and subclones which were consistently negative for CD7 in flow cytometric analysis were employed in further experiments.

Cell-free virus infection assays. In a 96 well round bottom plate, 5 X 10⁴ SupTl target cells/well were added to 100 TCID50 cell-free virus (HIV-1 III_B or HIV-lr_ø,) in a total volume of 100 μl. Plates were incubated for 1 hour at 37<"-C Cells were washed twice in IX Dulbecco's PBS, then trypsinized for 15 min. at 37oc to remove any bound virus that had not penetrated the cell. Cells were washed and resuspended in fresh media. Samples of culture supernatant were collected every five days for analysis by the "mini" reverse transcriptase assay described in Willey, R.L. , et al . (1988) J^". Virol . 62:139, which is incorporated herein by reference RESULTS: Identification of Antibodies Able to Inhibit SupTl Fusion by HIV-1

It is possible to measure one aspect of HIV-1 cytopathicity by assaying the ability of the viral envelope proteins, gpl20 and gp41, to mediate cell membrane fusion resulting in syncytium formation. Syncytium Inhibitory activity for HIV-1 isolate III_B was therefore assessed using SupTl target cells. To date, published reports of inhibitory antibodies against non-CD4 molecules have implicated cell surface proteases and LFA-1 as important for virally mediated fusion. The broadly reactive polyclonal anti-human cell membrane antisera could block HIV-driven syncytium formation has been reported (Weiner, D.B., et al . (1989) Vaccines Cold Spring Harbor Press:115, which is incorporated herein by reference) . Accordingly, a panel of more than 150 monoclonal antibodies and antisera was screened against human hematopoietic cell surface markers in this assay. Anti-CD7 antibody CD7-6B7 had significant anti-syncytial activity, inhibiting syncytium formation by 50% at a concentration of 8 μg/ml. (Figure 1, panel A) Characterization of Anti-CD7 Antibody Syncytium Inhibition.

A number of different isolates of HIV-1 have been described. At the amino acid level, variability between the sequences of the envelope proteins of isolates can be greater than 60% although considerably less in conserved regions. To test if the inhibitory activity of CD7-6B7 was restricted to the particular viral isolate, ascites were utilized in in vitro syncytial assays of HIV-lr_ø, on SupTl target cells. Significant reduction in the number of syncytia in the presence of CD7-6B7 but not control mouse immunoglobulin was seen at approximately 48 hours in replicate experiments (Figure 1, panel B) .

Additional anti-CD7 antibodies were tested for anti- syncytial activity against HIV-1 III_B. Eight anti-CD7 antibodies were tested in all. Interestingly, a range of inhibitory activity was seen, with CD7-6B7 having the greatest activity and antibody N-170A having no inhibitory activity in numerous experiments. In contrast, anti-LFA-1 antibodies MHM.24, TS1/18, TSl/22, and H52 do not inhibit syncytium formation in this system (Table I) , suggesting SupT 1 cell fusion does not depend upon this adhesion molecule. Different T Cell lines Also Show CD7 Dependence in Fusion T cell lines differ in their ability to form syncytia in vitro. In our hands, SupTl cells in the presence of virus infected cells form large syncytia distributed throughout the tissue culture plate within 24-48 hours of coculture. HuT-78 normally grow as small clumps and upon addition of H9/III_B cells will form more numerous medium-sized syncytia with similar kinetics as seen in SupTl cells. In contrast, CEM-SS cells grow as single cells and form fewer, very small syncytia at a slower rate, (48-72 hours) . All three cell lines show strong reactivity on FACS analysis with Leu3a and 3A1. Inhibition of HIV-1 III_B syncytium formation by anti-CD7 antibodies was observed in all three of these different cell lines at similar dilutions of antibodies (Table II) . CD7 Reagents Do Not Crossreact with CD4 CD7 is a member of the immunoglobulin supergene family, with a gene structure most similar to Thy-1. Structural analysis suggests that while the CD7 protein most closely resembles kappa light chain structures, it possesses both structural as well as limited amino acid homology to CD4. Experiments were designed to rule out the possibility that the anti-syncytial activity of the effective anti-CD7 antibodies was due to crossreactivity of the antibodies for CD4 and therefore an artefact of the concentrations of antibody used in the system. To assess the ability of the antibodies to bind CD4, two types of binding assays were employed. Solid-phase enzyme-linked immunosorbant assay (ELISA) against a recombinant soluble CD4 protein (Repligen; Cambridge MA) demonstrated no significant crossreactivity of the antibodies for CD4. Antibodies were also tested for their ability to bind to CD4 in flow cytometric assays. Antibody reactivity was compared with a mouse ly phoma cell line, SP2/0, and the stably transfected SP2-CD4 cell line (30) which expresses high levels of human CD4. No significant binding to the CD4+ cells was noted for any of the CD7 antibodies tested (inhibitory or non-inhibitory) whereas strong reactivity was seen for these cells with Leu3a. The converse situation was also examined: CD7+CD4-cells were used to verify that our antibody preparations recognized CD7 and that a CD4-like epitope (that recognized by Leu3a) with which the anti-CD7 antibodies might bind by crossreactivity was not present on CD7. A pCDM7-CD7 plasmid construct was transfected into COST cells by standard DEAE-dextran techniques. Strong reactivity with anti-CD7 antibodies 3A1 and 142-9 but not Leu3a was detected in transfected cells when compared to mock or vector (pCDM8) transfected control cells. CD7 Reagents Do Not Appear to Directly Bind Virion Components It was possible that the anti-CD7 antibodies were not binding the target cells in the fusion assay but were binding instead to either CD7 molecules expressed on the surface of the infected cells (H9/III_B or H9/MN cells) or to crossreactive epitopes located on the viral proteins. Comparison of the HIV- 1 III_B cells with the SupT 1 target cells (Table III) strongly suggests that the anti-syncytial activity of the anti-CD7 antibodies is due to interaction with CD7 on the target cells. Similarly, the antibodies did not show any reactivity with viral protein lysates on Western blot analysis. Evidence For At Least Two Fusion-Related Epitopes Having excluded potential confounding crossreactivities, portion(s) of the CD7 molecule which were functionally important for fusion were determined. As an initial effort in this direction, a panel of antibodies were mapped epitopically by competition studies. ¹²⁵I-labeled B-Fl 2 was seen to be effectively competed for binding to SupTl cells by all other anti-CD7 antibodies but not by Leu3a or by antibodies to beta-2-macroglobulin or transferrin receptor. This result is consistent with previous studies which suggested that the epitope recognized by the 3A1 antibody is immunodominant. On performing similar studies with FITC- labeled 3A1 antibody, competition for binding was seen for all anti-CD7 antibodies except for CD7-6B7 (Figure 2) . The failure of these two antibodies to compete with each other for binding is particularly intriguing as CD7-6B7 is able to inhibit syncytium formation at significantly higher dilutions than the other antibodies studied. These results suggest that at least two epitopes on the CD7 molecule are important for inhibition of fusion.

Analysis of CD7-Epitope Negative T Cells in Syncytium Formation To further examine the role of CD7 in cell fusion events, CD7-cell lines were constructed by EMS mutagenesis of SupTl cells, followed by antibody selection with antibody 3A1 and limiting dilution cloning. The CD7-status of the derived cell lines was confirmed by FACS analysis with anti-CD7 antibodies 3A1, B-F 12 and CD7-6B7 (Figure 3) . Further FACS analysis was used to examine the cell surface phenotype of the CD7-cell clones. CD7-clone F8.E5 clearly remained CD4+ CDlla+ CD18+ as determined by reactivity with monoclonal antibodies Leu3a, TS1/22, and TSl/18, respectively (Figure 3) . The morphology and rate of proliferation (Figure 4) of the CD7-cell line was indistinguishable from those of the parental SupT 1 cell line (Figure 4) .

Using this clonal cell line, the ability of CD7-SupT 1 cells to fuse with HIV-1 infected cell lines was assessed. Both CD7- and CD7+ cell lines fused with H9 cells chronically infected with isolates III_B, RF, and MN. The size and number of syncytia formed by the CD7+ and CD7-cells were comparable. The ability of anti-CD4 and anti-LFA-1 antibodies to inhibit syncytium formation in F8.E5 cells was assayed next. Although no anti-syncytial activity was observed for anti-LFA-1 antibodies TSl/22 and TSl/18 for the parental SupTl cell line (Figure 5, panel A) , significant inhibition of syncytium formation was observed for the CD7- F8.E5 clone at the same concentration of antibody (Figure 5, panel B) . Fusion inhibition was seen for both the CD7+ and CD7- cells cultured in the presence of Leu3a but not control mouse immunoglobulin (Figure 5) . This dramatic change in the activity of the anti- LFA-1 antibodies strongly supports the role of CD7 as an accessory molecule for HIV fusion. Analysis of CD7-Epitope Negative T Cells in Cell-Free Infection The role of CD7 in cell-free infection by HIV-1 was examined. If the role of CD7 in syncytium formation was simply to provide additional adhesion sites between cells, then, in to the LFA-1 findings, no effect upon cell-free infection would be expected. The increased dependence of syncytium formation on LFA-1 in the CD7- SupTl cell line would thus reflect the loss of CD7-mediated adhesion: syncytium formation in CD7+ SupTl target cells would not be inhibited by anti-LFA-1 antibody as the presence of an alternative adhesion molecule (CD7) on the cell surface would allows for sufficient contact points for fusion with the infected cells, thereby overriding participation by LFA-1. Surprisingly, the role of CD7 in HIV infection appears to be more complex than that of an adhesion molecule. Direct comparison of the abilities of CD7+ and CD7- cells to be infected by cell-free virus revealed striking differences in infectivity between the two lines. As measured by both an early (syncytium formation) and a late marker (reverse transcriptase activity in culture media) of infection, the CD7- SupTl cells demonstrated no evidence of infection using as much as 100 TCID50's of HIV-1 III_B or of HIV-lr_ø, at up to 15 days following addition of the virus. In contrast, the CD7+SupTl cell line was infectable with as little as 12.5 TCID50's with evidence of infection observed by 5 days. This dramatic change in the susceptibility of the T cell line to undergo cell-free infection by HIV suggests that CD7 is important direct infection of cells by cell-free virus whereas its role in syncytium formation is more complex.

TABLE I

ANTIGEN ANTIBODY FINAL CONCENTRATION GIVING > 50% INHIBITION (MG/ML)

CD4 Leu3a <0.004

CD7 CD7-6B7 0.008 CD7 B-F12 0.500 CD7 124-1D1 0.125 CD7 142-9 0.125 CD7 142-24 0.125

CD7 63.7 0.250 CD7 N-170A NI (0.500) CD7 3A1 0.031

LFA- •1 (CDlla) TS1/22 NI (0.500)

LFA- ^■1 (CDlla) MHM-24 NI (0.500)

LFA- ^■1 (CD18) TSl/18 NI (0.500)

LFA- ^■1 (CD18) H52 NI (0.500)

Table 1: Antibody inhibition of cell fusion. Lowest concentration (mg/ml) at which H9/IIIB induced syncytium formation in SupTl target cells is inhibited by monoclonal anti-lymphocyte antibodies. NI = no inhibition seen at any concentration; highest concentration tested given in parentheses.

TABLE II

ANTIBODY SUPT1 HUT- 78 CEM-SS

CD7-6B7 0.008 0 . . 008 0.004 63.7 0.250 0 . . 125 0.125 3A1 0.031 0 . . 031 0.015

Table II: Comparison of anti-CD7 antibody syncytium inhibition on different target cell lines. The lowest concentration (in mg/ml) at which > 50% inhibition of syncytium formation relative to mouse serum IgG control is observed in the CD4+ CD7+ human T cell lines SupTl, HuT-78, and CEM-SS are given for three anti-CD7 antibodies tested.

TABLE III

ANTIBODY SUPT1 H9/IIIB

Secondary only (neg. control) 9 11 Leu3a (anti-CD4) 415 13

BBMI (anti-beta2microglobulin) 72 125 3A1 (anti-CD7) 540 17 63.7 (anti-CD7) 255 15

Table III: FACS staining of target and infected cell lines. 2 X 10^s cells per sample were preincubated with antibody as listed above prior to treatment with goat anti-mouse IgG-FITC secondary antibody. After washing and paraformaldehyde fixation, cells were analyzed on a FACScan machine (Becton- Dickinson) . Mean channel number for each sample is shown.

Claims

Claims

1. Essentially pure soluble human CD7 protein.

2. A pharmaceutical composition comprising: a) the soluble human CD7 protein of claim 1; and b) a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2 wherein said pharmaceutical carrier is acceptable for intravenous administration.

4. A hybridoma cell line which produced monoclonal antibodies that bind to human CD7.

5. A monoclonal antibody that binds to human CD7.

6. A pharmaceutical composition comprising: a) a monoclonal antibody of claim 5; and b) a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6 wherein said pharmaceutical carrier is acceptable for intravenous administration.

8. A method of treating an individual suspected of being infected by human immunodeficiency virus comprising the steps of administering a pharmaceutical composition that comprises a) a pharmaceutically acceptable carrier; and b) an active ingredient selected from the group consisting of a soluble human CD7 protein; and an anti-CD7 antibody.

9. The method of claim 8 wherein the active ingredient is a soluble human CD7 protein.

10. The method of claim 8 wherein the active ingredient is an anti-CD7 antibody.

11. The method of claim 10 wherein the anti-CD7 antibody is a monoclonal antibody.

12. The method of claim 8 wherein said pharmaceutical composition is administered intravenously.

13. The method of claim 8 further comprising the step of administering a second anti-HIV therapeutic.

14. The method of claim 13 wherein said second anti-HIV therapeutic is AZT.

15. The method of claim 13 wherein said second anti-HIV therapeutic is soluble CD4.

16. The method of claim 8 further comprising the step of administering two additional anti-HIV therapeutics.

17. The method of claim 16 wherein said two additional anti-HIV therapeutics are AZT and soluble CD4.

18. An isolated nucleic acid molecule comprising a nucleotide sequence that is complementary to mRNA which encodes human CD7, said nucleotide sequence consisting of 5 to 200 nucleotides.

19. The nucleic acid molecule of claim 18 wherein said nucleotide sequence is 5 to 50 nucleotides.

20. The nucleic acid molecule of claim 18 wherein said nucleotide sequence is 8 to 25 nucleotides.

21. The nucleic acid molecule of claim 18 wherein said nucleotide sequence is 12 to 25 nucleotides.

22. A pharmaceutical composition comprising a nucleic acid molecule according to claim 18 and a pharmaceutically acceptable carrier.

23. A method of inhibiting transcription of a human CD7 gene in a human cell comprising contacting said human cell with a nucleic acid molecule according to claim 18.

24. A non-human transgenic mammal, essentially all of the somatic cells of which comprise a nucleotide sequence that encodes human CD7 operably linked to regulatory sequences required for expression of said nucleotide sequence in said cells, wherein said cells produce human CD7 protein.

25. The transgenic mammal of claim 24 wherein said mammal is a mouse.

26. The transgenic mammal of claim 25 wherein said mammal further comprises human CD4.